Side-chain Dynamics of the SAP SH2 DomainCorrelate with a Binding Hot Spot and a Region withConformational Plasticity

Patrick J. Finerty Jr1,2, Ranjith Muhandiram3 andJulie D. Forman-Kay1,2*

1Structural Biology andBiochemistry, The Hospital forSick Children, 555 UniversityAvenue, Toronto, Ont., CanadaM5G 1X8

2Department of BiochemistryUniversity of Toronto, TorontoOnt., Canada M5S 1A8

3Department of MedicalGenetics and MicrobiologyUniversity of Toronto, TorontoOnt., Canada M5S 1A8

X-linked lymphoproliferative disease is caused by mutations in theprotein SAP, which consists almost entirely of a single SH2 domain. SAPinteracts with the Tyr281 site of the T $ B cell signaling protein SLAMvia its SH2 domain. Interestingly, binding is not dependent on phos-phorylation but does involve interactions with residues N-terminal to theTyr. We have used 15N and 2H NMR relaxation experiments to investigatethe motional properties of the SAP SH2 domain backbone amides andside-chain methyl groups in the free protein and complexes with phos-phorylated and non-phosphorylated peptides derived from the Tyr281site of SLAM. The most mobile methyl groups are in side-chains withlarge RMSD values between the three crystal structures of SAP, suggestingthat fast time-scale dynamics in side-chains is associated with confor-mational plasticity. The backbone amides of two residues which interactwith the C-terminal part of the peptides experience fast time-scalemotions in the free SH2 domain that are quenched upon binding of eitherthe phosphorylated or non-phosphorylated peptide. Of most importance,the mobility of methyl groups in and around the binding site for residuesin the N-terminus of the peptide is significantly restricted in the com-plexes, underscoring the dominance of this interaction with SAP anddemonstrating a correlation between changes in rapid side-chain motionupon binding with local binding energy.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: SH2; NMR; relaxation; dynamics; plasticity*Corresponding author

Introduction

X-linked lymphoproliferative disease (XLP, alsoknown as Duncans disease) is a recessive immuno-deficiency disease affecting boys and leading toextreme vulnerability to infection by the Epstein-Barr virus (EBV) and frequently to fatal infectiousmononucleosis and lymphomas.1 The XLP geneproduct was identified as the protein SAP (SLAM-associated protein) in experiments investigatingT $ B cell stimulation mediated by SLAM

(signaling lymphocyte-activation molecule), a highaffinity self-ligand present on the surface of B andT cells.2 SAP is a 128 amino acid (ca 14 kDa) proteinconsisting almost entirely of an SH2 domain witha short C-terminal tail. The cytoplasmic domainof SLAM contains three tyrosine residues, Y281,Y307, and Y327, and binding studies showed thatthe Y281 site is the highest affinity binding site forSAP.2

Protein–protein interactions are frequentlymediated by small modular-binding domains,such as the SH2 domain found in SAP. Originallyfound in the Rous sarcoma virus protein Src andsimilar, cellular protein tyrosine kinases,3,4 SH2domains (,100 amino acid residues) generallyrecognize phosphotyrosine (pTyr) sites in the con-text of other residues. Extensive binding studieson the SAP SH2 domain with peptides derivedfrom the Y281 site of SLAM revealed that it isunique among characterized SH2 domains; it

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author:forman@sickkids.ca

Abbreviations used: SH2, Src homology 2; SAP, SLAM-associated protein; SLAM, signaling lymphocyte-activation molecule; (p)Y281, a (phospho)peptidederived from the Y281 site of SLAM; RMSD, root-mean-squared deviation; NOE, nuclear Overhauserenhancement.

doi:10.1016/S0022-2836(02)00803-3 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 322, 605–620

binds both phosphorylated and non-phosphoryl-ated peptides with only a fivefold difference inaffinity (Kd values are ,120 nM and ,650 nM forthe pY281 and Y281 peptides, respectively).5,6 Thiscontrasts with the binding properties of mostother SH2 domains, which show a ca 1000-folddifference in affinity between phosphorylatedand non-phosphorylated ligands.7 Additional datafrom binding studies on SAP, as well as therecently determined structures of the SAP SH2domain free and in complex with phosphorylatedand non-phosphorylated peptides derived fromthe Y281 site of SLAM,5,8 have established a three-pronged binding model for interaction withtargets. These include a hydrophobic group at theþ3 position of the ligand relative to the (p)Tyr,the (p)Tyr and a relatively unique site at the 22position relative to the (p)Tyr. Of these, only twoout of the three prongs are required for highaffinity binding6 with a minimal sequence suf-ficient for binding by SAP of …T/S-x-x-x-x-V/I…,where x represents any amino acid residue, T isT279 (Thr22) and V is V284 (Valþ3) in the peptidederived from the Y281 site.8

NMR dynamics experiments performed on otherSH2 domains have provided important insightsinto the role of rapid motion in protein interactionsand suggested that fast time-scale dynamics canact to modulate binding affinity.9,10 Additionally,similar experiments performed on other proteinshave yielded information about the effect of ligandbinding on motional properties of the proteinbackbone as well as side-chain methyl groups.11 –16

Even though the tertiary structures of many SH2domain complexes have been determined, it is notpossible to predict the affinity of target peptidesor to distinguish those residues at the bindinginterfaces that contribute energetically to bindingor to binding specificity from those that do not.In order to further probe the unusual bindingproperties of SAP, we have performed 15N and 2HNMR relaxation experiments to investigate therole of fast time-scale motions in the interactionwith phosphorylated and non-phosphorylatedSLAM-derived peptides.

Results and Discussion

Assignments

Chemical shift resonance assignments of the freeprotein were performed using standard triple-resonance experiments on a 1.7 mM sample ofuniformly 15N, 13C and randomly 50% 2H labeledfull-length SAP prepared as described in Materialsand Methods.17,18 Assignments for SAP complexedwith the Y281 peptide were reported previously6

and spectra of the complex with pY281 were verysimilar to those obtained for the non-phosphoryl-ated peptide. Ambiguities due to differencesbetween spectra of the two complexes wereresolved as described in Materials and Methods.

Sample preparation

Our preparations of full-length SAP containedtwo species by SDS-PAGE, likely due to C-terminalproteolysis. Therefore, we used a truncated form ofSAP lacking the C-terminal 24 amino acid residues,which appear to be unstructured in solution.8

This form (104 amino acid residues) was alsoemployed by Poy et al.5 for crystallographic struc-ture determination and is referred to hereafter asthe SAP SH2 domain. The SAP SH2 domainpreparation used in NMR experiments was fully15N, 13C labeled and randomly fractionallydeuterated to approximately 50% as described inMaterials and Methods. A single preparation wasused for experiments on the free protein andcomplexes with synthetic Y281 and pY281 peptidesderived from the Tyr281 site of SLAM. Samplescontaining the SAP SH2 domain complexed withthe two peptides contained a small excess of pep-tide to ensure complete binding. 15N and 2H NMRrelaxation experiments were performed on thesame samples.

