Structural Basis for Simvastatin Competitive Antagonism ofComplement Receptor 3*

Received for publication, April 13, 2016, and in revised form, June 4, 2016 Published, JBC Papers in Press, June 23, 2016, DOI 10.1074/jbc.M116.732222

Maria Risager Jensen‡1, X Goran Bajic§¶1, Xianwei Zhang‡1, Anne Kjær Laustsen�**, Heidi Koldsø�**‡‡2,X Katrine Kirkeby Skeby�**‡‡, X Birgit Schiøtt�**‡‡, X Gregers R. Andersen§¶, and Thomas Vorup-Jensen‡¶**§§3

From the Departments of ‡Biomedicine, §Molecular Biology and Genetics, and �Chemistry, the **Interdisciplinary NanoscienceCenter (iNANO), the ‡‡Center for Insoluble Protein Structures (inSPIN), the ¶Lundbeck Foundation Nanomedicine Center forIndividualized Management of Tissue Damage and Regeneration (LUNA), and the §§MEMBRANES Research Center, AarhusUniversity, DK-8000 Aarhus C, Denmark

The complement system is an important part of the innateimmune response to infection but may also cause severe com-plications during inflammation. Small molecule antagonists tocomplement receptor 3 (CR3) have been widely sought, but astructural basis for their mode of action is not available. Wereport here on the structure of the human CR3 ligand-binding Idomain in complex with simvastatin. Simvastatin targets themetal ion-dependent adhesion site of the open, ligand-bindingconformation of the CR3 I domain by direct contact with thechelated Mg2� ion. Simvastatin antagonizes I domain binding tothe complement fragments iC3b and C3d but not to intercellu-lar adhesion molecule-1. By virtue of the I domain’s wide distri-bution in binding kinetics to ligands, it was possible to identifyligand binding kinetics as discriminator for simvastatin antago-nism. In static cellular experiments, 15–25 �M simvastatinreduced adhesion by K562 cells expressing recombinant CR3and by primary human monocytes, with an endogenous expres-sion of this receptor. Application of force to adhering mono-cytes potentiated the effects of simvastatin where only a 50 –100nM concentration of the drug reduced the adhesion by 20 – 40%compared with untreated cells. The ability of simvastatin to tar-get CR3 in its ligand binding-activated conformation is a novelmechanism to explain the known anti-inflammatory effects ofthis compound, in particular because this CR3 conformation isfound in pro-inflammatory environments. Our report points tonew designs of CR3 antagonists and opens new perspectives andidentifies druggable receptors from characterization of theligand binding kinetics in the presence of antagonists.

The complement system is an important part of innateimmunity and serves both as a first line of defense against mul-

tiple infectious agents and in guiding the adaptive immuneeffector and memory responses. However, complement activa-tion also contributes to the morbidity of diseases involvingchronic and acute inflammatory responses (1). Inhibitors ofcomplement activation and its effector functions have beenwidely sought. Recent progress supports the clinical signifi-cance of these strategies. Nevertheless, there are still consider-able unmet clinical needs for short and long term modulation ofleukocyte responses to complement activation (1, 2).

Complement receptor (CR3)4 (also known as Mac-1, integrin�M�2, or CD11b/CD18) is a member of the �2 (CD18) integrinfamily. Integrins are heterodimeric cell surface receptors,composed of an � and a � subunit, each with a single transmem-brane-spanning domain and an intracellular tail. CR3 isexpressed in myeloid cells, notably neutrophil granulocytes andmacrophage subsets, with major roles in leukocyte extravasa-tion and phagocytosis (3). CR3 contributes to atherosclerosis(4), and the severity of acute ischemic stroke correlates with thelevel of CR3 expressed on monocytes (5). Inhibition of CR3expressed in microglial cells limits synaptic loss in animal mod-els of Alzheimer disease (6). Taken together, the significantinvolvement of CR3 in multiple pathogenic mechanisms pointsto this receptor as a promising target for anti-inflammatorytherapy.

The major ligand-binding site in CR3 is the �M subunit-in-serted domain (�MI), so named because it is inserted, insequence, between the N and C termini of the larger, seven-bladed �-propeller domain (7). For both CR3 and the other Idomain-carrying integrins, these domains have been useful incharacterizing ligand interactions (8). Recent structural studieshave shown that integrin I domains are tethered on the top ofthe head piece, formed by the � subunit �-propeller domainand domains of the � subunit (9). In the case of CR3, there isevidence that parts of the �-propeller domain may contributeto the recognition of certain large ligands, such as the comple-ment fragment iC3b (10, 11). The flexible attachment of the Idomain to the rest of the integrin ectodomain allows greateraccessibility for ligand recognition, which makes it a majordeterminant for binding (9, 10). The I domains adopt at least

* This work was supported by the Danish Multiple Sclerosis Association, theAarhus University Research Foundation (to T. V.-J.), the Lundbeck Founda-tion (to T. V.-J. and G. R. A.), the Danish Council for Independent Research:Technology and Production Sciences (to B. S.), and Danish NationalResearch Foundation Grant DNFR59 (to B. S.). The authors declare that theyhave no conflicts of interest with the contents of this article.

The atomic coordinates and structure factors (code 4XW2) have been depositedin the Protein Data Bank (http://wwpdb.org/).

1 These authors contributed equally to this work.2 Present address: D. E. Shaw Research, New York, NY 10036.3 To whom correspondence should be addressed: Biophysical Immunology

Laboratory, Dept. of Biomedicine, Aarhus University, Bartholin Bldg.(1240), Bartholins Alle 6, DK-8000 Aarhus C, Denmark. Tel.: 45-8716-7853;Fax: 45-8619-6128; E-mail: vorup-jensen@biomed.au.dk.

4 The abbreviations used are: CR3, complement receptor 3; �LI, �L chain Idomain; �MI, �M chain I domain; ECIS, electric cell-substrate impedancesensing; ICAM, intercellular adhesion molecule; LFA, lymphocyte function-associated antigen; MD, molecular dynamics; MIDAS, metal-dependentadhesion site; SPR, surface plasmon resonance.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 33, pp. 16963–16976, August 12, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

AUGUST 12, 2016 • VOLUME 291 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 16963

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

by guest on January 9, 2021

http://ww

w.jbc.org/

Dow

nloaded from

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

by guest on January 9, 2021

http://ww

w.jbc.org/

Dow

nloaded from

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

by guest on January 9, 2021

http://ww

w.jbc.org/

Dow

nloaded from

two conformations, the open and the closed, based on x-raycrystallographic studies of the �MI (12, 13). Mutagenesis laterconfirmed that the open conformation binds ligands with con-siderably higher affinity than the closed conformation (8).

The mode of CR3 ligand recognition is complex and includesthe binding to several protein and non-protein biomacromol-ecules (14). Experimental investigations point to a simple car-boxylic acid as minimal ligand for the �MI (15). In its carboxy-late form, it becomes a part of the Mg2� coordination sphere inthe metal ion-dependent adhesion site (MIDAS) of the �MIopen conformation (12, 13). In consequence, �MI binds manyligands through a heterogeneous ensemble of stronger andweaker interactions (15–18). This is possible through a rela-tively high affinity for carboxylates, further regulated by thepresentation of these carboxylates in folded macromolecules(15, 16). Simple equilibrium-based analyses will, however, onlyreveal binding heterogeneity under relatively extreme circum-stances, because they are typically dominated by the strongestinteractions (19, 20), as was also found by Bajic et al. (16) incharacterization of the �MI binding to C3d. By contrast, thebinding kinetics are a rich source of information on this prop-erty (19). As shown recently by Zhang et al. (17), at least forsmall ligands, such ensembles of weak and strong interactionscan be characterized both from injection of the �MI over ligand-coated surfaces or, in the opposite orientation, with the �MIimmobilized to capture injected ligands. This is strong evidencethat the �MI has an intrinsic ability to form multiple types ofcontacts with polycarboxylate ligands, such as most proteins.Even the ligand iC3b, which supports the strongest reportedbinding by the �MI at KD �0.5 �M, presents, in addition,weaker, but stoichiometrically more abundant, interactions atKD �200 –300 �M (16). This weaker affinity agrees quantita-tively with the affinity of the �MI for soluble glutamate (15).Although well established biophysical principles explain howsuch weak interactions support cell adhesion in the context ofthe membrane (21), their potential as antagonistic targetsremains undefined.

With respect to inhibiting the function of �2 integrins, sev-eral small molecule antagonists have been identified (22).Type-1 statins, such as lovastatin and simvastatin, inhibit bind-ing of lymphocyte function-associated antigen (LFA-1; alsoknown as integrin �L�2 or CD11a/CD18) to intercellular adhe-sion molecule (ICAM)-1 (23, 24). In vivo experiments showedthat lovastatin significantly changes the leukocyte distributionsin several lymphoid tissues, including those of the mucosalimmune system (25). These findings have a clinical correlate.Statins lower plasma cholesterol levels and are among the mostwidely used drugs. In treated patients, however, these com-pounds exhibit pleiotropic effects, including an anti-inflamma-tory capacity not directly related to the cholesterol-loweringactivity (26).

Simvastatin is administered as a lactone prodrug and con-verted to a hydroxy acid form through hydrolysis (Fig. 1A). Asnoted by Kallen et al. (23), the ability of �MI to form a contactwith �-carboxylate of glutamate (12, 13) encouraged thehypothesis that integrin I domains would bind statins throughsimilar contact between the hydrolyzed statin and the MIDASof the I domain (23). Surprisingly, as revealed by both x-ray

crystallography and nuclear magnetic resonance spectroscopy,statin prodrug bound the �L I domain (�LI) of LFA-1 far fromthe MIDAS, underneath the �7 helix (the L site). This lockedthe domain in the closed, low affinity ligand binding conforma-tion. Nevertheless, whether and how integrins may bind thehydroxy acid form of statins through the I domain MIDASremains unclear, and the ability of statins to inhibit CR3 func-tions is controversial. Indeed, simvastatin showed no antago-nistic effects on CR3 binding of ICAM-1 (24). More recently,however, simvastatin was found to interfere with CR3-medi-ated adhesion by neutrophil granulocytes in several experimen-tal and clinical settings (27–29).

