Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers

Recombinant Technology

Stabilization of soluble, low-affinity HLA-DM/HLA-DR1

complexes by leucine zippers

Robert Busch a,*,1, Achal Pashine a,1, K. Christopher Garcia b, Elizabeth D. Mellins a

aDepartment of Pediatrics, Stanford University Medical Center, Room 2120 CCSR Bldg., 269 Campus Drive, Stanford, CA 94305-5164, USAbDepartments of Structural Biology and Microbiology/Immunology, Fairchild Center, Stanford, CA 94305-5124, USA

Received 26 October 2001; received in revised form 7 February 2002; accepted 7 February 2002

Abstract

The ectodomains of interacting membrane-bound proteins, when expressed as recombinant soluble molecules, often have

low affinities for each other, hampering studies of their interaction. We reasoned that stabilization of unstable protein–protein

complexes should aid our understanding of the structural and functional consequences of complex formation. Here, we have

used fusion with leucine zipper (LZ) domains to stabilize a complex formed between the class II major histocompatibility

complex (MHC-II) protein, HLA-DR1 (which binds peptides for presentation to CD4 + T cells) and HLA-DM (which catalyzes

peptide exchange of MHC-II molecules). To this end, the DM h chain ectodomains were fused to acidic LZ domains (AcidP1 or

Fos); similarly, the DR1 h chain ectodomains were fused to basic LZ domains (BaseP1 or Jun). We expressed LZ-modified

soluble DM or DR1 ah dimers, or both, in insect cells and purified the secreted sDM-AcidP1 and sDR1-BaseP1 molecules as

well as the complex. LZ modification greatly enhanced DM-catalyzed peptide binding to DR1 compared to unmodified soluble

DM and DR1. We readily detected LZ-modified DM/DR complexes on native PAGE gels and by coimmunoprecipitation. Thus,

fusion with artificial LZ domains can stabilize unstable protein–protein complexes for biochemical and structural studies of

interactions within the complex. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: MHC class II proteins; X-ray crystallography; Insect cell expression; Peptide binding; Protein expression

1. Introduction

A vast array of biological functions is carried out

by means of proteins interacting with other proteins

with affinities, as measured by dissociation constants,

ranging over many orders of magnitude. As a prereq-

uisite for efficient interaction, the affinities of soluble

monomeric proteins for their interaction partners often

are of a similar order of magnitude as the concen-

tration of those partner molecules. Other rules apply

when higher-order complexes are formed or when

the interacting proteins are spatially confined, for

instance, by being anchored in a membrane. In these

settings, the avidity of the interaction can be much

higher than suggested by monomeric affinities. Thus,

low-affinity interactions are not necessarily biologi-

cally irrelevant though they are often difficult to study

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -1759 (02 )00034 -0

Abbreviations: Con A, concanavalin A; HLA-DM/DR, human

leukocyte antigen DM/DR; LZ, leucine zipper; mAb, monoclonal

antibody; MHC-II, major histocompatibility complex class II; NTA,

nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis;

s (prefix), soluble.* Corresponding author. Tel.: +1-650-498-7574; fax: +1-650-

736-0194.

E-mail address: [email protected] (R. Busch).1 Equal contributors.

www.elsevier.com/locate/jim

Journal of Immunological Methods 263 (2002) 111–121

experimentally. It can thus be desirable to stabilize

low-affinity protein–protein complexes artificially to

facilitate structural and biochemical studies.

As an example illustrating the potential utility of

this approach, we have been investigating a low-

affinity protein–protein complex between MHC-II

molecules and HLA-DM, whose formation is critical

for the induction of cellular immune responses. MHC-

II molecules bind diverse self and foreign peptides in

the endocytic pathway and present them at the surface

of antigen-presenting cells for inspection by CD4+ T

lymphocytes (Germain, 1994). HLA-DM (H2-M in

mice) is a key factor in endosomal peptide loading of

MHC-II molecules. It accelerates release of invariant

chain peptides from newly synthesized MHC-II mol-

ecules, edits the peptide repertoire, and stabilizes

empty MHC-II (reviewed in Busch and Mellins,

1996; Busch et al., 2000). To mediate these effects,

DM transiently forms a complex with MHC-II pro-

teins (Denzin et al., 1996; Sanderson et al., 1996).

At present, little is known about the structure of the

DM/MHC-II complex and about the mechanism by

which DM catalyzes peptide release. The crystal

structures of human and murine DM (Fremont et al.,

1998; Mosyak et al., 1998) and of various MHC-II/

peptide complexes (e.g., Stern et al., 1994; Ghosh et

al., 1995) have been elucidated; however, they have

yielded few clues to the structure of the DM/DR

complex. Kinetic experiments have been interpreted

to highlight the importance of breaking conserved

hydrogen bonds involving the peptide backbone dur-

ing DM catalysis (Weber et al., 1996), but other

studies implicate peptide side chains (Chou and

Sadegh-Nasseri, 2000). The DM-stabilized conforma-

tion may resemble a kinetically defined unstable

MHC-II conformation that binds peptides rapidly in

the absence of DM, whose structure also remains

unknown (Rabinowitz et al., 1998; Natarajan et al.,

1999). Mutants have identified a face of MHC-II

molecules that interacts with DM during peptide

exchange (Doebele et al., 2000). A complementary

face has been identified on DM (A.P., J.N. Munning,

R.B., R.C. Doebele, E.D.M., in preparation). Inspec-

tion of the crystal structures in light of these results

reveals some shape complementarity (Fig. 1A), but

does not yield an obvious docking solution.