Oligomerization of SH2 domains and overallcorrelation time

Some isolated SH2 domains self-associate, eitheras dimers or higher-order oligomers, but thisproperty may be attenuated or abrogated by ligandbinding.16,19,20 Nonetheless, it can complicate com-parisons of dynamics between the free and boundstates; as a consequence of oligomerization, theoverall correlation time used as a parameter to fitrelaxation data will be a weighted average derivedfrom the distribution of oligomers, and further,those residues involved in the interaction mayhave altered relaxation properties.

A series of 15N T1 and T2 experiments16 on dif-ferent concentrations of the SAP SH2 domain wasperformed to determine if there is a tendency foroligomerization. Data measured for 0.5 mM, 1 mMand 2 mM SAP SH2 domain samples yieldedapproximate molecular correlation times (tc)

21 of5.52 ns, 6.09 ns, and 7.72 ns, respectively, indicatinga concentration-dependent dimerization or aggre-gation. Relaxation experiments on the SAP SH2domain complexed with Y281 indicated similarbehavior. Sedimentation equilibrium data (notshown) on the free SAP SH2 domain fit a mono-mer–dimer distribution with a calculated Kd valueof about 15 mM, leading to approximately 18%dimer at a concentration of 2 mM and 6% dimer at0.5 mM. Protein concentrations 0.5 mM, 0.52 mMand 0.57 mM for the free SAP SH2 domain andthe complexes with Y281 and pY281, respectively,were selected that limited the fraction of dimerizedSAP SH2 domain for the free protein and bothcomplexes to below 7% (on the basis of the Kd

value for dimerization measured for the free SH2domain). At these concentrations overall rotationalcorrelation times measured for the free SAP SH2domain and the Y281 and pY281 complexes are

606 NMR Relaxation Studies on the SAP SH2 Domain

within 0.54 ns of each other (5.53 ns, 6.07 ns, and5.88 ns for the free protein and complexes withY281 and pY281 peptides, respectively, seeMaterials and Methods) and comparison of 15N T1

and T2 relaxation data reported here for the freeSH2 domain and the Y281 complex with datameasured for more concentrated samples did notidentify any residues with altered relaxationproperties (data not shown). Thus, the small frac-tion of dimer present in all three cases does notsignificantly affect comparisons of the relaxationparameters.

NMR relaxation experiments

Backbone 15N T1, T2 and steady-state 1H– 15Nnuclear Overhauser enhancement (NOE) relaxationexperiments16 were conducted at 600 MHz and30 8C as described in Materials and Methods. Intotal, 15N relaxation data for 84, 82 and 79 residueswere obtained for the free SAP SH2 domain, Y281and pY281 complexes, respectively (Figure 1 andTable 1). Comparison with average values for eachexperiment shows that deviations from the meanvalues are found primarily in loop regions and atthe N-terminus.

Side-chain 2H T1 and T1r relaxation parameters,shown in Figure 2, were measured for CH2Dmethyl groups at 600 MHz and 30 8C and analyzedaccording to published procedures9,10,22 (seeMaterials and Methods). The SAP SH2 domainhas 37 methyl-containing residues with a total of62 methyl groups. Relaxation parameters wereobtained for 25 (37), 31 (43) and 32 (42) of theseresidues (methyl groups) for the free protein, Y281and pY281 complexes, respectively. While datawere obtained for approximately two-thirds of themethyl groups in each case, due to spectral overlapthere were only 29 methyl groups for which datawere obtained in all cases. Methyl 2H T1 and T1r

relaxation times are shorter and have a broaderdistribution than the corresponding relaxationparameters measured for backbone amide groups(Table 1).

Model-free analysis

To facilitate comparisons of fast time-scaledynamics in proteins it is useful to employ model-free23,24 analysis in which an order parameter (S 2)and effective correlation time for internal motions(te) are calculated (see Materials and Methods).Order parameters describe the amplitude ofinternal pico–nanosecond (ps–ns) motions for aparticular bond vector and range from S 2 ¼ 0, fora bond vector rapidly sampling multiple orien-tations, to S 2 ¼ 1, for no internal motion. Besidesan order parameter and effective correlation time,in some instances an exchange contribution toT2 (Rex) that reflects dynamic processes on themicro–millisecond (ms–ms) time-scale was alsoincluded. In addition, data for some amidesrequired use of an extended model to describeinternal motions that take place on two distinct

Figure 1. 15N T1 times in seconds(maroon circles), T2 times inseconds (blue squares), and 1H–15NNOE ratios (green triangles) as afunction of residue number areshown (a) for the free protein,(b) complex with the Y281 peptide,and (c) complex with the pY281peptide with error bars showingthe uncertainties in the measure-ments. Approximate positions ofsecondary structural elements forthe SAP SH2 domain are shown atthe bottom of the Figure as well asin Figures 2–5.

Table 1. Average values of relaxation parameters

Free SAP SH2 Y281 complex pY281 complex

15N T1 (ms) 539 ^ 35.6 560 ^ 34.2 555 ^ 36.215N T2 (ms) 134 ^ 19.5 121 ^ 14.4 126 ^ 15.01H–15NNOE

0.755 ^ 0.084 0.785 ^ 0.074 0.779 ^ 0.074

2H T1 (ms) 48.8 ^ 16.7 49.7 ^ 18.5 52.8 ^ 19.12H T1r (ms) 14.5 ^ 5.9 13.8 ^ 5.5 15.0 ^ 6.8

NMR Relaxation Studies on the SAP SH2 Domain 607

time-scales and which differ by at least an order ofmagnitude.25 For these residues order parametersfor slow (Ss

2) and fast (Sf2) time-scale internal

motions and an effective correlation time (te) forslow internal processes are calculated.

The motional models selected for the model-freeanalysis of the 15N relaxation parameters are

summarized in Table 2. 15N relaxation parametersfor the majority of residues in the free protein andY281 and pY281 complexes were fit using the firsttwo, relatively simple, models. For the free protein,22 residues were fit using a model that includedan exchange term, while 18 residues for the Y281complex and nine residues for the pY281 complexrequired inclusion of an exchange term to fit therelaxation data. Rex values ranging from 2 to 4 s21

were calculated for only four residues in the freeprotein (Y76, D91, Q92, and I94), while neithercomplex had residues with Rex terms greater than2 s21. Additionally, relaxation dispersion experi-ments performed to detect such motions failed tofind ms–ms time-scale dynamics within the freeSAP SH2 domain (for CPMG field strengths from22 to 607 Hz, sensitive to exchange from ,10 s21

to faster than ,104 s21; data not shown). Theseresults, in combination with data from deuteriumexchange experiments performed by Hwang et al.,8

indicate that the free protein experiences slowertime-scale motions, especially in the BG and EF

Figure 2. 2H T1 (maroon circles) and T1r (blue squares) times in seconds as a function of methyl position are shownfor (a) the free protein, (b) complex with the Y281 peptide, and (c) complex with the pY281 peptide with error barsshowing the uncertainties in the measurements.