Using x-ray crystallography and molecular dynamics (MD)simulations, we reveal that the carboxylate in the hydroxy acidform of simvastatin engages in a ligand receptor-like complexwith the MIDAS of the �MI. In surface plasmon resonance(SPR)-based assays, simvastatin inhibited the binding of �MI toiC3b and C3d but not ICAM-1. We found that simvastatininhibits preferentially �MI interactions typified by slowly form-ing bonds to iC3b or C3d. We furthermore show that simvas-tatin blocked the adhesion of cell surface CR3 to complement-coated substrate. Our study provides a chemical rationale forsimvastatin as CR3 antagonist and novel ways of understandinghow antagonists may modulate the ligand binding kinetics oftargeted proteins.

Results

The Crystal Structure of the �MI-Simvastatin ComplexReveals the Molecular Basis for Simvastatin Binding—Toexamine the molecular basis for the effect of simvastatin onCR3, we determined the crystal structure of the CR3 I domainbound to simvastatin at 2.0 Å resolution (Fig. 1 and Table 1). Inthe crystal, the �MI, with a Mg2�-bound MIDAS, adopted theopen, ligand binding conformation (Fig. 1B). The initial elec-tron density indicated that one molecule of simvastatin in itshydroxy acid form (Fig. 1A) acted as a ligand to the MIDASMg2� ion, using its carboxylate group to complete the coordi-nation sphere of the Mg2� ion (Fig. 1, B and C). The majorcontacts between simvastatin and �MI involve polar contacts ofthe simvastatin carboxyl moiety with MIDAS residues Ser-142,Ser-144, and Thr-209. The carboxylate moiety is firmlyanchored to the �MI via its metal ion coordination. One of thesimvastatin carboxyl oxygens is directly hydrogen-bonding tothe hydroxyl of Thr-209. Two MIDAS-surrounding residues,Phe-246 and Arg-208, may contribute to the binding by form-ing �-� stacking interactions with the unsaturated decalin moi-ety of simvastatin. However, their orientation is not optimal forthe binding. Furthermore, although the electron density forthe simvastatin ring system is strong, it cannot be perfectlydescribed by the compound in a single conformation. Hence,the fitted simvastatin ligand is likely to approximate its averageconformation in the crystal rather than representing a detailedstructure of a single, stable conformation. A neighboring Idomain molecule in the crystal unit cell may provide an addi-tional buttress to the simvastatin decalin moiety but is evidentlyinsufficient to maintain the molecule in a unique conformation,as indicated by the electron density, as noted from Fig. 1, B andC, and the associated B-factor values (Table 1).

Simvastatin Antagonism of Complement Receptor 3

16964 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 33 • AUGUST 12, 2016

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

Molecular Dynamics Simulations Support the FlexibleNature of the �MI-Simvastatin Complex—Considering that thecrystal structure of the simvastatin complex was derived fromsoaking with the compound and the apparent lack of a single,predominant binding conformation of the unsaturated decalinmoiety, we sought to validate the stability of the observed com-plex through MD simulations (Fig. 2). Three independent100-ns simulations were performed using the crystal structureas the starting model. In these simulations, simvastatinremained firmly attached to the �MI MIDAS through the car-boxylate coordination of Mg2� (Fig. 2A). This was furtheremphasized by the small variations in the bond length from theMg2� ion to the carboxylate oxygen of simvastatin with fluctu-ations in bond length of a maximum of 0.15 Å (Fig. 2B). As inthe crystal structure, Ser-142, Ser-144, and Thr-209 formedhydrogen bonds to the simvastatin carboxylate group. Thesewere maintained as stable contacts during the simulation, alsowith minimal fluctuations in bond length (Fig. 2B). In additionto these contacts, the carboxylate was soliciting non-MIDASresidues for hydrogen bonding, namely the backbone carbonyloxygens of Gly-143, Gly-207, and Phe-246 (Fig. 2C). Impor-tantly, besides the polar interactions, simvastatin was able toestablish hydrophobic contacts with the side chains of Arg-208

Leu206

Arg208

Phe246

Ser144Asp242

Glu244

Simvastatin

Leu206

Arg208

Phe246

Ser144Asp242

Glu244 Mg2+Mg2+

Simvastatin

Simvastatin

MIDAS

C

O

O

H

O

OHO

OH

O

H

HO

OO-O

OH

Hydroxy-acid formLactone prodrugA B

FIGURE 1. The structure of the �MI-simvastatin complex. A, structures of simvastatin in the lactone prodrug and hydroxy acid forms. The unsaturateddecalin ring is indicated in cyan, and the carboxylate of the hydro-acid form is shown in red. B, simvastatin binds at the top of the I domain coordinatingthe MIDAS-bound Mg2� ion. The 2mFo � DFc electron density map contoured at 1.0 � is shown for simvastatin. C, stereo view of the simvastatinbinding interface. I domain residues within 4 Å of the simvastatin molecule are shown as sticks. Water molecules (shown in cyan) contribute to theinteraction.

TABLE 1X-ray crystallography data collection and refinement statisticsStatistics for the highest resolution shell are shown in parentheses.

CR3 I domain-simvastatin

Data collectionBeamline MAX-lab I911-3Wavelength (Å) 1Resolution range 28.84–2.00Space group P 21 21 21Unit cell parameters 56.38, 57.69, 58.4

a, b, c (Å) 90, 90, 90�, �, � (degrees)

Multiplicity 9.0 (8.8)Completeness (%) 100Mean I/�(I) 18.96 (3.91)Wilson B-factor 18.16Rmeas 0.1178 (0.6544)

RefinementReflections used in refinement 13,333 (1302)Rwork/Rfree 0.1885/0.2317No. of atoms 1587

Protein 1490Ligand 32

Root mean square bonds (Å)/angles(degrees)

0.007/1.05

Ramachandran favored/allowed/outlier (%)

96/4/0

Rotamer outliers (%) 0MolProbity clashscore 1.63Average B-factor 22.72

Protein 22.20Ligand 37.81Solvent 27.30

Simvastatin Antagonism of Complement Receptor 3

AUGUST 12, 2016 • VOLUME 291 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 16965

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

and Phe-246, although the latter was in contact with simvasta-tin only for 30 – 60% of the time during the simulations (Fig.2C). Leu-206, located on the opposite side of the MIDAS rela-tive to Phe-246, was also a possible interacting partner, contact-ing simvastatin for 60 – 80% of the time during the simulations(Fig. 2C).

Simvastatin Antagonism Targets �MI Ligand BindingKinetics—The crystal structure and MD simulations suggestedthat simvastatin may act as an �MI ligand binding antagonist.To test this hypothesis, we prepared surfaces carrying ligandsfor the �MI in a setting suitable for SPR monitoring of theinteractions.

As noted previously, the ligand binding kinetics of �MI pro-vide information on its interactions with various ligands. Theexperimental data were fitted using the EVILFIT algorithm (19,30). This analysis was used to characterize the interaction of�MI with native and denatured fibrinogen (15); myelin basicprotein and glatiramer acetate (18); the C3 fragments C3b,iC3b, and C3d (16); and the antimicrobial peptide LL-37 (17).Briefly, the algorithm solves the problem of calculating the min-imal distribution of association (ka) and dissociation (kd) rates,which is required to account for the experimentally observedbinding to ligand-coated surfaces. Therefore, rather than theusual characterization of protein-ligand interaction by a singlepair of ka and kd, the algorithm returns a two-dimensional dis-tribution of these constants. For easier interpretation of resultswith regard to affinity, results are shown as a distribution of kdand the equilibrium constant KD, the latter calculated from KD �kd/ka (19). Results are typically shown in a three-dimensionalcoordinate system, where the x and y coordinates indicate the

KD (in M) and kd (s�1), respectively. For each pair of KD and kd,defining a single 1:1 interaction, the z-coordinate indicates theabundance of such a 1:1 interaction with the visual aid of con-tour maps (20). In this analysis, I domains, which bind theirligand through a well defined, single type of 1:1 interaction,produce narrow distributions of binding constants with theappearance of a single, symmetric “island” in the contour map(15). By contrast, binding to most ligands by the �MI produces amore composite and wide distribution of binding constants,often appearing as an “archipelago” in contour maps (16 –18).

Initially, the �MI was injected in nine concentrations from 20to 10,000 nM over surfaces coated with iC3b, C3d, or ICAM-1(Fig. 3, A–C). To capture all relevant binding events within therange of the instrument, the algorithm probed interactions on agrid with 10 uniformly separated grid points in the interval10�9 M � KD � 10�2 M and 10 grid points in the interval 10�6

s�1 � kd � 100 s�1. The distribution of interactions for iC3b(Fig. 3A) and C3d (Fig. 3B) was essentially identical to our pre-vious report (16, 17). The ligands iC3b and ICAM-1 shared apopulation of binding sites with kd between �10�3 and 100 s�1

(Fig. 3, A and C). In addition, the iC3b presented another type ofinteraction with kd between �10�6 and 10�3 s�1 and KDbetween 10�5 and 10�2 M (Fig. 3A), discernible, albeit moreweakly, also for C3d (Fig. 3B) but not found for ICAM-1(Fig. 3C).

To investigate the influence of simvastatin on �MI ligandbinding, �MI with simvastatin and DMSO or, as a reference,with DMSO alone was injected over the surfaces mentionedabove. The SPR signal decreased with increasing simvastatinconcentrations, both for iC3b and C3d but not for ICAM-1 (Fig.

A

Ser144Phe246

Arg208

Glu244

Thr209

Leu206

Asp242Lys245Mg2+

Simvastatin

142143 144145 146 147 178 206 207 208 209244 245 246

Simul. 1Simul. 2Simul. 3

100

80

60

40

20

0

αMI residue

C

Cont

act o

ccur

ence

(%)

Mg-O (Ser142)

Mg-O (Ser144)

Mg-O (Thr209)

Mg-O (Simvastatin)

Time (ns)Bo

nd L

engt

h (Å

)

2.5

2.0

2.4

2.3

2.2

2.1

2.5

2.0

2.4

2.3

2.2

2.1

2.5

2.0

2.4

2.3

2.2

2.1

BSimul. 1

Simul. 2

Simul. 3

200 6040 10080

FIGURE 2. MD simulations of the �MI binding of simvastatin. A, from the MD simulations, the occupancy of simvastatin as calculated by the VolMap pluginfor the Visual Molecular Dynamics (65) is indicated as the covered volume (in gray) together with one simvastatin molecule (shown as sticks) at the positionfound in the crystal structure. B, occurrence of contacts between �MI residues and the simvastatin molecule during three independent MD simulations (Simul.).Primary structure-linked residues are indicated with bars. Residues forming parts of the primary Mg2� coordination sphere are indicated in boldface type.Residues in contact with simvastatin are indicated in italic type. C, bond lengths from Mg2� to oxygens in Ser-142, Ser-144, Thr-209, and simvastatin throughoutthe simulation in three MD simulations of the structure of the �MI domain in complex with simvastatin.