One obstacle to structural and biochemical studies

of the complex is the low apparent affinity of DM and

Fig. 1. Experimental design. (A) The putative orientation of DM and

DR in the DM/DR complex, as predicted from mutagenesis data.

Arrows indicate C termini of the DM and DR a and h chains.

Polypeptide chains are color-coded. (B) The experimental strategy

involves replacing the transmembrane and cytoplasmic regions of

DM and DR with complementary LZs. (C) Expression strategies:

DM and DR h chains fused to either Fos and Jun or artificial

AcidP1 and BaseP1 LZ domains, respectively, are expressed

together with their a chain partners in insect cells, yielding

individual zipper-modified DM and DR molecules. Alternatively,

both molecules are coexpressed.

R. Busch et al. / Journal of Immunological Methods 263 (2002) 111–121112

MHC-II for each other. Soluble ectodomains of the

human MHC-II molecule, HLA-DR1, require micro-

molar concentrations of soluble DM for efficient

peptide loading (Busch et al., 1998b; cf. Fig. 1B).

When full-length molecules are used, the interaction is

far more efficient (Busch et al., 1998b), probably

because the endosomal membrane (or in vitro a

detergent micelle) constrains the orientation of the

two molecules relative to each other, thus increasing

their effective concentration. In order to perform

structural studies without interference from lipid or

detergent, we sought alternative ways to stabilize the

DM/DR1 complex.

A common approach to stabilizing protein–pro-

tein dimers involves fusion with heterologous

domains that mediate dimerization such as engi-

neered immunoglobulin (Ridgway et al., 1996) or

leucine zipper (LZ) domains. First found in dimeric

transcription factors, LZs are coiled coil domains

comprised of two parallel, amphipathic a helices,

each of which features a characteristic heptad

leucine repeat (Alber, 1992). Among many other

applications, LZ fusion has been used to promote

assembly of heterodimeric proteins that fold ineffi-

ciently in heterologous expression systems (Chang

et al., 1994; Kalandadze et al., 1996; Scott et al.,

1996). However, once assembled, such heterodimers

are quite stable, in contrast to the instability of

complexes between soluble DM and DR1. Thus, it

remained an open question whether fusion with LZ

domains would stabilize the DM/DR1 complex

sufficiently to be useful. Here, we describe the

construction of soluble (s)DM and sDR1 molecules

fused to complementary LZ domains via their hchains (Fig. 1B). We demonstrate that this approach

results in correct assembly and expression of stable,

soluble, and functional DM/MHC class II com-

plexes. We propose that fusion with LZ domains

may be of general use in the characterization of

otherwise unstable protein–protein complexes.

2. Experimental procedures

2.1. Antibodies and affinity columns

The following antibodies were used: SU36, a rabbit

polyclonal antiserum against sDM (Cocalico, Reams-

town, PA); CHAMP, a rabbit polyclonal specific for

sDR1 (Stern and Wiley, 1992); M2, a mAb specific for

the FLAG epitope tag (Sigma, St. Louis, MO); 5C1, a

mAb specific for the DMa extracellular domains

(Sanderson et al., 1996); L243, a mAb specific for

the a chain of HLA-DR ah dimers (Lampson and

Levy, 1980); 2H11, a mAb specific for the artificial

AcidP1/BaseP1 LZ dimer (Chang et al., 1994; gift of

Dr. E. Reinherz). Concanavalin A (Con A)-sepharose

and M2-agarose (Sigma), Ni-NTA-agarose (Qiagen,

Valencia, CA) and protein A-sepharose (Amersham

Pharmacia Biotech, Piscataway, NJ) were obtained

commercially. L243 was conjugated to CNBr-activated

sepharose (Pharmacia) according to manufacturer’s

instructions.

2.2. Constructs, transfection, selection, and insect cell

culture

Constructs containing sDRA*0101, sDRB1*0101,

sDMA*0101 and sDMB*0101 cDNAs in the insect

cell expression vector, pRmHA-3, were a gift of Dr.

D. Zaller (Sloan et al., 1995). The cDNAs for Fos

and Jun zipper domains fused to DR2 were a gift of

Dr. K. Wucherpfennig (Kalandadze et al., 1996).

AcidP1 and BaseP1 artificial LZ domain cDNAs

were a gift of Dr. Luc Teyton (Scott et al., 1996).

Overlap extension PCR was performed essentially as

described (Doebele et al., 2000), yielding hybrid

cDNAs whose predicted amino acid sequence is

listed in Table 1. Hybrid cDNAs were digested with

appropriate restriction enzymes and reinserted into

pRmHA-3.

Schneider-2 Drosophila melanogaster cells were

transfected with 5 Ag of each cDNA construct and

0.5 Ag pUChs-Neo for drug resistance (Sloan et al.,

1995), using Ca3(PO4)2 precipitation (Life Technol-

ogies, Rockville, MD). Resistant cells were selected

using 1 mg/ml G418 in Schneider’s Drosophila

medium plus 10% FBS, 2 mM L-glutamine, and

antibiotics. Large-scale cultures were expanded to

0.5–2 l in spinner flasks, adapted to serum-free

medium (BaculoGold, Pharmingen, San Diego, CA)

and grown to maximal density. Expression of

soluble DM and DR molecules was induced by

adding 1 mM CuSO4 to activate the metallothionein

promoter of pRmHA-3 for another 7 days of

culture.