Table 2. Motional models selected in model-free analysis

Modelparameters Free SAP SH2

Y281complex

pY281complex

1: S 2 44 44 422: S 2 and te 12 14 243: S 2 and Rex 11 14 74: S 2, te and Rex 11 4 25a: Sf

2, Ss2 and te 5 6 4

Not fit 1 – –

Total 84 82 79

a Ss2 £ Sf

2 ¼ S 2.

608 NMR Relaxation Studies on the SAP SH2 Domain

loops, that are absent in either complex. For allthree cases, a similar number of residues were fitusing the last model, which incorporates motionsoccurring on two distinct time-scales.

For methyl dynamics, Saxis2 describes the ampli-

tude of ns–ps motions for the bond vector betweenthe methyl group and its directly attached carbonatom. Since only two experimental observables,2H T1 and T1r, are measured (besides the overallcorrelation time calculated using 15N data), onlythe simplest model-free formalism is applied, inwhich a methyl axis order parameter (Saxis

2 ) and aneffective correlation time for internal motions (te)are calculated.

Figures 3 and 4 show backbone amide andmethyl axis order parameters, respectively, andaverage values for amide S 2 and methyl Saxis

2 aresummarized in Table 3. Comparison of 15N and 2Horder parameters reveals that methyl Saxis

2 valueshave a much broader range than amide S 2 values.

Additionally, while 15N S 2 values vary dependingon the location of the amide group in the protein,with those in regular secondary structure elementslarger than those in loops, methyl Saxis

2 valuesshow no such correlation. We have previouslyobserved that methyl order parameters are notcorrelated with protein structure but generallydecrease as the separation between the methyland the protein backbone increases.26 Thus, com-parisons of methyl order parameters for methylgroups within a protein should be restricted tocomparisons among particular methyl types.The Saxis

2 values determined for the free SAP SH2domain and Y281 and pY281 complexes wereadded to a database of methyl order parameterspreviously assembled by Mittermaier et al.26 andthis expanded database was used for calculationof the mean values presented here. Table 4 showsaverage values for the free SAP SH2 domain andY281 and pY281 complexes by methyl type as well

Figure 3. The backbone amideorder parameters S 2 as a functionof residue number are shown (a)for the free protein, (b) complexwith the Y281 peptide, and(c) complex with the pY281 peptide.(d) The differences in S 2 valuesbetween the complex with the Y281and the free protein, (e) betweenthe complex with the pY281 andthe free protein, and (f) betweenthe two complexes are also shown.Error bars indicate error in themeasurements ((a)–(c)) or in thecalculated differences ((d)–(f)).

NMR Relaxation Studies on the SAP SH2 Domain 609

Figure 4 (legend opposite)

610 NMR Relaxation Studies on the SAP SH2 Domain

as average values for all methyl groups in ourdatabase (kSaxis

2 l) and demonstrates that our dataare similar to the average values (even when SAPdata are excluded from the database). Differencesbetween mean values and those calculated for thefree protein and both complexes are illustrated inFigure 5, with error bars indicating the standarddeviation of Saxis

2 values for particular methyltypes (not experimental uncertainty). Despite thelarge standard deviations, Saxis

2 values for somemethyl groups differ significantly from the mean.

Generally, these methyl groups with Saxis2 values

farthest from the mean (kSaxis2 l) are spatially clus-

tered (within 3 to 7 A of each other) in threeregions of the SAP SH2 domain (Figures 5 and 7).The most dynamic cluster consists of I80 Cd1 andCg2 in the FB loop, I84 Cd1 in the N-terminal partof helix aB, and L31 Cd1 and Cd2 on the face ofthe central b-sheet opposite the peptide-bindingsurface. These methyl groups are more mobilethan average and several are located near theN-terminus where the backbone is also relativelydynamic. The second cluster, I94 Cd1 and Cg2 andL98 Cd2, found in the turn following helix aBleading in to the last b-strand, are less dynamic

than average. The third cluster is comprised ofL30 Cd1 and V102 Cg1 and Cg2, and are also lessdynamic than average. These methyl groups arepositioned on the opposite side of the b-sheet asthose in the second cluster and pack against helixaA. Interestingly, while all of these methyl groupsexhibit dynamics that deviate from mean values(kSaxis

2 l), in nearly every case they have similarmotional properties in the free protein and bothcomplexes (see below).

Correlation between dynamics andstructural plasticity

To determine if there is a correlation betweenfast time-scale dynamics and conformationalplasticity, methyl Saxis

2 2 kSaxis2 l values were com-

pared with side-chain heavy atom root-mean-squared deviation (RMSD) values (see Materialsand Methods) for the three SAP crystal structures5

(Figure 6). Methyl groups that are significantlymore mobile than average are generally found inside-chains with larger RMSD values, whereas theconverse is observed for those that are less mobilethan average (Figure 6, black symbols).

With the exception of the I51 Cd1 methyl in thefree protein (I51 is discussed below in the sectionon the Thr22-binding site), the methyl groupsthat are more dynamic than average are proximalto the N-terminus and the short N-terminalb-strand (bA) in the SH2 domain (the first clusterdiscussed above). This region of the SAP SH2domain exhibits both larger than average dynamicson a ps–ns time-scale as well as structural dif-ferences among the three states, suggesting thatthis region has more conformational plasticitythan other areas of the SH2 domain. Amide orderparameters for residues preceding Y7 are low(Figure 3), indicating the backbone in this regionis relatively dynamic on a ps–ns time-scale. Evenso, the lack of negative 15N–1H steady-state NOEratios for these amide groups (Figure 1) demon-strates that the backbone in this region is notcompletely disordered. Possibly the motional dis-order of these methyl groups and the structuralfluctuations present in this region of the SH2domain are coupled. Although this region isdistal to the binding interface, such a featurecould facilitate subtle side-chain rearrangementsupon binding different ligands by reducing theenergetic cost of such conformational changes.

While most methyl groups have motionalproperties that are correlated with RMSD values,data for three groups (I11 Cd1, L19 Cd1, and I96 Cd1)deviate from this trend. Inspection of the crystal

Figure 4. The methyl axis order parameters Saxis2 as a function of methyl position are shown for (a) the free protein,

(b) complex with the Y281 peptide, and (c) complex with the pY281 peptide. (d) The differences in Saxis2 values between

the complex with the Y281 and the free protein, (e) between the complex with the pY281 and the free protein, and (f)between the two complexes are also shown. Error bars indicate error in the measurements ((a)–(c)) or in the calculateddifferences ((d)–(f)).