Simvastatin Antagonism of Complement Receptor 3

16966 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 33 • AUGUST 12, 2016

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

3, D–F). From this comparison, it seemed possible that onlysome types of interactions by �MI were susceptible to simvas-tatin inhibitions, whereas others were exempt. A simple, essen-tially model-independent testing of this hypothesis was madeby comparing the relative inhibition at two time points. In the�MI sensorgrams, the signal at t � 234 s (S234 s) reflects com-bined contributions from high affinity and a spectrum of loweraffinity interactions. By contrast, the signal at 210 s after the endof the injection period (i.e. t � 450 s (S450 s)) corresponds to thelate dissociation phase. Hence, only interactions with a half-time of dissociation (t1⁄2) on the order of 50 s or longer, andhence a dissociation rate of kd � ln 2/t1⁄2 � 10�2 s�1 or less,would be expected to influence the signal (S450 s). As shown bythe normalized inhibition (Fig. 3, G–I), simvastatin actedpotently on such slowly dissociating interactions (S450 s) withiC3b (Fig. 3G) and C3d (Fig. 3H). The combined interactions

(S234 s) were less affected. In the case of ICAM-1 ligand, only thecombined interactions were robustly measureable and showedlittle, if any, inhibition (Fig. 3I).

To conduct a more comprehensive analysis of the simvasta-tin antagonism, we used the EVILFIT methodology describedabove. We extracted the binding kinetics for �MI binding to itsligands from the single sensorgrams also shown in Fig. 3, D–F.These were produced with 10 �M �MI and a range of simvasta-tin concentrations from 0 to 100 �M. In this way, due to the highconcentration of �MI, the analysis better allowed the recordingof changes in weaker �MI interactions. For all analyses, irre-spective of ligand or simvastatin concentration, the modelaccounted tightly for the experimental data with small rootmean square deviations (Fig. 4). The types of binding with kdranging from 10�3 to 100 s�1 are indicated as Bin 1 in Fig. 5A.The other types of interactions, with kd between 10�6 and 10�3

10x10

B

−6

−4

−2

0

012345×103

20406080100

0

iC3b (0.0527 pmol/mm2)

A

1000

2000

3000

0

500

1000

1500

C3d (0.0569 pmol/mm2)

0

ICAM-1 (0.0109 pmol/mm2)

C

0

50

100

150

−8 −6 −4 −2log10KD

log 10

k d

012345×103

−6

−4

−2

0

log 10

k d

−8 −6 −4 −2log10KD

−6

−4

−2

0

log 10

k d

−8 −6 −4 −2log10KD

4000

3000

2000

1000

0

2000

1500

1000

500

0

300

200

100

0

0-3.16.3

12.52550

100

03.16.3

12.525

50-100

03.1

6.3-100

00503.1 6.3 12.5 25

S243s

S450s

0 100503.1 6.3 12.5 25

60

40

20

0

Inhi

bitio

n (%

)

0 100503.1 6.3 12.5 25

Simvastatin conc. (μM)

0 100 200 300 400 0 100 200 300 400 0 100 200 300 400

μM

0

6.312.5

2550

100

μM

3.1

03.16.3

12.525

50-100

μM

0-3.16.3-100

Time (s)

S (R

U)

S243s

S450s

S243s

S450s

S243s

S450s

S243s

S450s

S243s

S450s

E

H

10

FD

IG

FIGURE 3. Effects of simvastatin on the �MI binding to iC3b, C3d, or ICAM-1 measured by SPR. A–C, SPR sensorgrams for the �MI binding to flow cell-immobilizediC3b (A), C3d (B), and ICAM-1 (C). For iC3b and C3d (A and B), �MI were injected in nine concentrations from 20 to 10,000 nM (ascending order ofsensorgrams). For each ligand, the results shown were from one of two similar binding experiments. Fits to the experimental data were made withEVILFIT (30) as described elsewhere (20). The goodness of fit is indicated in A–C from a comparison between the data (solid black curve) and the model(solid red line). For ICAM-1, sensorgrams for �MI injections in the concentration range 20 –156 nM were omitted from the analysis due to insufficientsignal. The distribution in ligand binding kinetics is shown in the inset two-dimensional grids. Each grid point represents a 1:1 interaction typified by KDand kd. A third coordinate, indicated with a color scale, represents the abundance of these interactions. D–F, simvastatin was titrated against a fixedconcentration (10 �M) of �MI, followed by injection of the mixture over the surfaces coupled with iC3b (D), C3d (E), or ICAM-1 (F). Binding signals (S, inresonance units (RU)) were recorded at the terminal part of the association phase (S243 s) and within the late dissociation phase (S450 s). To visualize theorder of the sensorgrams, the S243 s and S450 s parts of the sensorgrams were magnified with the order of the associated simvastatin concentrationindicated. G–I, the percentage of inhibition by simvastatin was determined at t � 243 s and t � 450 s from the SPR signal at these time points (S243 s andS450 s, respectively). The level of inhibition was plotted as a function of the simvastatin concentration, using the response with no simvastatin added asreference (0% inhibition). Data represent the average of the two experiments, with error bars indicating the minimum and maximum levels.

Simvastatin Antagonism of Complement Receptor 3

AUGUST 12, 2016 • VOLUME 291 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 16967

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

s�1, are indicated as Bin 2. From integration of the signals in thebins, it appeared that only Bin 2 interactions in iC3b and C3dwere susceptible to simvastatin antagonism. By contrast, for allligands, including ICAM-1, Bin 1 interactions were unaffected(Fig. 5, A and B).

Antagonistic Effects of Simvastatin on the Binding of CR3 toiC3b—To investigate the effect of simvastatin on CR3 bindingto one of its natural substrates, complement iC3b, we employedthree different types of cell adhesion or contact assays.

In the electric cell-substrate impedance sensing (ECIS)assay, cells are applied in tissue culture wells with small,planar gold film electrodes deposited on the bottom surface(31). In this system, the alternating current impedanceincreases with cellular adhesion and thereby offers a label-free means of following this process over time (32). Theadhesion of recombinant K562 cells or primary humanmonocytes was compared, in the absence or in the presenceof simvastatin. The recombinant cells expressed �76% of theCR3 level found on monocytes, as judged from the meanfluorescence intensity when staining both cell types forCD11b (data not shown).

The binding to iC3b substrate by CR3/K562 cells was probedin the presence of 6.25–50 �M simvastatin with clear signs of

inhibition at 25 and 50 �M (Fig. 6A), whereas simvastatin had noinfluence on the parental K562 cell line (Fig. 6B). Simvastatinalso inhibited the adhesion by primary human monocytes to thesubstrate, either with an added activating antibody (Fig. 6C) orwithout such an addition (Fig. 6D). Half-maximum inhibitionfor the CR3-supported adhesion was reached with �25 �M sim-vastatin (Fig. 6E) when taking into account the backgroundbinding observed for parental K562 cells (Fig. 6B). In the case ofthe monocytes, which formed stronger contacts with the iC3b-coated substrate, half-maximum inhibition was reached at �35�M (Fig. 3F). For both CR3/K562 cells and monocytes, the inhi-bition persisted for hours.

Simvastatin also inhibited the ability of monocytes to bindiC3b-coated fluorescent particles, as investigated by flowcytometry (Fig. 7, A and B). The cellular gating was carefullyadjusted to accommodate the small changes in forward and sidescatter induced by the addition of simvastatin (Fig. 7A). As alsonoted in experiments with other bead-coupled CR3 ligands(18), distinct peaks in the fluorescence histograms suggested aquantized association or phagocytosis of the beads. This wasused to distinguish contacts with more than one bead from thetotal of bead-contacted monocytes. In both types of measures

35.53 RU

35.56 RU

iC3b C3d ICAM-1

0 μM

3.125 μM

6.25 μM

12.5 μM

25 μM

50 μM

100 μM

Simvastatinconc.

500

1500

2500

3500

500

1500

2500

3500

500

1500

2500

3500

500

1500

2500

3500

500

1500

2500

3500

1000

2000

3000

1000

2000

3000

0 100 200 300 400

400

800

1200

1600

400

800

1200

1600

400

800

1200

1600

400

800

1200

1600

400

800

1200

1600

200

600

1000

1400

200

600

1000

1400

0 100 200 300 400

50100150200

50100150200

50100150200

50100150200

50100150200

50100150200

50100150200

0 100 200 300 400

34.86 RU

42.28 RU

34.53 RU

33.61 RU

11.17 RU

13.49 RU

12.82 RU

15.99 RU

19.29 RU

16.85 RU

5.51 RU

9.16 RU

1.84 RU

1.67 RU

1.45 RU

1.59 RU

3.09 RU

3.55 RU

8.90 RU

Time (s)

S (R

U)

FIGURE 4. SPR sensorgrams (solid black lines) for injections of 10 �M �MI in the presence of 0 –100 �M simvastatin in flow cells coupled with iC3b, C3d,or ICAM-1. For each sensorgram, the ligand binding kinetics were extracted by fitting with the EVILFIT algorithm. In each panel, the goodness of fit is indicatedwith opaque gray lines (model) and the root mean square deviation (in resonance units (RU)), comparing experimental data and the model, as shown in italictype.

Simvastatin Antagonism of Complement Receptor 3

16968 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 33 • AUGUST 12, 2016

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

and unlike in the purely static cell adhesion assays (Fig. 6), aninhibitory influence was discernible even at �12 �M simvasta-tin (Fig. 7C).