R. Busch et al. / Journal of Immunological Methods 263 (2002) 111–121 113

2.3. Immunochemical analyses

Supernatants were loaded onto 12% SDS-PAGE

minigels or onto native PAGE gels with pH 8.2

separating gel buffer (Jardetzky et al., 1990). Alter-

natively, supernatants (0.5 ml) were immunoprecipi-

tated using concanavalin A (Con A)-sepharose or

antibodies adsorbed to protein A-sepharose (20 Al)prior to SDS-PAGE and Western blotting (Busch et

al., 1998a).

2.4. Protein purification

All steps were done at 4 jC. Unmodified sDM and

sDM-AcidP1, both containing FLAG epitope tags at

the sDMa C terminus, were purified from culture

supernatants by affinity chromatography on M2-agar-

ose and FLAG peptide elution as described (Busch et

al., 1998b). Further purification was achieved by high

performance size-exclusion chromatography (TSK

G3000SWXL, TosoHaas, Montgomeryville, PA) in

10 mM sodium phosphate, pH 7.0, 150 mM NaCl,

0.02% NaN3 with isolation of the sDM dimer peak.

Unmodified sDR1 and sDR1-BaseP1 were purified by

L243-sepharose chromatography as described (Sloan

et al., 1995).

Complexes formed between sDM-AcidP1 and

sDR1-BaseP1 were purified by Ni-NTA agarose

chromatography as described by the manufacturer

(Qiagen). Briefly, culture supernatants were dialyzed

against PBS and concentrated using an Amicon

pressure cell with a YM-30 membrane (Millipore,

Bedford, MA), adjusted to 300 mM NaCl, 10 mM

imidazole, pH 8.0, and filtered. Dialyzed proteins

were mixed with 0.01 volume of Ni-NTA-agarose,

rotated overnight at 4 jC, and the resin poured into

a column. After extensive washing in 50 mM

sodium phosphate buffer, pH 8.0, 300 mM NaCl,

10 mM imidazole, bound proteins were eluted

successively with 20, 100, and 250 mM imidazole

in the same buffer. The concentrated 100 mM eluate

was diluted in wash buffer and repurified using the

same protocol.

2.5. Peptide-binding assay

Purified sDR1 or sDR1-BaseP1 (10 nM) was

mixed with sDM or sDM-AcidP1 at the indicated

concentrations in 50 mM sodium acetate buffer, pH

4.7, 150 mM NaCl, 1% (w/v) BSA, 0.05% (w/v)

NaN3. Binding of biotinylated HA307–319 peptide

(PKYVKQNTLKLAT; 20 AM) was allowed to pro-

ceed at 37 jC for the indicated times before termi-

nating reactions with 2 volumes of ice-cold

neutralization buffer (50 mM Tris–HCl, pH 8.2,

150 mM NaCl, 1% (w/v) BSA, 0.5% (v/v) NP40,

0.05% (w/v) NaN3). DR-peptide complexes were

quantified by L243 capture of sDR1 on microtiter

plates and fluorescence detection of peptide-bound

streptavidin-Eu3 + (Sloan et al., 1995).

2.6. Formation of sDM-AcidP1/sDR1-BaseP1 com-

plexes in vitro

sDM-AcidP1 and sDR1-BaseP1 (ca. 1.5 AM each)

were mixed in 10 mM sodium phosphate, pH 7.0, 150

mM NaCl, 0.02% (w/v) NaN3 alone or with the

indicated additives and incubated for 40 min at 37 jC.

Table 1

LZ-modified DM and DR1 h chains

Name Structure

DM or DR Spacer Leu zipper His6 tag

sDMh-Fos leader-h1-h2-SPMQTLK GGGSGGGS LTDTLQAET-Fos-stop

sDR1h-Jun leader-h1-h2-SESAQSK VDGGGGG RIARLEEKV-Jun-stop

sDMh-AcidP1 leader-h1-h2-SPMQTLK GGGSGGGS TTAPSAQLE-AcidP1-LEKELAQ HHHHHH-stop

sDR1h-BaseP1 leader-h1-h2-SESAQSK GGGSGGGS TTAPSAQLK-BaseP1-LKKKLAQ HHHHHH-stop

Structures of zipper-modified sDM (DMB*0101) and sDR1 (DRB1*0101) h chains (one-letter code). Leader peptide (L) and extracellular

domains (h1/h2; Sloan et al., 1995) were fused to LZ domains via 7 or 8 amino acid flexible spacers. Fos and Jun zipper domains are derived

from the Fos/Jun transcription factor dimer (Kalandadze et al., 1996). AcidP1 and BaseP1 are artificial leucine zippers with a distribution of

charged residues engineered for optimal heterodimer stability (O’Shea et al., 1993; Scott et al., 1996). His6 tags were incorporated into the C

termini of the artificial zippers.


3. Results

Our model of the DM/DR complex predicts that

the C termini of the ectodomains of DM and DR hchains should be in closer proximity to each other

than to either of the a chains, and closer than the a

chains are to each other (Fig. 1A, arrows). Accord-

ingly, we fused complementary LZ domains to the

sDM and sDR1 h chains (Table 1). As the precise

orientation of DM and DR in the complex is not

known, we separated the zipper domains from the h2domains by flexible spacers. We compared two sets of

complementary LZ domains used previously to facil-

itate heterodimer assembly, the zipper domains of Fos

and Jun and the artificial zipper domains called

AcidP1 and BaseP1 (Table 1).