Table 4. Average Saxis2 values listed by methyl type

Methyltype

Free SAPSH2

Y281complex

pY281complex Databasea

Ala Cb 0.73 ^ 0.24 0.78 ^ 0.24 0.77 ^ 0.16 0.83 ^ 0.09Thr Cg 0.68 ^ 0.11 0.71 ^ 0.13 0.62 ^ 0.06 0.72 ^ 0.13Val Cg1 0.69 ^ 0.15 0.66 ^ 0.09 0.69 ^ 0.18 0.63 ^ 0.17Val Cg2 0.74 ^ 0.06 0.66 ^ 0.15 0.59 ^ 0.15 0.67 ^ 0.15Leu Cd1 0.71 ^ 0.18 0.63 ^ 0.21 0.65 ^ 0.30 0.50 ^ 0.22Leu Cd2 0.70 ^ 0.15 0.67 ^ 0.22 0.58 ^ 0.25 0.52 ^ 0.20Ile Cd1 0.42 ^ 0.28 0.41 ^ 0.23 0.48 ^ 0.26 0.49 ^ 0.19Ile Cg2 0.72 ^ 0.19 0.70 ^ 0.27 0.69 ^ 0.26 0.73 ^ 0.13

a Average values of Saxis2 shown in this column were calcu-

lated using all Saxis2 values measured for free SAP SH2 and

(p)Y281 complexes reported here and those used in the analysisby Mittermaier et al.26

Table 3. Average values of order parameters

Free SAP SH2Y281

complexpY281

complex

S2 0.83 ^ 0.08 0.85 ^ 0.09 0.84 ^ 0.09Saxis

2 a 0.67 ^ 0.20 0.65 ^ 0.20 0.63 ^ 0.22Saxis

2 (common)b 0.63 ^ 0.21 0.64 ^ 0.21 0.63 ^ 0.21

a Average Saxis2 values for all residues for which data were

available.b Average Saxis

2 values for which data were available in allthree cases.

NMR Relaxation Studies on the SAP SH2 Domain 611

structures does not reveal a straightforwardexplanation for these so it is difficult to say if thedeviation from the trend discussed above ismeaningful or if these groups are simply outliers.Nevertheless, the overall results are strongly sup-portive of a correlation between rapid time-scalemotion of side-chains and structural plasticity.

Changes in dynamics upon ligand binding

Main-chain atoms constitute about one-fifth ofthe area at an average protein interface with theremainder being comprised of amino acid side-chains.27 Consequently, there has been muchinterest in obtaining dynamics information forside-chain atoms at protein interfaces.22,26,28 The 2Hmethyl order parameters presented here providevaluable information about the role of these groupsin the binding of ligands by the SAP SH2 domainand, since the interactions mediated by methylgroups are generally hydrophobic, offer comple-

mentary data to that obtained for backboneamide groups. Plots of the changes in 15N S 2 and2H Saxis

2 values upon binding of the Y281 andpY281 complexes as well as differences betweenthe two complexes are shown in Figures 3 and 4.Additionally, a schematic illustration of the SAPSH2 domain complexed with the pY281 peptide5

is presented in Figure 7, highlighting methylgroups with the largest differences in mobilitybetween the free protein and the complexes.

For some proteins19,29,30 ligand binding leads to areduction in fast time-scale dynamics, while inother instances the degree and direction of changesin order parameters following ligand bindingvaries.14 – 16 In the SAP SH2 domain, there is anoverall decrease in backbone flexibility uponbinding of either peptide, but 15N order parametersfor the majority of residues do not exhibit largechanges (see average S 2 values in Table 3). Sur-prisingly, inspection of 2H order parametersreveals a similar trend; although there are methyl

Figure 5. Plots of Saxis2 2 kSaxis

2 l values as a function of methyl position are shown (a) for the free protein, (b) complexwith the Y281 peptide, and (c) complex with the pY281 peptide with error bars indicating the standard deviation in thedatabase of kSaxis

2 l values, not the error in measured Saxis2 values.

612 NMR Relaxation Studies on the SAP SH2 Domain

groups with relatively large changes in mobility,the net change in dynamics upon binding of eitherligand is negligible (Table 3). Note that the datafor the Y281 and pY281 complexes are very similarwith only two methyl groups having significantlydifferent Saxis

2 values between them. Nonetheless,

more residues show changes in 2H order para-meters upon ligand binding than show changes inbackbone order parameters. Despite the lack of alarge global reduction in flexibility of the SAP SH2domain upon ligand binding, it is notable that,with the exception of a few residues, those amidesand methyl groups showing the largest changesare found in and around the ligand-binding site.In the sections that follow, changes in 15N and2H order parameters upon ligand binding areexamined in terms of the tripartite-binding modelestablished by Li et al. for SAP,6 beginning withthe (phospho)tyrosine-binding pocket, continuingwith the hydrophobic-binding pocket for Valþ3and concluding with the unusual Thr22-bindingregion.

The (p)Tyr-binding pocket has complexmotional properties

Of the many residues in the (phospho)tyrosine-binding pocket (Figure 7), only R13, G24, S36, I51,and R55 show significant differences in dynamics(15NlDS 2l $ 0.05 or 2HlDSaxis

2 l $ 0.1) between thefree protein and the two complexes. Since themethyl groups from I51 for which 2H relaxationdata were measured are not near the phosphate-binding pocket, discussions of dynamics changesfor this region of the SAP SH2 domain are limitedto differences in 15N order parameters. Interest-ingly, amide groups for three of these residues,G24 and S36 in the Y281 complex, and R55 in thepY281 complex, show increased mobility, while

Figure 6. Saxis2 2 kSaxis

2 l values (as shown in Figure 5) asa function of side-chain heavy atom RMSD values.RMSD values were determined using the three crystalstructures of the SAP SH2 domain published by Poyet al.5 using the program MOLMOL (see Materials andMethods). Methyl groups with Saxis

2 2 kSaxis2 l values for

which the error does not overlap with 0 (Figure 5) areshown as black symbols with the remainder shownin gray. Values for the free protein, Y281 complex,and pY281 complex are shown as circles, squares, andtriangles, respectively.

Figure 7. Stereo backbone ribbon diagram of the crystal structure of the SAP SH2 domain complexed with aphosphorylated peptide derived from the protein SLAM (PDB code 1D4W).5 Methyl groups for which Saxis

2 valueswere measured are shown as spheres. Those that have .0.1 difference in Saxis

2 and are less (more) mobile in eithercomplex relative to the free protein are shaded blue (red). Methyl groups for which data are only available for thetwo complexes are also colored: L31 Cd1 (green; less mobile in the pY281 complex) and V72 Cg1 (yellow; more mobilein the pY281 complex). Secondary structural elements and methyl groups are labeled. This illustration was producedusing the program MOLMOL.48

NMR Relaxation Studies on the SAP SH2 Domain 613

the I51 amide shows reduced mobility in the Y281complex relative to the free protein (Figure 3(d)and (e)).