To directly test whether force load on adhering cells wouldalter the potency of simvastatin-mediated inhibition, weemployed a previously described centrifugation-based cell

Simvastatin conc.0 μM 3.125 μM 6.25 μM 12.5 μM 25 μM 50 μM 100 μM

−6

−4

−2

0

012345×103

−6

−4

−2

0

−9 −7 −5 −3−6

−4

−2

0

−9 −7 −5 −3 −9 −7 −5 −3 −9 −7 −5 −3 −9 −7 −5 −3 −9 −7 −5 −3

20406080100

−9 −7 −5 −30

012345×103

Bin 1

Bin 2

Bin 1

Bin 2

Bin 1

Bin 2

25

20

15

10

5

0

Bin2Bin1

5

4

3

2

1

010 00503.1 6.3 12.5 25

1.5

1

0.5

0

0 100503.1 6.3 12.5 250 100503.1 6.3 12.5 25

B

A

log10 KD

log 10

k d

S (RU)

∫∫S Bi

n (k

RU)

iC3b C3d ICAM-1

Simvastatin conc. (μM)

iC3b

C3d

ICA

M-1

FIGURE 5. Distribution of �MI ligand binding interactions in the presence of simvastatin. A, from fitting of the sensorgrams, as shown in Fig. 4, the distribution inkinetics of the �MI ligand binding was determined as described (19, 20, 30) for iC3b, C3d, and ICAM-1. Results are shown in two-dimensional grids as explained under“Results” in the section “Simvastatin Antagonism Targets �MI Ligand Binding Kinetics,” and in the legend to Fig. 3, A–C. To quantify the development in the distributionof binding kinetics, two bins (Bin 1 and Bin 2) were defined, guided by the major types of interactions observed for iC3b and C3d. B, from integration of signals (��SBin,in kilo resonance units (kRU)) in the bin areas, it was possible to plot these values as a function of the applied simvastatin concentration. Data represent the average ofthe two experiments, with error bars indicating the minimum and maximum levels.

12.5 μM

25 μM

50 μM

0.6

0.5

0.4

0.3

0.2

0.1

0.0

6.25 μM(none)

(none)12.5 μM

6.25 μM

25 μM

50 μM

2.0

1.5

1.0

0.5

0.00.1 1.1 2.10.2 0.7 1.2 1.7 2.2 2.7

Cell

inde

x

A C

Monocytes

0

20

40

60

80

0 10 20 30 40 50

Wt K562+KIM185

CR3/K562+KIM185

0 10 20 30 40 50

Time (h)

Simvastatin conc. (μM)

Nor

mal

. inh

ibiti

on (%

)

Monocytes+KIM185

E F

(none)12.5 μM6.25 μM25 μM

50 μM

B D+KIM185

3.10.2 0.7 1.2 1.7 2.2 2.7 0.1 1.1 2.1 3.1

CR3/K562 Wt K562

+KIM185 +KIM185 (No activation)

FIGURE 6. Simvastatin inhibition of cellular CR3 adhesion to iC3b measured by ECIS. A and B, the ECIS cell adhesion assay was applied to surfaces coupled withiC3b, using either K562 cells with a recombinant expression of CR3 (A) or the parental cells (B), which do not express CD18 integrins. C and D, similar studies were madewith primary human monocytes, either in the presence of the function-activating antibody KIM185 to CD18 (C) or without suchantibody(D). InA–D, theshowncell indexrecordings are representative of three independent experiments. E and F, the normalized inhibition was calculated from the cell index without the addition of simvastatin forCR3/K562 and the parental K562 cells (E) and monocytes (F). In each panel, the mean values for three experiments are indicated along with error bars showing the S.E.

Simvastatin Antagonism of Complement Receptor 3

AUGUST 12, 2016 • VOLUME 291 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 16969

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

adhesion assay (33). This methodology works by use of micro-titer wells with a conical shape, usually referred to as v-wells,and fluorescent dye-labeled cells. Following centrifugation, thepropensity of the cells to reach the bottom, or nadir, of the wellsis inversely proportional to the ability to adhere to the sides ofthe wells. By comparison of the fluorescent signals obtained inthe ligand-coated wells (Fcoated) with wells without ligand coat-ing (Funcoated), the percentage of cell adhesion (CA) is estab-lished through the following equation (33).

CA � 100% Funcoated Fcoated

Funcoated(Eq. 1)

Statin potentiated the detachment of adhering cells as a func-tion of increasing centrifugal force, clearly shown in the pres-ence of 20 �M simvastatin compared with the detachment with-out such an addition (Fig. 8A). With weak centrifugal force(10 � g), only a modest difference was found in the adhesion bysimvastatin-treated and -untreated cells when analyzed accord-ing to Equation 1. By contrast, with increased centrifugal forces(30 –50 � g), almost complete inhibition (94%) of the bindingwas obtained with statin, whereas the untreated controlsshowed robust binding. Nevertheless, even at low centrifugalforce (10 � g) it was possible to detect an influence of simvas-tatin (Fig. 8, B and C). As also shown in Fig. 8A, when usingEquation 1, only a modest reduction in cell adhesion wasobtained from application of 20 �M simvastatin at 10 � g.Lower concentration apparently did not reduce the binding(Fig. 8B). However, as shown from the inclusion of the rawsignals Fcoated and Funcoated (Fig. 8B), the simvastatin concen-tration significantly impacted the background binding inuncoated wells. This creates a difficulty in comparing the cellbinding through Equation 1, because there is a non-linearchange in CA with changes in Funcoated,

�CA

�Funcoated�

1

Funcoated

Funcoated Fcoated

Funcoated2 (Eq. 2)

We compared the influence of simvastatin through a simplerequation,

�F � Funcoated Fcoated (Eq. 3)

where �F is proportional to the iC3b-specific cell adhesion andchanges proportionally with changes in either Funcoated orFcoated. Using Equation 3 with normalization against no treat-ment with simvastatin (�F0), it was clear that simvastatin low-ered the iC3b-specific binding in a titratable fashion, at least inthe interval 100 –20,000 nM (Fig. 8C). In principle, inhibitionwas also observed at 50 nM, but the binding data were subject tomore variation.

Discussion

We report here on structural and ligand binding kineticsaspects of competitive antagonism of CR3. Structural insightwas gained from both x-ray crystallographic studies and MDsimulations of the complex between �MI and a small moleculeantagonist, simvastatin. Alongside these findings, cellular andbiochemical assays measured inhibition of CR3 ligand bindingby simvastatin. Our report is the first to structurally character-ize an �MI ligand-binding antagonist in complex with �MI.

As judged from the x-ray crystallographic data, the major sitefor interaction between simvastatin and the ligand-bindingactivated �MI is the MIDAS. In this way, the �MI antagonismdiffers markedly from how statins block binding by the �LI,where the primary interaction between statin and the I domainis distant from the MIDAS, in the so-called L site (24). In thestructure of the �MI-simvastatin complex, the carboxylate moi-ety of simvastatin forms a contact with the MIDAS Mg2�. This

01020304050607080

0 10 20 30 40 50

Multiple beads+ cells

Total beads+ cells

Nor

mal

. inh

ibit.

(%)

C

-103 0 103 104 105

Simvastatin conc. (μM)

13.0%

0 100K 200K0

50K

100K

150K

200K

250K

9.21%

0

50K

100K

150K

200K

250K

26.1%

0

50K

100K

150K

200K

250K

0

300

600

900

1.2K

Total Beads+

64.9%

Mutiple Beads+

53.8%

0

300

600

900

1.2K

44.7%

31.4%

0

300

600

900

1.2K

Side

scat

ter

Forward scatter

Coun

ts

FITC

(No

addi

tions

)Be

ads

+ D

MSO

veh

icle

Bead

s +

50 μ

M S

imva

stat

in

BA

FIGURE 7. Simvastatin inhibition of monocyte binding and phagocytosisof iC3b-coupled fluorescent beads. A, forward side scatter plots of mono-cytes either with no additions, with beads and DMSO vehicle, or with beadsand 50 �M DMSO-dissolved simvastatin. B, histograms show bead fluores-cence intensities either with no additions, with beads and DMSO vehicle, orwith beads and 50 �M DMSO-dissolved simvastatin. Gates were placed todistinguish the total number of cells with beads (solid line) or cells with mul-tiple (�1) beads (hatched line). C, normalized inhibition of bead adhesion orphagocytosis. The percentage of monocytes positive for bead contact (TotalBeads�) was calculated in the absence or presence of simvastatin. By normal-izing to the value of positive cells in the absence of simvastatin (0% inhibi-tion), the percentage of inhibition was calculated for each concentration ofsimvastatin. A similar calculation was also made for the gate only includingcells with multiple beads (Multiple Beads�). The mean values for three exper-iments are indicated along with error bars showing the S.E.

Simvastatin Antagonism of Complement Receptor 3

16970 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 33 • AUGUST 12, 2016

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from

contact agrees well with what was reported in the past, namelythat �MI reacts with simple carboxylic groups of free glutamateand even glutamate side chains in the context of the �MI itself(13, 15). The �LI binds such moieties with considerably weaker

affinity, even in the high affinity ligand binding conformation(15), which may explain why the �LI fails to bind statins throughthe MIDAS.

In addition, simvastatin also forms hydrogen bonds withmain chain carbonyl oxygens, as well as with the functionalgroups of the side chains of MIDAS-forming residues. In MDsimulations, the unsaturated decalin moiety of simvastatin wasfound to be highly mobile. Consistent with these data, simvas-tatin probably also adopted multiple conformations in the crys-tals. Although Leu-209 and Phe-246 are not in the immediatevicinity of the simvastatin in the crystal structure, the MD stud-ies suggested that these residues may form transient contacts tothe simvastatin decalin moiety. No significant electron densitywas observed within the putative �MI L site.