In order to generate soluble, zipper-modified HLA-

DR1 molecules, we transfected sDR1B-Jun or -

BaseP1 cDNAs together with sDRA into Drosophila

cells (Fig. 1C). The DR1 allele (DRB1*0101) was

chosen for its ability to form stable soluble ahheterodimers in insect cells without stabilizing mod-

ifications (Stern and Wiley, 1992). Similarly, we co-

transfected sDMB-Fos or -AcidP1 with sDMA to

express zipper-modified sDM ah dimers. In an

attempt to form zipper-modified DM/DR complexes

in vivo, we performed quintuple cotransfections using

genes for DM a and h, DR a and h, as well as for

drug resistance.

3.1. Expression of zipper-modified DM and DR in

insect cells

To examine whether LZ-modified DM and DR

molecules were properly expressed, assembled, and

secreted, culture supernatants were analyzed by West-

ern blotting (Fig. 2). LZ-modified DM chains were

detected by anti-DM antisera (Fig. 2A), and the a

chain was detected by an epitope tag-specific mAb

(Fig. 2B). Comparison with the sDM standard

revealed that the expression level was on the order

of 1 mg sDM per liter of supernatant. As observed

previously for unmodified sDM, the a chains showed

size heterogeneity, likely due to differential glycosy-

lation (Busch et al., 1998b). Interestingly, this hetero-

geneity was reduced when zippered DR molecules

were coexpressed with DM, suggesting that the coex-

pressed DM and DR molecule assemble with each

Fig. 2. Expression of zipper-modified DM and DR heterodimers in

insect cells. Insect cells were transfected with cDNA constructs

encoding zipper-modified soluble DM and/or DR1 h chains

together with soluble a chain constructs. Gene expression was

induced in small-scale cultures. For comparison, purified sDR1 and

sDM standards were added to supernatants from untransfected cells

at 1 Ag/ml (A–C) or loaded directly on the gel (D, E). Band

assignments are based on internal comparisons and other data (Fig.

5A; Busch et al., 1998b). (A) Detection of DM on Western blots of

SDS-PAGE gels, using an antiserum against recombinant soluble

DM. (B) Detection of DM a chain using anti-Flag mAb, M2. Note

differences in mobility between samples containing zipper-modified

DM alone and those that coexpress zipper-modified DR. (C) Dimer

region of a native PAGE gel probed with the anti-DM antiserum.

The nonspecific band migrating slightly slower than DM is bovine

serum albumin. Note that the dimer band is comparatively weak for

the sDM-AcidP1/sDR1-BaseP1 complex. Some anti-DM-reactive

material in this lane was supershifted into a higher molecular weight

complex (see Fig. 3). (D) Expression of LZ-modified sDR1

molecules detected by immunoblotting of SDS gels using an anti-

DR antiserum after glycoprotein capture on Con A-sepharose beads.

(E) Capture of LZ-modified, soluble DR1 ah dimer using a DR

dimer-specific mAb. Detection as in panel D.


other intracellularly prior to carbohydrate processing

(Fig. 2B). Proper assembly of LZ-modified DM into

ah heterodimers was demonstrated by Western blot-

ting of native PAGE gels with anti-DM antisera (Fig.

2C). As expected, zipper-modified DM migrated more

slowly than unmodified DM. Disproportionately low

amounts of sDM-AcidP1 dimer were detected when

sDR1-BaseP1 was coexpressed; as shown in Fig. 3,

some sDM-AcidP1 is supershifted in this situation.

When LZ-modified DR molecules from superna-

tants or glycoprotein preparations were blotted with

an anti-DR a and h chain antiserum, only a single

band was detected (Fig. 2D and data not shown). This

is most likely due to comigration of the LZ-modified

sDR1h chains with sDRa because a lower molecular

weight sDR1h band was detected when unmodified

sDR1 was used. Similar results were obtained under

reducing (Fig. 2D) and nonreducing (not shown)

conditions. To confirm the presence of DR ah heter-

odimers, we demonstrated that DR molecules could

be immunoprecipitated with a dimer-specific mAb

(Fig. 2E). Comparison to quantitative standards indi-

cated high levels of expression, in excess of 1 mg LZ-

modified sDR1 per liter of supernatant (data not

shown) although expression was less in cells that also

expressed LZ-modified DM. This may reflect lower

efficiency of quintuple than of triple cotransfection,

promoter competition for limiting transcription fac-

tors, or an effect of DM on DR1 stability. Nonethe-

less, DR1 was present in excess over DM in cells that

expressed both molecules.

3.2. Biochemical evidence for association of zippered

DM and DR in vivo

To examine whether zipper-modified DM assem-

bles with zipper-modified DR1, we immunoprecipi-

tated DR and detected any associated DM molecules

by Western blotting (Fig. 3A). Specific association

was detected by this assay for both Fos/Jun and Acid/

Base LZs. Thus, LZ-modified DM and DR form

complexes in vivo that survive coprecipitation.

Does coprecipitation reflect proper assembly of the

LZ heterodimer, or do zipper-modified DM and DR

associate by other mechanisms such as binding of the

DM zipper domain to the antigen-binding groove of

MHC class II? The availability of a mAb to the

artificial AcidP1/BaseP1 LZ heterodimer (Chang

et al., 1994) allowed us to examine this question.