Three residues, R13, S36, and R55, also showinteresting differences when comparing the twocomplexes to each other (Figure 3(f)). R13 is moremobile in the pY281 complex than in the Y281complex consistent with it not being well orderedin the pY281 crystal structure.5 Although R13 doesnot make hydrogen-bonding interactions with thepTyr, several NOEs were observed between it andthe pTyr ring protons in the pY281 complex, mostof which were absent in the complex with theunphosphorylated ligand.8 Located in the BC loopof the SH2 domain, S36 exhibits one of thelargest differences in 15N S 2 values between thepY281 and Y281 complexes, with an S 2 value thatis larger by 0.13 in the pY281 complex. In thecrystal structure of the Y281 complex, S36 interactswith a water network occupying the space filledby phosphate in the pY281 complex. However,deuterium exchange data show that the N-terminalpart of the BC loop, and S36, in particular, issignificantly more flexible in the Y281 complexthan in a complex with a truncated, phosphoryl-ated peptide that has identical interactions in thisregion as the pY281 peptide.8 Thus, the greaterps–ns motions of S36 in the absence of interactionswith the pTyr phosphate group may facilitateincreased slow time-scale motions (measured by2H exchange) in the BC loop of the Y281 complex.R55 also exhibits a significant structural differencebetween the two complexes and interacts with thewater molecule network that replaces interactionswith the pTyr in the complex with the non-phosphorylated peptide. However, unlike S36, theamide group of R55 is more dynamic on theps–ns time-scale in the pY281 complex. This sur-prising result is supported by hydrogen exchangemeasurements that also show R55 is more flexiblein the pY281 complex than the Y281 complex.8

These dynamics measurements indicate thatthe motional properties of the phosphotyrosine-binding pocket are complex; some backbonepositions become more flexible, while othersbecome more rigid in the presence of the tyrosineand phosphate group. These results are consistentwith binding studies revealing that, in thecontext of the full-length Y281 peptide, there is nopreference for a particular residue at the Tyrposition, suggesting that the Tyr does not contri-bute significant binding energy in the Y281complex.8

Motions in the Val 1 3 hydrophobic-bindingsite are quenched upon ligand binding

Val þ 3 (V284) of the Y281 peptide has importantinteractions with the SAP SH2 domain that helpdefine the specificity of binding. The side-chain ofV284 binds in a pocket formed by residues fromthe EF (A66, E67, T68, A69, and K74) and BG (G93and I94) loops. The residues with the largest

changes in 15N S 2 values, T68 and A69, are foundin the EF loop of the SH2 domain (Figure 7)which, in the free protein, occlude the hydro-phobic-binding site for the C-terminal part of theligand and must move considerably to allowligand binding. In the free SH2 domain, both T68and A69 have unusually low 15N order parametersbut have S 2 values that are slightly above averagein the Y281 and pY281 complexes, indicating thatsmall amplitude, fast time-scale motions found inthe backbone of the free protein are quenched byligand binding. The presence of fast time-scaledynamics in a loop that is displaced by ligandbinding suggests that rapid motions may act as alubricant to weaken hydrophobic interactions inthis part of the free protein thus facilitating slower,large amplitude motions required to accommodatethe ligand. Consistent with this, deuteriumexchange experiments performed to characterizethe flexibility of the SAP SH2 domain complexedwith the n-pY peptide (a ligand without the Valþ3 prong) showed that this region of the SH2domain is very flexible relative to the rest of theprotein on a much slower time-scale (seconds)with mobility reduced in the presence of the Y281peptide.8 However, ms–ms time-scale motionswere not detected in the free SH2 domain usingrelaxation dispersion experiments (see above) and,although relaxation data for T68 and A69 in thefree protein was fit using a two-time-scale model,Rex terms were not necessary for these residues.

2H relaxation data were obtained for methylgroups from three of the residues interacting withVal þ 3 (A66, T68 and I94) and, of these, only themobility of the T68 Cg2 shows a significant changeupon ligand binding (lower portion of Figure 7).However, the direction of the change, as the T68Cg2 methyl is more mobile in the complexes, issurprising. Conceivably, the large structuralchange that stabilizes the EF loop also places thismethyl group in an environment that is not aswell ordered as in the free protein. Overall, thedynamics data for the Val þ 3-binding site suggestthat interactions with the ligand only provide suf-ficient energy to stabilize the open conformationof the EF and BG loops in the SH2 domain butdo not contribute significant binding energy.

Dynamics in the Thr22-binding site suggest anetwork of interacting methyl groups

The SH2 domain in SAP is the first describedto have significant interactions with residuesN-terminal of the central Tyr in the ligand. Tertiarystructures show that the majority of contacts withthis portion of the peptide are made with Thr22,which binds in a pocket formed by residues R13,E17, I51, and T53.8 While amide relaxation data donot indicate a significant change in dynamicsupon binding for the protein backbone in thisregion of the SH2 domain, the methyl dynamicsdata reveal the stabilizing effects of the interactionwith Thr22 and suggest that the decreased

614 NMR Relaxation Studies on the SAP SH2 Domain

mobility of methyl groups interacting with thepeptide are propagated throughout this region ofthe SH2 domain.

The I51 Cg2 and Cd1 methyl groups have 16 NOEsto atoms from residues in the 21 (I280), 22 (T279)and 23 (L278) positions of the peptide8 and bothmethyl groups show a significant reduction inmobility upon binding to the peptides (Figures 4and 7). Relaxation data for the T53 Cg2 methylgroup were only available for the Y281 complexwhere it is less mobile than average (Figure 5).

Interestingly, a nearby methyl group, L46 Cd1,also shows a significant reduction in mobility afterligand binding despite the fact that only a singleNOE was observed between it and the peptide.L46 is located near the C-terminal end of strandbC where it is positioned on the opposite sideof the I51 side-chain as the peptide (Figure 7).Compared to the free protein, I51 Cd1 is rotated byabout 1208 towards the peptide in both complexes,while the L46 side-chain maintains a similarorientation after ligand binding.5 In both com-plexes, the mobility of L20 Cg2, another nearbymethyl, is restricted after ligand binding althoughit shows a larger change in the pY281 complex(DSaxis

2 (Y281-free) ¼ 0.06 and DSaxis2 (pY281-free) ¼

0.13). The L20 side-chain is sandwiched betweenhelix aA and strand bB and is relatively far awayfrom the peptide suggesting changes in its mobilityare, like those for L46, indirect (Figure 7).