To efficiently study antagonistic effects, we used an �MImutated to break an isoleucine-based allosteric switch and per-mit the �MI �7 helix to preferentially occupy its open confor-mation position (34, 35). It is widely acknowledged in the liter-ature that such stabilized, or locked, integrin I domains havebeen useful in ligand binding studies, including both x-ray crys-tallographic studies and biochemical investigations of ligandaffinity (8). Nevertheless, the induced conformational rear-rangements in these constructs are likely to destroy the putativeL site (i.e. the site of contact between statin and the �LI). Hence,we cannot exclude the possibility that statins also might bindthis site in the closed conformation of �MI. Clearly, the tightlink between conformation and affinity presents a challengewith regard to choosing appropriate �MI constructs in studieswith antagonists. There can be little doubt, however, about therelevance of the open �MI conformation in these studies. In apro-inflammatory environment, a large population of CR3 mol-ecules is in their ligand-binding conformation (36). In the past,the influence of statins on conformationally activated �2 integ-rins or their I domains was never tested. We now show thatsimvastatin is able to antagonize receptors in this biologicallyrelevant conformation.

It is increasingly evident that binding kinetics are importantdeterminants of antagonist efficacy (37). Our study adds to thisconcept by showing that the complex �MI ligand binding kinet-ics are affected in a non-trivial way by the presence of an antag-onist. As noted above, equilibrium-based analyses of �MI ligandbinding are a poor source of information on its multiple inter-actions with ligands. This compromises the use of the classicSchild regression (38) for understanding the mode of simvasta-tin antagonism to �MI ligand binding. We now propose extract-ing the relevant information directly from the observed associ-ation and dissociation phases, using a methodology previouslydescribed by Schuck and co-workers (19, 30). We noted thatonly some of the interactions in the ensemble, namely thosereferred to as Bin 2, were affected by simvastatin, whereas thosebelonging to Bin 1 were not. Mass transport effects influencingthese data are unlikely because of the goodness of fit for theapplied model of the �MI binding kinetics, and for the vastmajority of all interactions, the kd is faster than 10�5 s�1, athreshold value for this phenomenon (39).

From the structural analysis shown in the present study, theselective quenching of Bin 2 interactions is not easily reconciledwith an allosteric mechanism similar to the lovastatin antago-

0

20

40

60

80

100

0

20

40

60

80

100

0

50

100

150

200

250

300

0 50 100 200 400 20,000

Cell adhesion

FiC3b

FNo IC3b

F ×1

0-3 (c

ount

s/s)

Cell Ahesion (%

)

101 102 103 104 105

100%

× Δ

F/ΔF

0

(No statin)

Simvastatin conc. (nM)

A

B

0 5 10 15 20 25 30 35 40 45 500

10

20

30

40

50

60

70

Cell

adhe

sion

(%)

Centrifugal force ( ×g)

Ξ100%

Ξ100%65%

51% 47%

30%

10%6%

No statin

20 μM simvastatin

Simvastatin conc. (nM)

C

FIGURE 8. Centrifugal force-based cell adhesion assay with primaryhuman monocytes. A, monocyte adhesion, calculated according to Equation1, is shown as a function of the applied centrifugal force, either with no addi-tion of simvastatin (filled circles) or in the presence of 20 �M simvastatin (opencircles). The relative decline in binding was indicated as a percentage fromnormalizing the adhesion at 20 –50 � g to the results obtained for 10 � g. B,recording of the fluorescent signal from labeled monocytes in iC3b-coatedwells (FiC3b) and in uncoated, control wells (FNo iC3b) following spinning at10 � g. The cell adhesion (indicated in gray bars) was calculated from thesevalues using Equation 1 as above. C, from FiC3b and FNo iC3b shown in B, �F wascalculated according to Equation 3 and normalized to �F in experiments withno statin (�F0). In A and C, the mean values for three independent experi-ments are indicated together with error bars showing the S.E. Data in B arefrom one experiment representative of a total of three independentexperiments.

Simvastatin Antagonism of Complement Receptor 3

AUGUST 12, 2016 • VOLUME 291 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 16971

nism to �LI ligand binding (23, 24). We suggest that at least twostructurally different mechanisms can account for the observedselectivity. First, it is known from RGD-binding integrins, suchas the �3 integrins, that small RGD motif-containing com-pounds may exert both ligand binding antagonistic and agonis-tic effects. Binding of these ligand mimetics induces changes inthe tertiary structure of the �3 chain I-like domain (40, 41),which persist after dissociation of the compound (42). Idomains are structurally highly similar to I-like domains (43). Ithas already been reported that the �LI may exist in several dis-tinct tertiary structures with low, intermediate, or high ligand-binding affinities (44). By acting as a ligand mimetic with sev-eral MIDAS contacts, we propose that simvastatin imprints theconformation of the simvastatin-dissociated �MI to permit theBin 1, but not Bin 2, interactions. Second, as an alternativeexplanation, it is possible that some protein ligands (i.e. in ourstudy iC3b and C3d) form types of contacts with the �MI thatpermit the simvastatin to writhe itself inside the complex andgain access to the MIDAS, causing the complex to dissociate. Inother ligands (i.e. ICAM-1), the contacts formed with �MI donot permit such intrusion by simvastatin and are hence exemptfrom simvastatin antagonism toward �MI binding. Unlike forthe ability of small drugs to make conformational imprints inintegrin I-like domains, to our knowledge, there are currentlyno experimental data available to support this model. In anyevent, both mechanisms proposed here add a chemical ration-ale to an earlier report on the inability of simvastatin to blockCR3 binding of ICAM-1 (24) while still exerting antagonismtoward complement binding.

The ability of simvastatin to inhibit intact CR3 was shown inthree distinct types of cellular assays; however, all used the sameligand, iC3b. Two of these, namely the ECIS and bead-basedassays, involved no external force load on the cell-ligand con-tact, whereas the v-well assay works by application of centrifu-gal force. The ECIS assays, which measure time-resolved cell-substrate contacts, revealed a persistence of the simvastatinantagonism to CR3-mediated adhesion, lasting for hours, bothfor recombinant cell lines and primary monocytes. Under con-ditions similar to those in our cell culture experiments, it waspreviously found that the simvastatin lactone prodrug quicklyhydrolyzed to the hydroxy acid form (45). Consequently, thepersistent antagonizing effect on CR3 adhesion suggests thatthe hydroxy acid form of simvastatin is the antagonist ratherthan the lactone prodrug.

To enable a study with CR3 uniformly activated and with aclear link to conformational change, we used the KIM185 anti-body to activate CR3 (46) through an allosteric mechanism,which ultimately involves the �MI in the open conformation(47). Also, in this case, simvastatin inhibited cell adhesion. Nat-ural targets for CR3 binding, such as complement-opsonizedmicrobes, were mimicked with iC3b-coated beads. Althoughwe did not formally distinguish between engulfed beads andthose merely attached to the membrane, it is noteworthy thatsimvastatin was able to block particle interactions with thesephagocytic cells. The simvastatin concentration required toobtain robust (�20%) inhibition in both of these assays was onthe order of �25 �M. Half-maximum inhibition (50%) wasobtained with �50 �M, almost identical to what was reported in

the past for inhibition of LFA-1-mediated adhesion to ICAM-1(23). In the cell adhesion assay based on centrifugal force, sim-vastatin increased the sensitivity to cell detachment. This wasclearly demonstrated from the almost equal cell adhesion fol-lowing centrifugation at 10 � g, irrespective of simvastatinaddition, versus near ablation of cell adhesion at 30 –50 � g forcells treated with 20 �M simvastatin. In contrast to the twostatic adhesion assays mentioned above, further comparisonswith the force-based assay revealed that such differences couldbe found even at a concentration of 100 nM statin, whichreduced the cell adhesion by �20% compared with cells treatedonly with the vehicle DMSO.

Considerable evidence exists that simvastatin acts as animmunosuppressant independently of its cholesterol loweringcapacity (26). However, with this and other pleiotropic effects, ageneral concern is the plasma concentration of statins found tobe in the low nanomolar range (48, 49). Such concentrationswould seem to preclude any influence of statin on, for instance,LFA-1, expressed by leukocytes in blood, because past measure-ments made in both cellular and purely molecular assaysreported effects only in the micromolar range (23). We are nowable to point to at least two principles that may alter the view of�2 integrin sensitivity to simvastatin inhibition and hence theimmunomodulatory capacity of statins.

First, a special feature of integrin ligand binding is thedynamic influence of force loads on the adhesive bonds to sub-strates, which involves significant conformational alterations inthe integrin ectodomains (50). Weitz-Schmidt (51) suggestedthat such forces increase the integrin susceptibility to statinantagonism of ligand binding, but experimental evidence hasbeen lacking. Our findings now support this idea. Adding to therole of integrin conformation, it seems likely that force load onthe structurally labile integrin ectodomain supports conforma-tional changes, which potentiate the antagonism of simvastatin.This also points to a limitation in quantifying the simva-statin ligand-binding antagonism, however, because availablemethodologies do not capture the full plethora of forces affect-ing CR3 substrate adhesion in vivo.

Second, the oral delivery and later absorption over the intes-tinal epithelium is a topic often neglected in the discussion ofthe pharmacokinetics of statins vis-a-vis an influence on theimmune system. Statins reach the mucosal immune systembefore the liver or even the albumin-rich plasma, both factorsbeing critical to the low availability of statins in blood. Althoughenzymes responsible for degradation of simvastatin are alsoexpressed in the intestines, the resulting concentration is likelyto be far higher in important secondary lymphoid tissue, such asthe Peyer patches and lamina propria, and in the environmentof intraepithelial leukocytes, compared with the concentrationin plasma. Little is known, however, about the actual statin con-centration in these tissues. An important study by Bergman etal. (52) showed that rosuvastatin accumulates in the bile inconcentrations �5,000-fold higher than in plasma, clearlyimplicating the statins as potentially active in the intestinalenvironment. Indeed, experimental evidence already showsthat the leukocyte distribution is altered in the Peyer patchesfollowing lovastatin administration (25); simvastatin directlyaffects the intraepithelial lymphocytes (53). With the emerging

Simvastatin Antagonism of Complement Receptor 3

16972 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 33 • AUGUST 12, 2016

focus on the role of the mucosal immune system in inflamma-tory diseases (54), an important part of the statin immuno-modulation is likely to relate to the action of these compoundsin the gut-associated lymphoid tissues.