Insect cell supernatants were immunoprecipitated with

this mAb and analyzed for the presence of zippered

DM and DR by immunoblotting (Fig. 3B). Both

sDR1-BaseP1 and sDM-AcidP1 were immunopreci-

pitated when coexpressed. Precipitation was specific

Fig. 3. Association of zippered DM/DR complexes in vivo. Insect

cell supernatants were prepared as in Fig. 2. (A) Zipper-modified

DR ah dimers were immunoprecipitated as in Fig. 1E, and

associated zipper-modified DM was detected using the anti-DM

polyclonal antiserum, SU36. Soluble DM and DR1 standards (1 Ag/ml) were loaded directly onto the gel. (B, C) Immunoprecipitation

of sDM-AcidP1/sDR1-BaseP1 complexes using a mAb reactive

with the AcidP1/BaseP1 LZ heterodimer. DM (B) and DR (C)

molecules were detected by Western blotting with appropriate

polyclonal antisera. (D) Detection of sDM-AcidP1/sDR1-BaseP1

complexes by Western blotting of native PAGE gels using the

AcidP1/BaseP1 heterodimer-specific mAb. The upper third of the

gel is shown; no other bands were observed.


for the Acid/Base heterodimer because DM/DR com-

plexes stabilized by the Fos/Jun heterodimer were not

precipitated. We also detected complexes of coex-

pressed sDM-AcidP1 and sDR1-BaseP1 on native

PAGE gels blotted with the AcidP1/BaseP1-specific

mAb (Fig. 3C). This technique simplified screening of

culture supernatants and purification fractions for the

presence of properly assembled DM/DR complexes.

We concluded that both Fos/Jun and AcidP1/

BaseP1 LZ domains mediate specific association

between sDM and sDR1. The AcidP1/BaseP1 com-

plexes were chosen for purification because their

fusion to His6 tags allowed simple enrichment, proper

assembly of the zipper domains could be verified, and

purification with the LZ dimer-specific mAb might

allow specific isolation of complexes in the presence

of the uncomplexed individual molecules (Chang et

al., 1994).

3.3. Purification and biological activity of sDM-

AcidP1 and sDR1-BaseP1 in vitro

We purified sDM-AcidP1 and sDR1-BaseP1 by

methods that have been used previously for purifica-

tion of unmodified sDR1 and sDM. Immunoaffinity

chromatography was used to capture sDM-AcidP1 via

a FLAG epitope tag fused to the sDMa C terminus.

Elution with FLAG peptide resulted in material of

good purity, which in some experiments was purified

further by HPSEC. Similarly, we purified sDR1-

BaseP1 in good yield and purity using affinity chro-

matography on immobilized anti-DR ah mAb (not

shown; cf. Fig. 5A below).

High concentrations of sDM increase the efficiency

of biotinylated peptide binding to sDR1 in fluorescent

plate-binding assays, providing a convenient measure

of sDM activity (Sloan et al., 1995; Busch et al.,

1998b). This may reflect catalysis of conformational

changes that limit the rate of stable peptide/sDR1

complex formation (Zarutskie et al., 2001) or possibly

release of insect cell-derived peptides or polypeptides

from sDR1 (Busch et al., 1996; Aichinger et al.,

1997). Stabilization of sDM/sDR1 complexes by

LZs might be expected to result in increased efficacy

and altered concentration dependence of sDM catal-

ysis. To examine this, we measured the time course of

binding of excess biotinylated HA307–319 peptide to

10 nM purified sDR1 or sDR1-AcidP1 in the presence

of an equimolar amount of sDM or sDM-AcidP1.

Under these conditions, the rate of peptide binding to

sDR1 in the presence of unmodified sDM (Fig. 4A)

was indistinguishable from the uncatalyzed reaction

(not shown; cf. Busch et al., 1998b) because sDM was

too dilute for measurable catalysis. A strikingly differ-

ent result was obtained when sDM-AcidP1 and sDR1-

BaseP1 were allowed to interact (Fig. 4A): A sub-

stantial amount of peptide binding was detected

within 5 min, and the reaction was complete within

30 min of peptide addition. Both sDM and sDR1 are

needed to be modified with complementary LZs to

achieve this effect. When unmodified sDR1 was

exposed to sDM-AcidP1, the same progress curve

was obtained as with unmodified sDM (Fig. 4A).

Unexpectedly, a rate increase also was observed when

sDR1-BaseP1 was exposed to unmodified sDM, but

this was modest compared to the enhancement seen

when both molecules contained LZ domains.

To quantify the degree of stabilization of DM/DR

complexes by the AcidP1/BaseP1 LZ pair, a constant

amount of sDR1 or sDR1-BaseP1 (10 nM) was tested

for peptide binding in the presence of varying

amounts of sDM or sDM-AcidP1 in a 3-h assay

(Fig. 4B). As expected, both sDR1 and sDR1-BaseP1

bound similar quantities of biotinylated HA peptide in

the absence of sDM. Peptide binding to unzippered

sDR1 was enhanced only slightly in the presence of

up to 100 nM zipper-modified or -unmodified sDM.

Consistent with the rate enhancement seen when only

sDR1 was fused to a LZ, sDR1-BaseP1 required

about 100 times less sDM (c 1 nM) for detectable

enhancement of peptide binding than unmodified

sDR1. Extremely efficient peptide binding was

observed when both sDM and sDR1 were fused to

LZs, with near-maximal peptide binding to 10 nM

sDR1-BaseP1 observed at sDM-AcidP1 concentra-

tions as low as 100 pM. In other experiments, in

which even lower concentrations of sDM-AcidP1

were used, as little as 1 pM sDM-AcidP1 resulted in

detectable enhancement of peptide association (A.P.,

J.N. Munning, R.B., R.C. Doebele, E.D.M., in prep-

aration). These results showed that sDM-AcidP1 can

act catalytically to promote peptide loading of multi-

ple sDR1-BaseP1 molecules, implying that associa-

tion between sDM-AcidP1 and sDR1-BaseP1 is

reversible. We concluded that complementary LZs

fused to the h chain C termini of sDM and sDR1


greatly improve functional interaction between DM

and DR in vitro.