The I51 Cd1 is one of the few methyl groups thathas both decreased mobility upon ligand bindingbut that is also more mobile than average (Figures5 and 6). In addition, the comparison of Saxis

2 valuesthat differ from mean values (Saxis

2 2 kSaxis2 l) and

side-chain heavy atom RMSD values (see above)placed this group in the same region of the plot asthe L31, I80, and I84 methyl groups. However,unlike those groups, the difference in side-chainorientation for this position can be attributed to aconformational change that occurs upon ligandbinding. Nonetheless, the lower than averageSaxis

2 value for the I51 Cd1 methyl indicates that itexperiences significant motional disorder on theps–ns time-scale and suggests that the confor-mational change of this residue upon ligand bind-ing may be facilitated by the absence of stronginteractions with surrounding atoms in the freeprotein. NOE data indicate that the I51 methylgroups comprise two of the primary contacts forthe Thr22 residue of the ligand, yet bindingstudies demonstrate that the SAP SH2 domain canalso bind ligands containing a Ser at the 22position.8 Possibly the conformational plasticity inligand recognition involving I51 is facilitated bythe higher than average mobility of the Cd1 methylgroup (Figure 5).

While the dominant change upon binding in theThr22 binding region is a reduction of motion,some nearby methyl groups, the L21 and L25 Cd2

methyl groups, become more mobile in the com-plexes (no data was available for L21 in the pY281complex). Unlike the L20, L46 and I51 side-chains,

which are largely buried in the SH2 domain,the L21 side-chain is significantly more solventexposed (Figure 7). Still this side-chain has severalNOEs to atoms from the Leu-3 position (L278),indicating it makes some interactions with theligand. The L25 methyl groups, on the other hand,are nearly completely solvent-accessible and donot interact with the peptide (Figure 7). Since theL21 and L25 methyl groups are largely solventaccessible, their interactions with, and thereforetheir ability to influence, nearby residues is small.In contrast, I51, L46, and L20 appear to form anetwork (upper portion of Figure 7) in which areduction in mobility of the I51 methyl groupsupon ligand binding is transmitted from one side-chain to the next thus leading to an overall stabiliz-ation of this region of the SH2 domain. This sig-nificant dynamic quenching correlates with theenergetic importance of the N-terminal region ofthe peptide for binding (see below).

Concluding remarks

Although structures of the free SAP SH2 domainand complexes with the Y281 and pY281 peptidesare presented as static pictures, proteins are notrigid, but undergo a variety of motions on differenttime-scales. Comparison of the dynamics datameasured for the SAP SH2 domain with thatmeasured for other SH2 domains reveals thatsome show reduced dynamics upon ligandbinding, while others have little or nochange.9,10,16,19,20,29,31 – 33 For example, as with theSAP SH2 domain, residues in the phosphotyro-sine-binding pocket of the C-terminal SH2 domainof phospholipase Cg1 (PLCC) show a mixture ofincreased and decreased motion in the complexedstate.34 Although SH2 domains have a commonfold and some conserved residues, the variety ofchanges in dynamics upon ligand bindingobserved to date precludes generalizations aboutchanges in protein motions upon ligand bindingand points to the need to measure dynamics forindividual proteins.

One of the primary purposes for investigatingmotions within proteins and at protein interfacesis to gain an understanding of the atomic basisfor the thermodynamic properties of proteins andprotein interactions. Certain backbone positionsand methyl groups in the SAP SH2 domain haveincreased mobility in the complexes relative to thefree state, suggesting these changes may compen-sate for entropy that is lost upon ligand binding.NMR relaxation experiments investigatingdynamics changes in other proteins upon ligandbinding found increased dynamics in some cases,while in others side-chain methyl group orderparameters showed both increased and decreasedvalues relative to the free state.11 – 13 In the free SAPSH2 domain, except for two residues (T68 andA69) that exhibit a large reduction in fast time-scale dynamics upon ligand binding, amide groupsof most residues show relatively modest changes

NMR Relaxation Studies on the SAP SH2 Domain 615

in flexibility. As well, assuming the methylSaxis

2 values reported here are representative ofdynamics throughout the SH2 domain, the netdifference in conformational entropy due to fasttime-scale motions between the free protein andeither complex is small (Table 3).

Unlike most other SH2 domains, the SAP SH2domain shows a preference for particular residuesN-terminal to the central Tyr in the ligand.The dominance of interactions with the Thr22prong relative to the Val þ 3 site is apparent uponanalysis of the methyl dynamics data in the contextof the binding data reported by Li et al.6 N-terminaltruncation of the ligand reduces the bindingaffinity to 16% of that measured for the pY281peptide, the largest decrease observed in thebinding study, indicative of this region being a“hot spot”35 at the peptide-binding interface.Consistent with the importance of interactionswith this part of the ligand, methyl groups inthis region of the SAP SH2 domain (I51, L46)show the largest restriction of motion upon bind-ing. In contrast, truncating the ligand to removethe þ3 prong (Val þ 3) results in only a 50%reduction in affinity relative to the pY281 peptide.Although there is a significant reduction in thebackbone dynamics of two residues in the EF loop(T68 and A69), the methyl groups that interactwith the ligand show only small reductions inmobility and one methyl group is actually moredynamic in the Y281 and pY281 complexes(Figure 7). The lack of a decrease in the mobilityof side-chain methyl groups in this region is con-sistent with the energetics of these groups beingsimilar in the free and bound states. Both thebinding and methyl dynamics data expose theasymmetric distribution of binding energy acrossthe SAP-binding interface and highlight theenergetic dominance of the N-terminal-bindingregion of the SH2 domain. These results aresupportive of a correlation between the methyldynamics data presented here and local bindingenergy as previous methyl dynamics studies ofthe PLCC SH2 domain and N-terminal SH2domain of Shp2 (previously called Syp) have alsodemonstrated.9,10

Recently, the binding properties and structureof the EAT-2 SH2 domain were characterized.36

EAT-2 has a similar tertiary structure (RMSD forCa atoms of 0.69 A) and also binds to some of thesame ligands as SAP, including the Y281 site ofSLAM in the phosphorylated state. While EAT-2has a similar three-pronged mode of interactionwith the pY281 peptide, it is not able to bind tothe non-phosphorylated state or to other non-phosphorylated ligands, suggesting the distri-bution of binding energy is not similar. Applicationof NMR relaxation experiments similar to thosepresented here that probe side-chain motion in theEAT-2 complex with the pY281 peptide shouldenable a mapping of this local distribution of bind-ing energy, providing important complementaryinformation to structural and binding studies.

Clearly, the methyl dynamics experiments utilizedhere and by others are powerful tools for investi-gating so-called hot spots at protein interfaces,35

not requiring mutations or other perturbationsthat alter the native interaction. These results, inaddition to the correlation between side-chainrelaxation data and structural plasticity for theSAP SH2 domain, point to the utility of NMRrelaxation experiments for probing the energeticlandscape of proteins and their binding interfaces.