Experimental Procedures

Cells and Biologic Reagents—Codon-optimized C3d cDNAsequence (encoding protein residues 993–1288 of human C3,mutant C1010A) and a human �MI (subunit �M residues 127–321), mutated as C128S/I316G (34), were expressed in BL21(DE3) Escherichia coli cells and purified to �95% purity asjudged by SDS-PAGE, as shown by Bajic et al. (16) in Fig. 5E ofthat paper. The same constructs were used for structural, bio-chemical, and cellular studies. iC3b was purchased as 1.0 mg/mlstock solution (A115; CompTech), and ICAM-1 was boughtfrom R&D Systems (ADP4). Simvastatin (S6196) was pur-chased from Sigma-Aldrich. The monoclonal KIM185 anti-body (46) recognizes the fourth, membrane-proximal cysteine-rich domain of the �2 subunit (47) and activates integrin �2ligand binding (46, 47). Manufacture and purification ofKIM185 antibody was performed by GenScript.

K562 cells with an engineered expression of CR3 (CR3/K562)and their parental cell line have been described elsewhere (55).Before use in the experiments, the cells were cultivated in RPMI1640 medium (BE12-702F, Lonza) supplemented with 10%(v/v) FCS, 100 units/ml penicillin, 100 �g/ml streptomycin, and292 �g/ml L-glutamine (PenStrep Glutamine, Life Technolo-gies, Inc.), and 10 mM HEPES, pH 7.5, in an incubator (Thermo-Fisher) set at 37 °C, 5% (v/v) CO2, and 80% relative humidity.For maintenance of recombinant CR3 expression, 4 �g/mlpuromycin was added to the culture medium.

Human monocytes were prepared from the buffy coat of cen-trifuged donor blood, kindly supplied by the Blood Bank at Aar-hus University Hospital Skejby. The buffy coat was depleted oferythrocytes by Ficoll-PaqueTM PLUS (GE Healthcare) gradi-ent centrifugation, followed by negative isolation of monocytesby use of Invitrogen’s UntouchedTM human monocyte kit(REF11350D, LifeTech). Cells were stored as frozen in RPMI1640 medium (BE12-702F, Lonza) supplemented with 20%(v/v) FCS and 10% (v/v) DMSO (Sigma-Aldrich). The cell via-bility was �90% after thawing.

Crystallization and Structure Determination—The I domainwas purified in 20 mM HEPES, pH 7.5, 200 mM NaCl. Five mM

MgCl2 was added to the protein solution before crystallization.The protein crystallized by vapor diffusion at 19 °C. Crystalswere grown in a few days by mixing the CR3 I domain sample (1�l at 12 mg/ml) with an equal volume of reservoir solution (0.2M sodium malonate, pH 7.0, 20% (w/v) PEG 3350). Rod-shapedcrystals appeared after a couple of days. The crystals were thensoaked for 1–5 h in mother liquor containing 1 mM simvastatin.Before data collection, the crystals were cryoprotected inmother liquor, supplemented with 1 mM simvastatin and 30%(v/v) glycerol, and flash-frozen in liquid nitrogen. The datawere collected at 1.0 Å with a rotation of 0.5°/image on theI911-3 beamline at MAX-lab (Lund, Sweden). Data reductionwas carried out with XDS (56). The structure was solved withmolecular replacement in PHASER (57), using the previouslypublished structure of �MI (Protein Data Bank entry 1IDO (12))

as the search model. The initial electron density maps revealedthe presence of a ligand in the MIDAS, after which one mole-cule of simvastatin was placed manually. Rebuilding with Coot(58) and refinement with phenix.refine (59) were carried out inan iterative manner, whereas model quality was assessed withMolProbity (60). All figures were prepared with the PyMOLMolecular Graphics System version 1.5.0.4 (Schrodinger LLC,New York).

Molecular Dynamics Simulations—MD simulations basedon the �MI crystal structure were prepared by adjusting atomtypes in the ligand by using Protein Preparation Wizard in theSchrodinger software package (22). In Protein PreparationWizard, the protonation states of the four histidine residueswere chosen based on surrounding residues. His-148 and His-210 were modeled as the N�-tautomer, whereas His-183 andHis-295 were modeled as the flipped N�-tautomer. None of thehistidines were close to the binding site of simvastatin. Thesystem was solvated with the TIP3P water model (61) and sub-sequently neutralized with NaCl to a concentration of 150 mM,which produced a system containing 28,801 atoms. All simula-tions were performed in Desmond (Desmond MolecularDynamics System version 3.1, Schrodinger, LLC) using theOPLS force field (62) as embedded in the Schrodinger 2012suite of programs (Protein Preparation Wizard; Epik version2.3; Impact version 5.8; Prime version 3.1) and by applying theMultisim method. The simulations were made using a RESPAintegrator (63), with bonded and nearly non-bonded interac-tions calculated every 2 fs, the non-bonded interactions trun-cated at a cut-off of 9 Å. Far interactions were calculated every6 fs, using smooth particle mesh Ewald (64) to treat full electro-statics beyond 9 Å. Here, the Multisim method minimizes thesystem with restraints on solute, followed by a minimizationwithout any restraints. First, a short simulation of 12 ps was runin the NVT ensemble at 10 K. Afterward, another short simu-lation was run in the NPT ensemble at 10 K. Short simulationswere then run in the NPT ensemble for 12 ps, with restraints onsolute heavy atoms at 310 K, followed by a similar simulationwithout restraints, which was run for 24 ps. Then the MD pro-duction runs were initiated and continued for 100 ns. Constantpressure of 1 atm was maintained by the Martyna-Tobias-Kleinbarostat with a relaxation time of 2 ps. A constant temperatureof 310 K was achieved utilizing the Nose-Hoover chain thermo-stat method when simulated in the NPT ensemble. Snapshotswere saved every 4.8 ps, and snapshots for every 192 ps wereused for analysis. The trajectories were analyzed using the pro-gram VMD (65).

Surface Plasmon Resonance-based Assays—Preparation ofCM4 sensor chips (GE Healthcare) and recording of sensor-grams was carried out on a BIAcore 3000 instrument (GEHealthcare). C3d, iC3b, and ICAM-1 were immobilized at0.057, 0.053, and 0.011 pmol/mm2, respectively, through aminecoupling together with a reference prepared without protein asdescribed (20). Surfaces were initially probed with �MI dilutedin running buffer (150 mM NaCl, 1 mM MgCl2, 50 mM HEPES,pH 7.4) to concentrations of 20 nM, 39 nM, 78 nM, 156 nM, 312nM, 625 nM, 1.25 �M, 2.5 �M, 5 �M, and 10 �M. The sampleswere injected over the reference and ligand-coupled surfaceswith a flow rate of 10 �l/min and a contact time of 240 s, fol-

Simvastatin Antagonism of Complement Receptor 3

AUGUST 12, 2016 • VOLUME 291 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 16973

lowed by a dissociation phase of 210 s. The sensor chips wereregenerated in 1.5 M NaCl, 50 mM EDTA, 100 mM HEPES (pH7.5). Following subtraction of the reference cell signal, theresulting sensorgrams were carefully aligned with the BIAe-valuation software (GE Healthcare) and analyzed using EVIL-FIT version 3 software (19, 20, 30) to determine the distributionof binding kinetics. The operator-set boundaries for the distri-butions were uniformly set to limit KD values in the intervalfrom 10�9 to 10�2 M, and kd values in the interval from 10�6 to100 s�1. For experiments with simvastatin, the procedures forSPR measurements were the same as for characterization of the�MI binding to ligands described above. In all experiments withsimvastatin, the �MI concentration was fixed at 10 �M. Fromstocks of simvastatin diluted in DMSO to 10 mg/ml (23.9 mM),a fresh dilution of simvastatin for each experiment was made toa final concentration of 200 �M, corresponding to a final con-centration of 0.42% (v/v) DMSO. From this dilution, furtherserial dilutions of simvastatin were made to 100, 50, 25, 12.5,6.25, and 3.13 �M simvastatin in running buffer supplementedwith 0.42% (v/v) DMSO together with a reference containing nosimvastatin. This buffer was also used as running buffer. Thedistribution in binding kinetics was extracted for each sensor-gram resulting from application of simvastatin or the referencewith no simvastatin.

Electric Cell-substrate Impedance Sensing Cell AdhesionAssay—ECIS measurements were made essentially as described(17). E-plate L 8 plates (ACEA Biosciences, Inc., San Diego, CA)were activated by 4 mg/ml dithiobis(succinimidyl propionate)for 0.5 h and then treated with 100 �l/well of 10 �g/ml iC3bdissolved in PBS or PBS only as coating reagents for 1 h at roomtemperature. The plates were then washed with PBS threetimes. Meanwhile, the monocytes were thawed, added to PBSsupplemented with 20% (v/v) FCS, and harvested by centrifu-gation. Freshly cultured K562 cells were simply harvested fromculture medium. Cells were subsequently washed once andthen resuspended in binding buffer (10 mM HEPES, pH 7.4, 150mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) supple-mented with 0.1 mg/ml human serum albumin and 5 mM D-glu-cose. Monocytes were used at 1 � 107 cells/ml, whereas theK562 cells were used at 1.5 � 106 cells/ml. CR3 was activated bythe addition of KIM185 antibody at 10 �g/ml. DMSO-dissolvedsimvastatin was applied in a fixed 100-fold dilution to the cellsuspensions for reaching a final concentration of 6.25–50 �M.The mixtures were added to the E-plate L 8, which were subse-quently mounted on an iCELLigence device (ACEA) for datacollection over 3 h in an incubator at 37 °C and with 5% (v/v)CO2. All measurements for both coated and control wells weredone at least three times.

Fluorescent Bead Binding Assay—Samples of 50 �l of fluores-cent beads (F-13081, Life Technologies) were activated by 1.5mg/ml NHS and 0.3 mg/ml N-(3-dimethylaminopropyl)-N -ethylcarbodiimide in 50 mM MES, pH 6.1, for 0.5 h and thenwashed twice with 50 mM MES, pH 6.1. Immediately afterward,beads were incubated with 100 �g of iC3b diluted in 50 mM

MES, pH 6.1, for 1 h. Finally, the beads were washed twice in 50mM MES, pH 6.1, and diluted in binding buffer with humanserum albumin and glucose as described above to 1 ml (5 � 108

beads/ml). Frozen monocytes were thawed as described for the

ECIS assay. Simvastatin stock solutions were added into flowcytometry tubes, followed by the addition of 100 �l of mono-cyte suspension and a brief mixing. Quickly following this step,35 �l of iC3b-coated beads were added and mixed well. Themixture was transferred to the incubator at 37 °C and with 5%(v/v) CO2 for 15 min. Afterward, cells were washed with 2 ml of0.1% (w/v) human serum albumin, 2 mM EDTA in PBS, pH 7.4,buffer and then with 2 ml of 0.5% (w/v) BSA in PBS, pH 7.4,buffer. Finally, the cells were fixed with 400 �l of 0.9% (v/v)formaldehyde in PBS, pH 7.4, and used in flow cytometry anal-ysis with the BD Bioscience LSRFortessaTM cell analyzer.