Intriguingly, when equimolar or higher amounts of

sDM-AcidP1 were used, the amount of peptide bind-

ing to sDR1-BaseP1 after 3 h was lower than when

catalytic amounts of sDM-AcidP1 were used (Fig.

4B). A likely explanation is that under these condi-

tions, the peptide forms unstable complexes with

sDM-AcidP1/sDR1-BaseP1 complexes rather than

the stable complexes formed with free sDR1-BaseP1

(not shown).

The observation that coexpression of sDR1-

BaseP1 and sDM-AcidP1 resulted in altered post-

translational modification of sDM-AcidP1 suggested

that the properties of complexes formed by coexpres-

sion might differ from those of complexes formed in

vitro. To examine this possibility, the two populations

of DM/DR complexes were compared by native

PAGE and Western blotting with the AcidP1/BaseP1

zipper-specific mAb (Fig. 4C). Complexes were

detected after mixing equimolar quantities of DM

and DR in vitro under a variety of conditions; their

electrophoretic mobility was similar to that seen for

zippered DM/DR complexes formed in vivo. Anti-

DM antisera demonstrated the presence of a popula-

tion of free sDM-AcidP1, suggesting that whether

formed in vitro or in vivo, a fraction of DM/DR

complexes dissociated during native PAGE (not

shown). Taken together, these results suggested that

the stability of the DM/DR complexes formed in vitro

and in vivo was not drastically different.

3.4. Purification and peptide-binding activity of sDM-

AcidP1/sDR1-BaseP1 complexes formed in vivo

The techniques used for purification of the indi-

vidual sDM and sDR1 molecules were poorly suited

for purification of sDM-AcidP1/sDR1-BaseP1 com-

plexes assembled in vivo. Immunoaffinity purification

of sDR1 uses relatively harsh elution conditions (pH

11) that may disrupt DM/DR interactions. In addition,

small-scale attempts to purify complexes using M2-

agarose affinity chromatography revealed that sDR1-

BaseP1 bound M2-agarose beads even in the absence

of sDM-AcidP1 (not shown). To overcome these

difficulties, we purified sDM-AcidP1/sDR1-BaseP1

complexes by Ni-NTA-agarose chromatography via

C terminal His6 tags (Fig. 5). Bands in the expected

Fig. 4. Interaction of sDM-AcidP1 and sDR1-BaseP1 in vitro. (A)

Rate of peptide binding to unmodified and BaseP1 zipper-modified

sDR1 in the presence of unmodified or Acid-P1-modified sDM.

Equimolar concentrations of sDM and sDR1 were mixed with

biotinylated HA peptide and incubated at pH 4.7 for various times.

DR/biotinylated peptide complexes were captured on microtiter

plates coated with an anti-DR mAb and detected using streptavidin-

Eu3+. o, sDR1 plus sDM; D, sDR1 plus sDM-AcidP1; E, sDR1-

BaseP1 plus sDM; ., sDR1-BaseP1 plus sDM-AcidP1. (B) Effect

of DM concentration on peptide loading. Fixed concentrations

of sDR1 or sDR1-BaseP1 (10 nM) were mixed with varying

concentrations of sDM or sDM-AcidP1 as indicated and tested for

binding of biotinylated HA307–319 peptide in a 3-h assay as

described above. Symbols as in A. (C) Stable complexes between

sDR1-BaseP1 and sDM-AcidP1 are formed in vitro. Mixtures of

zipper-modified sDR1 and sDM were prepared in PBS alone (‘nil’)

or plus 1 mM CuSO4, 100 mM citrate buffer (final pH= 5.3), 1 M

NaCl, or 10% serum-free insect medium (SFM) as shown. Complex

formation was detected by native PAGE and Western blotting using

the LZ dimer-specific mAb.


molecular weight range eluted at 100 mM imidazole

(Fig. 5A), and purity was increased by a second round

of Ni chromatography (Fig. 5B). This eluate reacted

with antibodies to DM, DR (not shown), and the

AcidP1/BaseP1 LZ heterodimer (Fig. 5B). Interest-

ingly, the amounts of sDM AcidP1 and sDR1-BaseP1

recovered were comparable (Fig. 5A, B; data not

shown) even though the latter was expressed in excess

(Fig. 2). Anti-DR immunoblots suggested that excess

sDR1-BaseP1 eluted at 20 mM imidazole after initial

Ni capture (not shown). Like complexes formed by

mixing in vitro, the purified in vivo assembled

AcidP1/sDR1-BaseP1 complexes were capable of

efficient peptide binding (Fig. 5D). Peptide-binding

activity was recovered mostly in the 100 mM imida-

zole fraction.

4. Discussion

In this study, we have used LZs to force association

between two different weakly interacting molecules,

HLA-DR1 and HLA-DM. Our functional and bio-

chemical data show that fusion with complementary

LZs more than compensates for the c 200-fold drop

in the efficiency of sDM catalysis of peptide/sDR1

binding seen when the transmembrane region and

cytoplasmic tail are truncated (Busch et al., 1998b).

Thus, specific structural features of the truncated

regions are not required for efficient catalysis.