Materials and Methods

Expression, purification, and preparation of 15N, 13C,2H (50%) SAP

The T7 expression plasmid pRSET containing residues1–104 of Human SAP,5 kindly provided by Dr MichaelJ. Eck, was used to overexpress the SAP SH2 inEscherichia coli strain BL21(DE3) for use in NMR relax-ation experiments. Cells were grown at 34 8C in twoliters of M9 minimal media, 50% 2H2O/50% H2O with15NH4Cl and [13C6]glucose as the sole nitrogen andcarbon sources. Following induction with IPTG, thetemperature was reduced to 23 8C and the culturegrown overnight. Cells were harvested by centrifugationthen lysed by sonication in 35 ml of P6(0) buffer (100 mMNaHPO4 (pH 6.0), 2.5 mM EDTA, 2.5 mM DTT) contain-ing Complete (Roche) protease inhibitors. The lysatewas adjusted to pH 5.6 by addition of 15 ml of a buffercontaining 50 mM sodium acetate, 25 mM NaCl and2.5 mM EDTA, applied to a 20 ml Mono-S column(Pharmacia) at 3 ml min21 and eluted using a 0 to 1 MNaCl gradient over five column volumes. SAP fractionswere pooled, dialyzed against four liters of P6(0) buffer,concentrated to 1.5 ml and applied in 200 ml aliquots toa 20 ml Superdex 75 (Pharmacia) column. SAP fractionswere pooled, concentrated to 5 ml and dialyzed over-night against four liters of argon purged SAP NMRbuffer (20 mM NaHPO4 (pH 6.0), 100 mM NaCl, 50 mMEDTA) plus 100 mM DTT. The purity of the protein wasverified by the presence of a single band on a Coommas-sie-stained SDS/polyacrylamide gel. The protein concen-tration was determined by absorbance at 280 nm using atheoretical extinction coefficient of 17,270 M21 cm21.37

Full-length SAP was expressed using the T7expression plasmid SAP-pET (provided by Dr Shun-Cheng Li). Uniformly labeled 15N, 13C, and 50% 2H pro-tein was prepared as described above for the SAP SH2except that a value of 17,300 M21 cm21 was used for theextinction coefficient.

Preparation of Y281 and pY281 peptides

The sequences of Y281 and pY281 are RKSLTIY-AQVQK and KKSLTIpYAQVQK, respectively.6 PurifiedY281 used in NMR experiments was a gift from S. C. Li.pY281 was kindly synthesized by Dr Gerald Gish in thelaboratory of Dr Tony Pawson, purified by HPLC asdescribed6 and confirmed by mass spectrometry. Priorto addition to the SAP SH2, peptides were dissolved inargon purged SAP NMR buffer (see above).

616 NMR Relaxation Studies on the SAP SH2 Domain

Preparation of NMR samples

Assignment of backbone and side-chain resonancesfor free, full-length SAP was performed using a sampleof 1.7 mM uniformly labeled 15N, 13C, and 50% 2H pro-tein in SAP NMR buffer, 90% H2O/10% 2H2O. A 0.5 mMsample of free, uniformly labeled 15N, 13C, and 50% 2HSAP SH2 in SAP NMR buffer, 90% H2O/10% 2H2O wasused in NMR relaxation experiments to minimize oligo-merization. Complexes of SAP with Y281 and pY281peptides were prepared by adding purified peptides touniformly labeled 15N, 13C, and 50% 2H SAP SH2 in SAPNMR buffer, 90% H2O/10% 2H2O to yield a small excessof peptide relative to the SAP SH2. The concentrationof each complex was determined by measuring theabsorbance at 280 nm using a theoretical extinctioncoefficient of 18,550 M21 cm21. The SAP SH2 complexeswith Y281 and pY281 were 0.57 mM and 0.52 mM,respectively. All NMR samples were blanketed withargon to prevent oxidation.

Chemical shift assignment experiments

NMR experiments for chemical shift assignments wereperformed at 30 8C on Varian Inova 500 MHz and600 MHz spectrometers with triple-resonance, pulsed-field gradient probes with an actively shielded z-gradientcoil. 1HN, 15N, 13Ca, 13Cb, 13C-carbonyl, and side-chain13C-methyl assignments for free, full-length SAP wereobtained from CBCA(CO)NH, HNCACB, HNCO,(H)CC(CO)NH-TOCSY, H(CC)(CO)NH-TOCSY spectrarecorded using enhanced sensitivity pulsed-fieldgradient methodology.17,18 Assignment of the SAP Y281complex was reported previously6 and differences inchemical shifts between the SAP Y281 and SAP SH2pY281 complexes were resolved using HNCACB,(H)CC(CO)NH-TOCSY and H(CC)(CO)NH-TOCSYspectra of each complex. Stereospecific assignment ofVal and Leu methyl groups in free, full-length SAP wereobtained using 10% 13C-enriched protein and a constant-time 1H–13C HSQC experiment as described.38,39 Similarassignments for the SAP Y281 complex had been madepreviously8 and differences in the 13C-methyl chemicalshifts between the SAP SH2 Y281 and pY281 complexeswere resolved as above. Assignments were made usingthe Linux version of NMRView package40 and NMRresonance assignments have been deposited in theBioMagResBank.

15N relaxation experiments

NMR relaxation experiments were performed at 30 8Con a Varian Inova 600 MHz spectrometer equipped witha triple-resonance, pulsed-field gradient probe with anactively shielded z-gradient coil. The 15N T1, T1r andNOE values were measured using previously publishedpulse schemes.16,41 Steady-state 1H–15N NOE valueswere obtained from 1H–15N correlation spectra withthree seconds of 1H saturation and a five second delaybetween scans and without 1H saturation using an eightsecond delay between scans for the free protein andboth complexes. 15N T1 values were measured fromspectra recorded with eight different values of the relaxa-tion delay of 10.1, 50.4, 121.1, 191.7, 272.4, 363.2, 474.1and 605.3 ms, 10.1, 55.5, 136.2, 206.8, 277.5, 378.4, 494.4and 630.6 ms, 10.1, 55.5, 136.2, 206.8, 277.4, 378.3, 494.3,and 630.5 ms for the free SAP SH2, Y281, and pY281complexes, respectively. 15N T1r values were measuredfrom eight different spectra recorded with delays of 8,

16, 24, 32, 40, 56, 80, and 96 ms for the free protein andboth complexes. 15N T2 values for each residue wereobtained by correction of the observed relaxation rateR1r for the offset Dn of the applied spin-lock rf field (n1)to the resonance using the relation R1r ¼ R1 cos2u þR2 sin2u, where u ¼ tan21(n1/Dn). n1 was 1683.5, 1686.9,and 1689.2 Hz for free SAP SH2 and the Y281, andpY281 SAP SH2 complexes, respectively. All spectrawere recorded as 128 £ 576 complex matrices with spec-tral widths of 9000.9 Hz and 1338 Hz employed in the1H and 15N dimensions, respectively. All data sets wereprocessed using the Linux version of the NMRPipesuite42 with Lorentzian-to-Gaussian apodization in bothdimensions. Peak intensities were obtained using thenlinLS routine of NMRPipe and used to fit a twoparameter function of the form I(t ) ¼ I0

e2t/T1,2 by least-squares non-linear regression using a conjugate gradientalgorithm.43 Errors in relaxation rates were estimated byMonte Carlo analysis.44 Steady-state NOE values weredetermined from the ratios of peak heights with andwithout proton saturation. Errors in peak heights wereestimated from the root-mean-square value of back-ground noise in the spectra.