Centrifugation-based Cell Adhesion Assay with Monocytes—The centrifugation-based cell adhesion assay was conductedessentially as described (15, 33). Briefly, 96-well polystyrenemicrotiter plates with V-formed wells (CostarTM, Corning)were coated in 100 �l/well of 1 �g/ml iC3b dissolved in coatingbuffer (150 mM NaCl, 20 mM Tris, pH 9.4) and incubated for 1 hat 37 °C. The plates were blocked in PBS with 0.05% (v/v)Tween 20, pH 7.2, at room temperature for 1 h. Meanwhile,monocytes were thawed and added to PBS supplemented with20% (v/v) FCS. The cells were centrifuged and resuspended inRPMI 1640 supplemented with 2% (v/v) FCS. Cells were fluo-rescently labeled by incubation with 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (catalog no.14562, Sigma-Aldrich) at 37 °C and 5% (v/v) CO2 for 15 min.The cells were washed twice and resuspended in the bindingbuffer described above supplemented with 2.5% (v/v) FCS to1 � 106 cells/ml. Fifty microliters of binding buffer supple-mented with 2.5% (v/v) FCS and 0, 2, 10, or 20 �M simvastatinwere added to each well. Finally, to each well, 50 �l of the fluo-rescence-labeled monocyte suspension was added. Followingincubation at 37 °C and 5% (v/v) CO2 for 15 min, the plates werecentrifuged for 5 min at 10 � g, 20 � g, 30 � g, and 50 � g.Following each centrifugation, signals were read in a fluores-cence plate reader (Victor 3, Wallac Oy), and the fraction ofbinding cells was estimated by comparison with the signal fromuncoated wells blocked with Tween 20. All measurements forboth coated and control wells were done in triplicate and wereaveraged and used to calculate the cell binding.

Author Contributions—M. R. J. performed S. P. R. and cell adhesionexperiments; G. B. performed the x-ray crystallographic studies;X. Z. performed the cell adhesion studies; and M. R. J., K. K. S.,A. K. L., and H. D. performed the MD studies. B. S., G. R. A., andT. V.-J. designed and planned studies. M. R. J. and T. V.-J. wrote thepaper. All authors contributed to the writing of the draft and finalmanuscripts.

Acknowledgments—We thank Drs. Thorsten J. Maier and Niels Jessenfor advice and Bettina W. Grumsen and Anne Marie Bundsgaard forexcellent technical assistance. Allocations of time for supercomputingwere generously offered by the Center for Scientific Computing, Aar-hus University.

References1. Bajic, G., Degn, S. E., Thiel, S., and Andersen, G. R. (2015) Complement

activation, regulation, and molecular basis for complement-related dis-eases. EMBO J. 34, 2735–2757

Simvastatin Antagonism of Complement Receptor 3

16974 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 33 • AUGUST 12, 2016

2. Morgan, B. P., and Harris, C. L. (2015) Complement, a target for therapy ininflammatory and degenerative diseases. Nat. Rev. Drug Discov. 14,857– 877

3. Fossati-Jimack, L., Ling, G. S., Cortini, A., Szajna, M., Malik, T. H., Mc-Donald, J. U., Pickering, M. C., Cook, H. T., Taylor, P. R., and Botto, M.(2013) Phagocytosis is the main CR3-mediated function affected by thelupus-associated variant of CD11b in human myeloid cells. PLoS One 8,e57082

4. Curley, G. P., Blum, H., and Humphries, M. J. (1999) Integrin antagonists.Cell Mol. Life Sci. 56, 427– 441

5. Tsai, N. W., Chang, W. N., Shaw, C. F., Jan, C. R., Huang, C. R., Chen, S. D.,Chuang, Y. C., Lee, L. H., and Lu, C. H. (2009) The value of leukocyteadhesion molecules in patients after ischemic stroke. J. Neurol. 256,1296 –1302

6. Hong, S., Beja-Glasser, V. F., Nfonoyim, B. M., Frouin, A., Li, S., Ra-makrishnan, S., Merry, K. M., Shi, Q., Rosenthal, A., Barres, B. A., Lemere,C. A., Selkoe, D. J., and Stevens, B. (2016) Complement and microgliamediate early synapse loss in Alzheimer mouse models. Science 352,712–716

7. Springer, T. A. (1997) Folding of the N-terminal, ligand-binding region ofintegrin �-subunits into a �-propeller domain. Proc. Natl. Acad. Sci.U.S.A. 94, 65–72

8. Liddington, R. C. (2014) Structural aspects of integrins. Adv. Exp. Med.Biol. 819, 111–126

9. Xie, C., Zhu, J., Chen, X., Mi, L., Nishida, N., and Springer, T. A. (2010)Structure of an integrin with an �I domain, complement receptor type 4.EMBO J. 29, 666 – 679

10. Yalamanchili, P., Lu, C., Oxvig, C., and Springer, T. A. (2000) Folding andfunction of I domain-deleted Mac-1 and lymphocyte function-associatedantigen-1. J. Biol. Chem. 275, 21877–21882

11. Li, Y., and Zhang, L. (2003) The fourth blade within the �-propeller isinvolved specifically in C3bi recognition by integrin �M�2. J. Biol. Chem.278, 34395–34402

12. Lee, J. O., Bankston, L. A., Arnaout, M. A., and Liddington, R. C. (1995)Two conformations of the integrin A-domain (I-domain): a pathway foractivation? Structure 3, 1333–1340

13. Lee, J. O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Crystalstructure of the A domain from the � subunit of integrin CR3 (CD11b/CD18). Cell 80, 631– 638

14. Vorup-Jensen, T. (2012) On the roles of polyvalent binding in immunerecognition: perspectives in the nanoscience of immunology and the im-mune response to nanomedicines. Adv. Drug Deliv. Rev. 64, 1759 –1781

15. Vorup-Jensen, T., Carman, C. V., Shimaoka, M., Schuck, P., Svitel, J., andSpringer, T. A. (2005) Exposure of acidic residues as a danger signal forrecognition of fibrinogen and other macromolecules by integrin �X�2.Proc. Natl. Acad. Sci. U.S.A. 102, 1614 –1619

16. Bajic, G., Yatime, L., Sim, R. B., Vorup-Jensen, T., and Andersen, G. R.(2013) Structural insight on the recognition of surface-bound opsonins bythe integrin I domain of complement receptor 3. Proc. Natl. Acad. Sci.U.S.A. 110, 16426 –16431

17. Zhang, X., Bajic, G., Andersen, G. R., Christiansen, S. H., and Vorup-Jensen, T. (2016) The cationic peptide LL-37 binds Mac-1 (CD11b/CD18)with a low dissociation rate and promotes phagocytosis. Biochim. Biophys.Acta 1864, 471– 478

18. Stapulionis, R., Oliveira, C. L., Gjelstrup, M. C., Pedersen, J. S., Hokland,M. E., Hoffmann, S. V., Poulsen, K., Jacobsen, C., and Vorup-Jensen, T.(2008) Structural insight into the function of myelin basic protein as aligand for integrin �M�2. J. Immunol. 180, 3946 –3956

19. Svitel, J., Balbo, A., Mariuzza, R. A., Gonzales, N. R., and Schuck, P. (2003)Combined affinity and rate constant distributions of ligand populationsfrom experimental surface binding kinetics and equilibria. Biophys. J. 84,4062– 4077

20. Vorup-Jensen, T. (2012) Surface plasmon resonance biosensing in studiesof the binding between �2 integrin I domains and their ligands. MethodsMol. Biol. 757, 55–71

21. Dustin, M. L., Ferguson, L. M., Chan, P. Y., Springer, T. A., and Golan,D. E. (1996) Visualization of CD2 interaction with LFA-3 and determi-nation of the two-dimensional dissociation constant for adhesion re-

ceptors in a contact area. J. Cell Biol. 132, 465– 47422. Shimaoka, M., and Springer, T. A. (2003) Therapeutic antagonists and con-

formational regulation of integrin function. Nat. Rev. Drug Discov. 2, 703–71623. Kallen, J., Welzenbach, K., Ramage, P., Geyl, D., Kriwacki, R., Legge, G.,

Cottens, S., Weitz-Schmidt, G., and Hommel, U. (1999) Structural basisfor LFA-1 inhibition upon lovastatin binding to the CD11a I-domain. J.Mol. Biol. 292, 1–9

24. Weitz-Schmidt, G., Welzenbach, K., Brinkmann, V., Kamata, T., Kallen, J.,Bruns, C., Cottens, S., Takada, Y., and Hommel, U. (2001) Statins selec-tively inhibit leukocyte function antigen-1 by binding to a novel regulatoryintegrin site. Nat. Med. 7, 687– 692

25. Wang, Y., Li, D., Jones, D., Bassett, R., Sale, G. E., Khalili, J., Komanduri,K. V., Couriel, D. R., Champlin, R. E., Molldrem, J. J., and Ma, Q. (2009)Blocking LFA-1 activation with lovastatin prevents graft-versus-host dis-ease in mouse bone marrow transplantation. Biol. Blood Marrow Trans-plant. 15, 1513–1522

26. Jain, M. K., and Ridker, P. M. (2005) Anti-inflammatory effects of statins:clinical evidence and basic mechanisms. Nat. Rev. Drug Discov. 4, 977–987

27. Canalli, A. A., Proenca, R. F., Franco-Penteado, C. F., Traina, F., Sakamoto,T. M., Saad, S. T., Conran, N., and Costa, F. F. (2011) Participation ofMac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle celldisease neutrophils to endothelial layers, and reversal of adhesion by sim-vastatin. Haematologica 96, 526 –533