Given the low affinity of the soluble ectodomains

for each other, the continued presence of the LZ

domain is probably critical for maintenance of a stable

complex. This differs from the use of LZs to facilitate

expression of otherwise misassembled proteins, such

as soluble MHC class II or T cell receptor ah dimers,

where the zippers can be proteolytically removed

from the secreted product without loss of dimer

stability (e.g., Scott et al., 1996).

Two strategies for complex formation were

explored. The first was to express the zipper-modified

molecules separately and to form complexes in vitro;

the second was to coexpress all four polypeptide

chains and to purify complexes formed in vivo. Both

strategies yield functional DM/DR complexes capable

of rapid peptide binding, but there are important

differences. Separate expression and purification of

the two molecules allows the use of well-established

Fig. 5. Purification and characterization of sDM-AcidP1/sDR1-

BaseP1 complexes isolated from Schneider cells coexpressing both

molecules. (A) Complexes were captured from insect cell super-

natants, eluted in 100 mM imidazole, and compared to purified

sDM or sDR1 with or without zippers. Samples (A, sDM-AcidP1;

B, sDR1-BaseP1; AB, complex; 200–500 ng each) were analyzed

by 12% reducing SDS-PAGE and silver staining. Band assignments

are based on internal comparisons and previous work (Busch et al.,

1998b). (B) A second round of Ni-NTA-agarose chromatography

improves purity of the 100 mM imidazole eluate as analyzed by

silver staining. (C) Elution of intact complexes demonstrated by

native PAGE and immunoblotting with a mAb against the AcidP1/

BaseP1 LZ dimer. Wash and eluate fractions are used as shown in

panel B. (D) Peptide binding activity of fractions from Ni-NTA-

agarose repurification. Fractions (diluted 1:50, except for the

starting material, which was diluted 1:100) were exposed to 20

AM biotinylated HA peptide in reaction buffer (pH 4.7) for 3 h,

neutralized, and peptide/DR complex formation was measured by

capture immunoassay. Shown are means and SD of duplicate

measurements.


protocols, control of the relative amounts of DM and

DR during complex formation, as well as detailed

comparisons between zipper-modified and -unmodi-

fied soluble molecules. Coexpression of both mole-

cules required a different purification scheme because

methods used to purify individual molecules could not

readily be adapted for use with the complex. How-

ever, standard protocols for His6-tagged proteins sim-

plified this problem. The expression system used

allowed little control over the relative expression

levels of the two proteins; nonetheless, we recovered

active complexes containing similar molar amounts of

the two proteins. Coexpression resulted in different

patterns of posttranslational modification; although

this did not drastically change the behavior of the

DM/DR complex, some biochemical assays and struc-

tural studies may be affected.

The functional effects of LZ modification could be

explained by a substantial stabilization of a functional

DM/DR complex. In association assays at endosomal

pH, the amount of sDM needed to enhance peptide

binding to sDR1 was decreased by four orders of

magnitude when both molecules were fused to com-

plementary zipper domains. The rate of peptide dis-

sociation was accelerated by several orders of

magnitude (not shown). The apparent association rate

was also accelerated although quantitative compari-

sons of true on-rates are difficult due to mechanistic

complexities encountered with MHC-II molecules and

the plate-binding assay used is not well suited for

rapid kinetic studies. Zipper modification rendered the

complexes stable enough to be detected by native

PAGE and coimmunoprecipitation. However, despite

the boost in affinity, association probably remained

reversible because substoichiometric amounts of

sDM-AcidP1 catalyzed maximal peptide loading of

sDR1-BaseP1, and because unbound sDM-AcidP1

was detected on native PAGE gels of DM/DR mix-

tures containing comparable or excess amounts of

sDR1-BaseP1. This is consistent with previous obser-

vations showing that LZ dimers and monomers can

exchange readily (Patel et al., 1996). Although diffi-

cult to rule out, we found no evidence that fusion with

LZ domains via flexible spacers distorts the geometry

of the DM/DR1 complex. For instance, the pH

dependence of DM action was similar with and with-

out LZ modification (data not shown; cf. Sloan et al.,

1995).

Intriguingly, sDR1-BaseP1 was more sDM-suscep-

tible than unmodified sDR1, even when there was no

complementary LZ on sDM. One explanation is that

sDR1-BaseP1 has a weak tendency to homodimerize

(not shown), perhaps facilitated by a tendency of the

sDR1 ectodomains to form dimers of heterodimers

(Brown et al., 1993). It is conceivable that a DR (ah)2dimer is a better substrate for sDM than a monomer.

Consistent with this view, HPSEC analysis of DM/DR

complexes suggests the presence of higher-order

structures (not shown). However, another explanation

is that the BaseP1 LZ tail interacts with the negatively

charged FLAG tag on the sDM a chain. A weak

tendency to homodimerize was also observed for

sDM-AcidP1 although this did not seem to affect

activity (Fig. 4).

In conclusion, fusion with LZs represents a straight-

forward approach for generating soluble, stable com-

plexes between DM and MHC-II molecules. This tool

promises to be useful for studying the mechanism of

DM chaperoning and catalysis of peptide exchange,

structural requirements for peptide binding to DM/DR

complexes, the structure and stoichiometry of the

complexes, and active MHC-II conformations. Correct

assembly was achieved despite possible complications

in this system such as inappropriate binding of LZ

peptides to the MHC-II groove or mispairing between

the structurally related DM and DR a and h chains.