Relaxation parameters were analyzed according to theiterative procedure described by Mandel et al. using theLinux version of the program Modelfree v4.01.45,46 Initialvalues for the overall rotational correlation time (tc)were calculated on a per residue basis by fitting 15N T1/T2 ratios assuming isotropic tumbling21 using softwarewritten in-house. For the free protein and complexeswith Y281 and pY281, tc values were 5.53, 6.07, and5.88 ns, respectively. These values are typical for a pro-tein of this size (ca 11.7 kDa).47 The quadric_diffusionprogram, from the Palmer group web site†, was used tofit an anisotropic diffusion tensor to the tc values afterexcluding residues with steady-state NOE ratios below0.70 and 0.75 for the free protein and both complexes,respectively. Optimized rotational diffusion tensors forthe free protein and both complexes were obtainedusing selected 15N T1 and T2 data and the previouslypublished crystal structures of free SAP SH2, Y281, andpY281 complexes (RCSB PDB codes 1D1Z, 1D4T, and1D4W, respectively)5 after adding protons using theprogram MOLMOL.48 For all three cases, an axially sym-metric rotational diffusion tensor was found to offer astatistically better fit of the data than the fully isotropicmodel, while the anisotropic model did not significantlyimprove the fit (comparison of the axially symmetricand anisotropic models yields P values of 0.17, 0.07, and0.34 for the free, Y281, and pY281 complexes, respec-tively). After an initial round of fitting, residues with S 2

values less than 0.75 or Rex terms greater than 1 s21 wereexcluded from fitting of the diffusion tensor. The finaloptimized values of Diso (Diso ¼ (Dxx þ Dyy þ Dzz)/3,where D is the diagonalized diffusion tensor) obtainedfrom these analyses correspond to tc ( ¼ (6Diso)

21) valuesof 5.60, 6.19, and 5.98 ns for the free protein and com-plexes with Y281 and pY281, respectively, and thesebest-fit values of the diffusion tensor for each state wereused to calculate 15N order parameters. These values arewithin 2% of the times obtained by fitting 15N T1/T2

ratios (above). Axial ratios, Dpar/Dper (Dpar ¼ Dzz andDper ¼ Dxx ¼ Dyy), obtained in each case were 1.22, 1.15,and 1.22 for the free protein, Y281, and pY281 complexes,respectively, indicating a low degree of anisotropy in

† http://cpmcnet.columbia.edu/dept/gsas/biochem/labs/palmer/software.html

NMR Relaxation Studies on the SAP SH2 Domain 617

each case. While the samples used for measurement of15N relaxation parameters were uniformly 15N and 13Clabeled, effects due to dipolar relaxation via 13Ca and13C0 were not included in our calculations. We calculatethat these pathways would only lead to a nearly homo-genous 2–2.8% error in the 15N S 2 values presented herefor the free protein and both complexes.

2H relaxation experiments

2H NMR relaxation experiments to obtain relaxationrates of the spin-operator terms HzCz

2Hz, HzCz2Hy, and

HzCz were recorded on the same 600 MHz spectrometeras the 15N data sets using previously described pulseschemes.22 HzCz

2Hz rates were obtained from 2D 1H–13Ccorrelation spectra with relaxation delays of 0.05, 4.1,8.6, 13.5, 19, 25.1, 32.1, 40.3, and 50 ms for the free proteinand both complexes. HzCz

2Hy rates were obtained from2D 1H–13C correlation spectra with relaxation delays of0.2, 2.06, 4.3, 6.8, 9.5, 12.6, 16.1, and 21 ms for the freeprotein and both complexes. HzCz rates were subtractedfrom HzCz

2Hz and HzCz2Hy to yield 2H 1/T1 and 1/T1r

values. All spectra were recorded as 128 £ 576 complexmatrices with spectral widths of 9000.9 Hz and 5000 Hzemployed in F1 and F2, respectively. Processing andanalysis of spectra were as described for 15N relaxationexperiments. Saxis

2 and te values were obtained from fitsto 2H T1 and T1r measurements assuming isotropictumbling with correlation times of 5.53, 6.07, and 5.88 nsfor the free protein, Y281, and pY281 complexes, respec-tively, obtained from 15N T1 and T2 values. Due to thesmall degree of anisotropy in each state, the Saxis

2 valuesreported here may differ from actual values by a maxi-mum of ^0.04 to 0.07.

Calculation of RMSD values

RMSD values presented in Figure 6 were calculatedusing the Linux version of the program MOLMOL.48

PDB files 1D1Z (free SAP SH2 domain), 1D4T (Y281peptide complex) and 1D4W (pY281 peptide complex)were used for the calculations. Initially the three struc-tures were superimposed using the “Fit” command andthe result used to generate a mean structure with the“MeanMol command”. RMSD values were calculatedon a pairwise basis for each structure compared withthe mean structure using the “CalcRmsd” commandand are “local displacements” (MOLMOL nomenclature)of side-chain heavy atoms for a given residue comparedwith the same residue in the mean structure.

Acknowledgements

We thank S.-C. Li for the plasmid used forexpressing full-length SAP, the Y281 peptide andchemical shift assignments for SAP complexedwith the Y281 peptide; G. Gish and the laboratoryof T. Pawson for synthesis of the pY281 peptide;M. J. Eck for the plasmid used for expressing theSAP SH2 domain; L. Hicks, the laboratory ofC. Kay and the PENCE facility for sedimentationequilibrium analysis; F. Mulder for assistance withthe Modelfree analysis; T. Pawson, L. E. Kay, A.Mittermaier, O. Millet and P. Hwang and members

of the Forman-Kay and Kay laboratories forvaluable discussions. This research was supportedby the National Cancer Institute of Canada withfunds from the Canadian Cancer Society.

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Edited by P. Wright

(Received 15 May 2002; received in revised form 25 July 2002; accepted 30 July 2002)

620 NMR Relaxation Studies on the SAP SH2 Domain