28. Silveira, A. A., Dominical, V. M., Lazarini, M., Costa, F. F., and Conran, N.(2013) Simvastatin abrogates inflamed neutrophil adhesive properties, inassociation with the inhibition of Mac-1 integrin expression and modula-tion of Rho kinase activity. Inflamm. Res. 62, 127–132

29. Chello, M., Mastroroberto, P., Patti, G., D’Ambrosio, A., Morichetti,M. C., Di Sciascio, G., and Covino, E. (2003) Simvastatin attenuates leu-cocyte-endothelial interactions after coronary revascularisation with car-diopulmonary bypass. Heart 89, 538 –543

30. Gorshkova, I. I., Svitel, J., Razjouyan, F., and Schuck, P. (2008) Bayesiananalysis of heterogeneity in the distribution of binding properties of im-mobilized surface sites. Langmuir 24, 11577–11586

31. Giaever, I., and Keese, C. R. (1993) A morphological biosensor for mam-malian cells. Nature 366, 591–592

32. Wegener, J., Keese, C. R., and Giaever, I. (2000) Electric cell-substrateimpedance sensing (ECIS) as a noninvasive means to monitor the kineticsof cell spreading to artificial surfaces. Exp. Cell Res. 259, 158 –166

33. Weetall, M., Hugo, R., Friedman, C., Maida, S., West, S., Wattanasin, S.,Bouhel, R., Weitz-Schmidt, G., and Lake, P. (2001) A homogeneous fluo-rometric assay for measuring cell adhesion to immobilized ligand usingV-well microtiter plates. Anal. Biochem. 293, 277–287

34. Xiong, J. P., Li, R., Essafi, M., Stehle, T., and Arnaout, M. A. (2000) Anisoleucine-based allosteric switch controls affinity and shape shifting inintegrin CD11b A-domain. J. Biol. Chem. 275, 38762–38767

35. Vorup-Jensen, T., Ostermeier, C., Shimaoka, M., Hommel, U., andSpringer, T. A. (2003) Structure and allosteric regulation of the �X�2integrin I domain. Proc. Natl. Acad. Sci. U.S.A. 100, 1873–1878

36. Orr, Y., Taylor, J. M., Cartland, S., Bannon, P. G., Geczy, C., andKritharides, L. (2007) Conformational activation of CD11b without shed-ding of L-selectin on circulating human neutrophils. J. Leukoc. Biol. 82,1115–1125

37. Nunez, S., Venhorst, J., and Kruse, C. G. (2012) Target-drug interactions:first principles and their application to drug discovery. Drug Discov. Today17, 10 –22

38. Kenakin, T. P. (1982) The Schild regression in the process of receptorclassification. Can. J. Physiol. Pharmacol. 60, 249 –265

39. Zhao, H., Gorshkova, I. I., Fu, G. L., and Schuck, P. (2013) A comparison ofbinding surfaces for SPR biosensing using an antibody-antigen system andaffinity distribution analysis. Methods 59, 328 –335

40. Arnaout, M. A., Goodman, S. L., and Xiong, J. P. (2007) Structure andmechanics of integrin-based cell adhesion. Curr. Opin. Cell Biol. 19,495–507

41. Mahalingam, B., Van Agthoven, J. F., Xiong, J. P., Alonso, J. L., Adair, B. D.,Rui, X., Anand, S., Mehrbod, M., Mofrad, M. R., Burger, C., Goodman,S. L., and Arnaout, M. A. (2014) Atomic basis for the species-specificinhibition of alphaV integrins by monoclonal antibody 17E6 is revealed by

Simvastatin Antagonism of Complement Receptor 3

AUGUST 12, 2016 • VOLUME 291 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 16975

the crystal structure of �V�3 ectodomain-17E6 Fab complex. J. Biol.Chem. 289, 13801–13809

42. Gao, C., Boylan, B., Bougie, D., Gill, J. C., Birenbaum, J., Newman, D. K.,Aster, R. H., and Newman, P. J. (2009) Eptifibatide-induced thrombocy-topenia and thrombosis in humans require Fc�RIIa and the integrin �3cytoplasmic domain. J. Clin. Invest. 119, 504 –511

43. Huang, C., Zang, Q., Takagi, J., and Springer, T. A. (2000) Structural andfunctional studies with antibodies to the integrin �2 subunit: a model forthe I-like domain. J. Biol. Chem. 275, 21514 –21524

44. Shimaoka, M., Xiao, T., Liu, J. H., Yang, Y., Dong, Y., Jun, C. D., McCor-mack, A., Zhang, R., Joachimiak, A., Takagi, J., Wang, J. H., and Springer,T. A. (2003) Structures of the �L I domain and its complex with ICAM-1reveal a shape-shifting pathway for integrin regulation. Cell 112, 99 –111

45. Alvarez-Lueje, A., Valenzuela, C., Squella, J. A., and Nunez-Vergara, L. J.(2005) Stability study of simvastatin under hydrolytic conditions assessedby liquid chromatography. J. AOAC Int. 88, 1631–1636

46. Andrew, D., Shock, A., Ball, E., Ortlepp, S., Bell, J., and Robinson, M. (1993)KIM185, a monoclonal antibody to CD18 which induces a change in theconformation of CD18 and promotes both LFA-1- and CR3-dependentadhesion. Eur. J. Immunol. 23, 2217–2222

47. Lu, C., Ferzly, M., Takagi, J., and Springer, T. A. (2001) Epitope mapping ofantibodies to the C-terminal region of the integrin �2 subunit revealsregions that become exposed upon receptor activation. J. Immunol. 166,5629 –5637

48. Bjorkhem-Bergman, L., Lindh, J. D., and Bergman, P. (2011) What is arelevant statin concentration in cell experiments claiming pleiotropic ef-fects? Br. J. Clin. Pharmacol. 72, 164 –165

49. Chataway, J., Schuerer, N., Alsanousi, A., Chan, D., MacManus, D.,Hunter, K., Anderson, V., Bangham, C. R., Clegg, S., Nielsen, C., Fox, N. C.,Wilkie, D., Nicholas, J. M., Calder, V. L., Greenwood, J., et al. (2014) Effectof high-dose simvastatin on brain atrophy and disability in secondaryprogressive multiple sclerosis (MS-STAT): a randomised, placebo-con-trolled, phase 2 trial. Lancet 383, 2213–2221

50. Alon, R., and Ley, K. (2008) Cells on the run: shear-regulated integrinactivation in leukocyte rolling and arrest on endothelial cells. Curr. Opin.Cell Biol. 20, 525–532

51. Weitz-Schmidt, G. (2002) Statins as anti-inflammatory agents. TrendsPharmacol. Sci. 23, 482– 486

52. Bergman, E., Forsell, P., Tevell, A., Persson, E. M., Hedeland, M., Bondes-son, U., Knutson, L., and Lennernas, H. (2006) Biliary secretion of rosuv-astatin and bile acids in humans during the absorption phase. Eur.J. Pharm. Sci. 29, 205–214

53. Zhang, J., Osawa, S., Takayanagi, Y., Ikuma, M., Yamada, T., Sugimoto, M.,Furuta, T., Miyajima, H., and Sugimoto, K. (2013) Statins directly suppresscytokine production in murine intraepithelial lymphocytes. Cytokine 61,540 –545

54. Weiner, H. L., da Cunha, A. P., Quintana, F., and Wu, H. (2011) Oraltolerance. Immunol. Rev. 241, 241–259

55. Petruzelli, L., Luk, J., and Springer, T. A. (1995) Adhesion structure sub-panel 5, leukocyte integrins: CD11a, CD11b, CD11c, CD18. in LeucocyteTyping V: White Cell Differentiation Antigens (Schlossman, S. F., Boum-sell, L., Gilks, W., Harlan, J., Kishimoto, T., Morimoto, T., Ritz, J., Shaw, S.,Silverstein, R., Springer, T. A., Tedder, T., and Todd, R., eds) pp.1581–1585, Oxford University Press, New York

56. Kabsch, W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–13257. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Sto-

roni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J. Appl.Crystallogr. 40, 658 – 674

58. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molec-ular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126 –2132

59. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols,N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., Mc-Coy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., et al.(2010) PHENIX: a comprehensive Python-based system for macromolec-ular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221

60. Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J., Wang,X., Murray, L. W., Arendall, W. B., 3rd, Snoeyink, J., Richardson, J. S., andRichardson, D. C. (2007) MolProbity: all-atom contacts and structure val-idation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383

61. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., andKlein, M. L. (1983) Comparison of simple potential functions for simulat-ing liquid water. J. Chem. Phys. 79, 926 –935

62. Kaminski, G. A., and Friesner, R. A. (2001) Evaluation and reparametriza-tion of the OPLS-AA force field for proteins via comparison with accuratequantum chemical calculations on peptides. J. Phys. Chem. B 105,6474 – 6487

63. Tuckerman, M. E., Berne, B. J., and Martyna, G. J. (1992) Reversible mul-tiple time scale molecular dynamics. J. Chem. Phys. 97, 1990 –2001

64. Darden, T., York, D., and Pedersen, L. (1993) Particle mesh Ewald: anNlog(N) method for Ewald sums in large systems. J. Chem. Phys. 98,10089 –10092

65. Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: visual moleculardynamics. J. Mol. Graph. 14, 33–38, 27–28

Simvastatin Antagonism of Complement Receptor 3

16976 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 33 • AUGUST 12, 2016

Katrine Kirkeby Skeby, Birgit Schiøtt, Gregers R. Andersen and Thomas Vorup-JensenMaria Risager Jensen, Goran Bajic, Xianwei Zhang, Anne Kjær Laustsen, Heidi Koldsø,

Receptor 3Structural Basis for Simvastatin Competitive Antagonism of Complement

doi: 10.1074/jbc.M116.732222 originally published online June 23, 20162016, 291:16963-16976.J. Biol. Chem. 

  10.1074/jbc.M116.732222Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/291/33/16963.full.html#ref-list-1

This article cites 64 references, 17 of which can be accessed free at

by guest on January 9, 2021http://w

ww

.jbc.org/D

ownloaded from