Thus, it is likely that LZ fusion will be useful in other,

often simpler situations where low complex stability

precludes detailed structural and biochemical studies.

The only requirements are that the oligomerization

domains do not interfere with folding, stability and

function of the protein, and that any such difficulties

can be tested for using appropriate functional or

biochemical assays.

Acknowledgements

We thank Drs. Lawrence J. Stern and Harden M.

McConnell for review of the manuscript; Drs. Dennis

Zaller, Kai Wucherpfennig, and Luc Teyton for

cDNAs; Drs. John Trowsdale, Lawrence Stern, and

Ellis Reinherz for antibodies; Wendy Liu and Bari

Holm for technical assistance; and Dr. Pehr Harbury

for helpful discussions. This work was supported by

funding from the National Institutes of Health (AI-


28809 to E.D.M., 1R01-AINS48540 to K.C.G.) and

the MS Society (to K.C.G.). R.B. was supported by

the Siegelman fellowship. A.P. was supported by a

fellowship from the Cancer Research Institute.

References

Aichinger, G., Karlsson, L., Jackson, M.R., Vestberg, M., Vaughan,

J.H., Teyton, L., Lechler, R.I., Peterson, P.A., 1997. J. Biol.

Chem. 272, 29127.

Alber, T., 1992. Curr. Opin. Genet. Dev. 2, 205.

Brown, J.H., Jardetzky, T.S., Gorga, J.C., Stern, L.J., Urban, R.G.,

Strominger, J.L., Wiley, D.C., 1993. Nature 364, 33.

Busch, R., Mellins, E.D., 1996. Curr. Opin. Immunol. 8, 51.

Busch, R., Cloutier, I., Sekaly, R.P., Hammerling, G.J., 1996.

EMBO J. 15, 418.

Busch, R., Doebele, R.C., von Scheven, E., Fahrni, J., Mellins,

E.D., 1998a. J. Immunol. 160, 734.

Busch, R., Reich, Z., Zaller, D.M., Sloan, V., Mellins, E.D., 1998b.

J. Biol. Chem. 273, 27557.

Busch, R., Doebele, R.C., Patil, N.S., Pashine, A., Mellins, E.D.,

2000. Curr. Opin. Immunol. 12, 99.

Chang, H., Bao, Z., Yao, Y., Tse, A.G.D., Goyarts, E.C., Madsen,

M., Kawasaki, E., Brauer, P.P., Sacchettini, J.C., Nathenson,

S.G., et al., 1994. Proc. Natl. Acad. Sci. U. S. A. 91, p. 11408.

Chou, C.L., Sadegh-Nasseri, S., 2000. J. Exp. Med. 192, 1697.

Denzin, L.K., Hammond, C., Cresswell, P., 1996. J. Exp. Med. 184,

2153.

Doebele, R.C., Busch, R., Scott, H.M., Pashine, A., Mellins, E.D.,

2000. Immunity 13, 517.

Fremont, D.H., Crawford, F., Marrack, P., Hendrickson, W.A., Kap-

pler, J., 1998. Immunity 9, 385.

Germain, R.N., 1994. Cell 76, 287.

Ghosh, P., Amaya, M., Mellins, E., Wiley, D.C., 1995. Nature 378,

457.

Jardetzky, T.S., Gorga, J.C., Busch, R., Rothbard, J., Strominger,

J.L., Wiley, D.C., 1990. EMBO J. 9, 1797.

Kalandadze, A., Galleno, M., Foncerrada, L., Strominger, J.L., Wu-

cherpfennig, K.W., 1996. J. Biol. Chem. 271, 20156.

Lampson, L.A., Levy, R., 1980. J. Immunol. 125, 293.

Mosyak, L., Zaller, D.M., Wiley, D.C., 1998. Immunity 9, 377.

Natarajan, S.K., Assadi, M., Sadegh-Nasseri, S., 1999. J. Immunol.

162, 4030.

O’Shea, E.K., Lumb, J.J., Kim, P.S., 1993. Curr. Biol. 3, 658.

Patel, L.R., Curran, T., Kerppola, T.K., 1996. Proc. Natl. Acad. Sci.

U. S. A. 91, 7360.

Rabinowitz, J.D., Vrljic, M., Kasson, P.M., Liang, M.N., Busch, R.,

Boniface, J.J., Davis, M.M., McConnell, H.M., 1998. Immunity

9, 699.

Ridgway, J.B., Presta, L.G., Carter, P., 1996. Protein Eng. 9, 617.

Sanderson, F., Thomas, C., Neefjes, J., Trowsdale, J., 1996. Im-

munity 4, 87.

Scott, C.A., Garcia, K.C., Carbone, F.R., Wilson, I.A., Teyton, L.,

1996. J. Exp. Med. 183, 2087.

Sloan, V.S., Cameron, P., Porter, G., Gammon, M., Amaya, M.,

Mellins, E., Zaller, D.M., 1995. Nature 375, 802.

Stern, L.J., Wiley, D.C., 1992. Cell 68, 465.

Stern, L.J., Brown, J.H., Jardetzky, T.S., Gorga, J.C., Urban, R.G.,

Strominger, J.L., Wiley, D.C., 1994. Nature 368, 215.

Weber, D.A., Evavold, B.D., Jensen, P.E., 1996. Science 274,

618.

Zarutskie, J.A., Busch, R., Zavala-Ruiz, Z., Rushe, M., Mellins,

E.D., Stern, L.J., 2001. Proc. Natl. Acad. Sci. U. S. A. 98,

12450.


Documents

Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers