of April 13, 2018.This information is current as

Ligand Molecular FlexibilityComplementary Matching of Receptor and TCR Scanning of Peptide/MHC through

M. Kranz, K. Christopher Garcia and Brian M. BakerK. Nicholson, Jaroslav Zajicek, Evgenii L. Kovrigin, David William F. Hawse, Soumya De, Alex I. Greenwood, Linda

ol.1302953http://www.jimmunol.org/content/early/2014/02/12/jimmun

published online 12 February 2014J Immunol 

MaterialSupplementary

3.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/02/12/jimmunol.130295

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on April 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on April 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

TCR Scanning of Peptide/MHC through ComplementaryMatching of Receptor and Ligand Molecular Flexibility

William F. Hawse,*,† Soumya De,‡ Alex I. Greenwood,‡ Linda K. Nicholson,‡

Jaroslav Zajicek,*,† Evgenii L. Kovrigin,x David M. Kranz,{ K. Christopher Garcia,‖,#,**

and Brian M. Baker*,†

Although conformational changes in TCRs and peptide Ags presented by MHC protein (pMHC) molecules often occur upon bind-

ing, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. In this study, we used nuclear

magnetic resonance to study TCR–pMHC binding, examining recognition of the QL9/H-2Ld complex by the 2C TCR. Although

the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3b loop remains mobile within the TCR–pMHC

interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3b is also mobile in the free pMHC and in the

TCR–pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3b and QL9 are

closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the

thermodynamic and kinetic stability of the TCR–pMHC complex and provides a mechanism for facile binding. We thus propose

that matching of structural fluctuations is a component of how TCRs scan among potential ligands for those that can bind with

sufficient stability to enable T cell signaling. The Journal of Immunology, 2014, 192: 000–000.

Tcells use the ab TCR to recognize peptide Ags presentedby MHC proteins (pMHC) on the surfaces of APCs. Crys-tallographic structures have demonstrated that TCR en-

gagement of pMHC often proceeds with conformational changesin TCR CDR loops, peptides, and MHC proteins (reviewed inRef. 1, see Refs. 2–6 for specific examples). However, beyondtheir influence on binding affinities and kinetics (7), the impactof these conformational changes is not fully understood. In gen-eral, protein conformational changes are associated with enhancedflexibility, as the lower energy barriers that facilitate structuralalterations translate into faster rates of motion (8). For TCRs, CDRloop motion has been directly linked with receptor cross-reactivity,allowing the loops to optimize structural complementarity withdifferent ligands (9). Although the magnitudes of conformationalchanges and the underlying motional properties can vary (4, 10,11), TCR loop motion is believed to be an important component

of the process through which TCRs scan for compatible ligandson the surfaces of APCs (12–14).Peptide and MHC conformational changes have generally re-

ceived less attention than those occurring in TCRs, but do occur,particularly for peptides (e.g., Refs. 5, 10, 11, 15). In some cases,conformational changes in both TCR and pMHC occur uponbinding, a process we have termed conformational melding (6, 16).The occurrence of flexibility in both receptor and ligand adds to thecomplexity of the TCR–pMHC interaction and raises structural andenergetic questions about how a TCR and pMHC can productivelyengage if regions of both molecules are moving and sampling con-formations with varying degrees of compatibility (1).Although a myriad of approaches have been used to study the

motional properties of TCRs and pMHC complexes, includingcomputation and various forms of spectroscopy (e.g., Refs. 5, 9,17–21), nuclear magnetic resonance (NMR) is advantageous inthat it can yield experimental insight into motion at atomic reso-lution without requiring the introduction of potentially interferinglabels (22). When combined with structural information, NMRcan yield information unattainable by other techniques.To gain new insight into how receptor and ligand motion impacts

TCR recognition of pMHC, in this study, we used NMR to examinethe interaction between the murine 2C TCR and the QL9 peptidepresented by H-2Ld (Ld). The 2C-QL9–Ld interaction is an ar-chetypal TCR–pMHC interaction, studies of which have providedkey details about the structural and physical nature of TCR rec-ognition (23–27). Conformational changes occur in both the TCRand pMHC upon formation of the 2C-QL9–Ld complex (24, 28,29), which, together with available immunological, biochemical,and biophysical data, make it an ideal system for investigatingTCR and pMHC flexibility at an atomic level.In examining 2C recognition of QL9–Ld, we found that the CDR

loops of the 2C TCR generally undergo a reduction in flexibilityupon binding, confirming prior inferences from various crystal-lographic and binding experiments. However, a surprising exceptionwas seen for residues in the CDR3b loop, which retains significantmobility in the TCR–pMHC complex. Reciprocal analysis of the

*Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,IN 46556; †Harper Cancer Institute, University of Notre Dame, Notre Dame, IN 46556;‡Department of Molecular Biology, Cornell University, Ithaca, NY 14853; xDepartmentof Chemistry, Marquette University, Milwaukee, WI 53201; {Department of Biochem-istry, University of Illinois at Urbana-Champaign, Urbana, IL 61801; ‖Department ofMolecular and Cellular Physiology, Program in Immunology, Stanford UniversitySchool of Medicine, Stanford, CA 94305; #Department of Structural Biology, Pro-gram in Immunology, Stanford University School of Medicine, Stanford, CA 94305;and **Howard Hughes Medical Institute, Stanford University School of Medicine,Stanford, CA 94305

Received for publication November 4, 2013. Accepted for publication January 9,2014.

This work was supported by Grants GM067079, GM084884, and RR025761 from theNational Institutes of Health, the Walther Cancer Research Foundation, and GrantPF-11-143-01 from the American Cancer Society.

Address correspondence and reprint requests to Brian M. Baker, Department ofChemistry and Biochemistry, 251 Nieuwland Science Hall, University of NotreDame, Notre Dame, IN 46556. E-mail address: brian-baker@nd.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: HSQC, Heteronuclear Single-Quantum Correla-tion; NMR, nuclear magnetic resonance; pMHC, peptide Ags presented by MHCprotein; sc2C, single-chain 2C; scLd, single-chain H-2Ld.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302953

Published February 12, 2014, doi:10.4049/jimmunol.1302953 by guest on A

pril 13, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

pMHC indicated the region of the peptide that interacts withCDR3b is similarly mobile in both the free pMHC as well as theTCR–pMHC complex. Remarkably, the rates at which CDR3band the peptide move are similar in both the free proteins and thecomplex. The matching of receptor and ligand flexibility and itspersistence in the TCR–pMHC complex provides a solution forthe structural and energetic challenges posed when two flexiblemolecules engage and is evocative of behavior recently described ininterfaces formed with other proteins that engage multiple targets(30). Considering the high incidence of TCR and pMHC structuralrearrangements that have been observed crystallographically andtheir links to protein flexibility (8), we suggest that dynamic com-plementarity, or the matching of conformational exchange in TCRand pMHC, is an element of how TCRs finely discriminate amongligands for those that can bind with sufficient kinetic and thermo-dynamic stability to enable T cell signaling.

Materials and MethodsProtein expression and purification

Unlabeled or labeled single-chain 2C (sc2C) and single-chain H-2Ld (scLd)was refolded from bacterially expressed inclusion bodies grown with richor 15N-labeled M9 minimal media as previously described (28, 31–33).Protein was purified via ion-exchange and size-exclusion chromatography.Unlabeled or labeled QL9 peptide was chemically synthesized (Genscriptor Tufts University Core Facility). All samples for NMR were exchangedinto a buffer containing 25 mM Bis-Tris (pH 7.1), 25 mM NaCl, 0.03%NaN3, and 90% H2O/10% D2O.

NMR resonance assignments

Spectra were recorded at 298 K (25˚C) on a Bruker Advance II 800 MHzspectrometer equipped with a TCI cryogenic probe (Bruker). Spectra wereprocessed with Topspin 2.1 pI6 and analyzed with the Sparky and CcpNmrsoftware packages (34). Spectra from Heteronuclear Single-QuantumCorrelation (HSQC), HNCO, HNCA, CBCA(CO)NH, and HNCACBexperiments (35–39) were used to sequentially assign the 2C TCR.Assignments for the TCR have been submitted to the Biological MagneticResonance Data Bank (identification number 19239).

NMR titrations

Two NMR titrations were performed: [15N]-sc2C titrated with unlabeledQL9-scLd and [15N]–QL9-scLd titrated with unlabeled sc2C. [1H, 15N]-HSQCspectra were recorded for each titration point at 298 K on a Bruker Ad-vance 800 II MHz spectrometer (Bruker). In each titration, the concen-tration of 15N-labeled protein used was 300 mM. The concentration rangeof the unlabeled binding partner ranged between 0 and 1.5 times theconcentration of the 15N-labeled protein, yielding .95% saturation at thehighest concentration used. Peptide dissociation from scLd was ignored asall concentrations of QL9-scLd were several thousand–fold higher than the7 nM KD the QL9 peptide has for Ld (27).

NMR line-shape analysis

Quantitative analysis of the NMR line shape data for Gly97b of the TCR andPhe7 of the peptide was performed as described in Greenwood et al. (40).These residues were selected as they showed the greatest complexity in thetitrations and were still compatible with the constraints required for line-shape analysis (well resolved or multiple peaks that shift in at least onedimension without the presence of intervening resonances). Briefly, one-dimensional 1H line shapes were extracted from [1H-15N]-HSQC spectrarecorded during the titrations using the BiophysicsLab 1D NMR Pythonextension in Sparky and composited for fitting. For compositing, one-dimensional slices through the center of each two-dimensional peakwere normalized by peak volume. Line shape fitting was performed usingthe BiophysicsLab software package (currently, Integrative Data AnalysisPlatform; http://kovrigin.chem.mu.edu/IDAP/) (41). Spectra were firstnormalized within their respective titration series. BiophysicsLab was thenused with the Bloch-McConnell equations (42) modified to reflect thevarious binding models. The population matrix, which describes thepopulations of species present, was formulated based on the total con-centration of TCR and pMHC and the equilibrium constants that describeconversion between the various states. The corresponding matrix of rateconstants was formulated from the rate equations governing exchangebetween the states. Separate matrices were constructed for the chemical

shifts for each state, the apparent transverse relaxation rate constants, andthe frequency variables. Data were fit using a Newton interior point method,with convergence and fit quality evaluated by the x2 statistic.

For each fit, a global minimum was ensured through the use of a gridsearch, in which the initial guess for each parameter was systematicallyvaried over a 10–20-fold range (8–10 initial guesses for each parameter).The overall KD in each fit was constrained to the value of 2.20 mM pre-viously determined using isothermal titration calorimetry (43). Parameterspace was explored by refitting with individual parameters fixed at valuesnear their best fit value, whereas other parameters were floated, and re-cording the x2 statistic for each new fit. Error space was further exploredby varying the constraining KD within the error range of the calorimetri-cally determined value and repeating the fitting procedure.

ResultsReduction of conformational mobility in the 2C CDR loopsupon binding

We began by titrating [15N]-labeled TCR with unlabeled pMHCusing [1H-15N]-HSQC NMR spectra collected as a function ofpMHC concentration to monitor binding. Because the chemicalshifts of the [1H] and [15N] nuclei are sensitive to changes in mo-lecular environment, the advantage of this experiment is that eachbackbone NH serves as a potential binding probe. This advantagewas recently demonstrated through the use of chemical shift per-turbation to identify residues within TCR–pMHC binding interfaces(31). However, because motion also alters molecular environment,careful analysis of [1H-15N]-HSQC spectra can provide insightinto flexibility and changes in flexibility that occur as binding pro-ceeds. In ideal cases, quantitative analyses can provide informa-tion on the kinetics of conformational exchange and how thisinfluences recognition (40–42, 44, 45).NMR experiments were facilitated through the use of recently

developed single-chain, minimized variants of 2C and Ld (referredto as sc2C and scLd, respectively), which, because of their smallersize, reduced experimental complexity and improved the signalcompared with the full-length molecules (28, 31). The single-chain variants and their full-length counterparts interact withsimilar affinities, kinetics, and structural topologies (23, 24, 28),indicating that minimization of the proteins has not significantlyaltered the recognition properties of either TCR or pMHC. HSQCspectra were well-dispersed and consistent with a stable, well-folded protein (Supplemental Fig. 1A). NMR assignments wereperformed using standard sequential strategies as described in theMaterials and Methods. Concentrations in the titrations werevaried to permit collection of data over a range of saturation, fromzero to .95%.We focused our studies on residues in the various CDR loops

of the 2C TCR, following changes in [1H-15N] cross-peaks as theTCR was titrated with pMHC. In most cases, the resonances forthe residues of the CDR loops of the unliganded TCR were broad,coalescing into sharper, more defined peaks in the TCR–pMHCcomplex. Data illustrating this behavior for each TCR residue thatcontacts peptide or MHC are shown in Fig. 1 and SupplementalFig. 1B. NMR peak broadening can be attributed to conforma-tional exchange, in which intrinsic molecular flexibility moves thebackbone NH between different environments. Peak broadening isalso expected upon the formation of a larger complex as the rate ofoverall molecular tumbling decreases. As our observations are tothe contrary, we interpret our results to indicate that the backbonesof the various CDR loops are structurally loose (or mobile) in thefree TCR and that this mobility is restricted in the TCR–pMHCcomplex. A role for loop motion in Ag recognition by the 2C TCRhas been inferred from biophysical binding data (27), as well asthe various crystal structures of the free and bound receptor (2, 24,29), which demonstrate that shifts in backbone conformationas large as 7 A are required for the TCR to engage ligand

2 TCR SCANNING BY DYNAMIC COMPLEMENTARITY

by guest on April 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

(Supplemental Fig. 2A) (note that reduced amide proton exchangewith solvent could also lead to sharper peaks in the complex;however, the solvent accessible surface areas of the backboneamides of the various residues in Fig. 1 and Supplemental Fig. 1 arenot always reduced, and in some cases even increase upon binding).

The flexible CDR3b loop retains mobility within theTCR–pMHC complex

Unlike other positions, the triple-glycine motif of the sc2C CDR3bloop (Gly96, Gly97, and Gly98; see Supplemental Fig. 1B) showedgreater complexity in the NMR titrations. For these residues, thebroad resonances in the free TCR evolved into additional cross-peaks upon titration with pMHC, with discrete peaks persistingeven when the TCR was saturated. This behavior was most evidentfor the backbone nitrogen of Gly97 at the apex of CDR3b (Fig. 2A,2B). The simplest interpretation of these data is that at least two dis-tinct bound states are formed with Gly97b, with the tip of the CDR3bloop moving between the different conformations on a slow time-scale (as aromatic ring currents can alter proton chemical shifts,the existence of multiple peaks for Gly97b in the TCR–pMHCcomplex could also arise from motion of the side chain of Phe7 ofthe QL9 peptide, which is adjacent to Gly97 of CDR3b, as shownin Fig. 2A. However, given the packing in the 2C-QL9-Ld inter-face, any Phe7 ring flips or substantial motion would still neces-sitate compensatory motion in CDR3b to avoid steric clashes).

To gain further insight into the behavior of Gly97b, we extractedone-dimensional 1H line shapes from the [1H-15N]-HSQC titra-tion data for Gly97b. By extracting the rate constants that un-derlie the chemical shift data, quantitative analysis of such NMRline shapes can yield the kinetics that govern the movement ofNMR-visible nuclei between bound, free, and other conforma-tional states (40–42, 44, 45). NMR line-shape analysis has beenrecently used, for example, to examine the isomer-selectivebinding mechanism of the Pin1 cis-trans prolyl isomerase (45)and identify the site-specific mechanisms through which the Srchomology 2 domain of PI3K binds flexible peptides (44). In de-termining kinetics, the line shape data as a function of concen-tration are fit to modified Bloch equations that incorporate themass action laws and corresponding rate constants for the modelunder investigation.The simplest model consistent with the spectra for Gly97b is one

in which the backbone can move between two conformationsin the free state of the TCR, both of which are competent to bindligand and remain accessible in the TCR–pMHC complex (Fig.2C). The line shape data as a function of concentration were wellfit by this model (Fig. 2D). The kinetic parameters indicatedthat movement between the two conformations in the free TCRoccurred with rate constants near 0.2 s21, with the two confor-mations equally populated (Table I). The rates of motion wereelevated in the complex, but neither conformation was signifi-cantly favored, indicating that neither of the states can be con-sidered a binding intermediate on the path toward a final complex.The rates of TCR binding and dissociation were similar for bothconformations and close to those determined previously by sur-face plasmon resonance (23, 28). Overall, the results suggest thatGly97b moves slowly in both the free and bound TCR, samplingdiscrete conformations without significantly impacting TCR bind-ing affinity or kinetics.

FIGURE 1. Excluding the third hypervariable loop of the b-chain,

residues in the CDR loops of the 2C TCR exhibit reduced conformational

mobility upon engaging QL9-Ld. (A) The positions of the apexes of each

CDR loop in the 2C-QL9-Ld complex. (B) HSQC data for the residues in

(A) in the free sc2C TCR (blue spectra) and the sc2C-QL9–scLd complex

(red spectra). The reduced widths upon binding are indicative of reduced

mobility. Data for additional residues in each loop are shown in

Supplementary Fig. 1B.

FIGURE 2. Gly97 in CDR3b moves slowly between multiple con-

formations in free and bound sc2C. (A) The position of Gly97b and its

interactions with Phe7 of the QL9 peptide in the 2C-QL9-Ld complex. (B)

HSQC data for Gly97b in the free sc2C TCR (blue spectrum) and the sc2C-

QL9–scLd complex (red spectrum). (C) The least complex model consis-

tent with the spectra for Gly97b. In the free TCR, Gly97b interconverts

between two conformations, both of which are competent to bind and

kinetically linked in the bound state. The two states are designated TCR

and TCR*. k2cf and kcf are the rates of forward and reverse conformational

exchange in the free protein, whereas k2cb and kcb are the rates of forward

and reverse exchange in the complex. k1 and k21 are the rates of binding

and dissociation of TCR, and k2 and k22 are the rates of binding and

dissociation of TCR*. (D) Fit of HSQC slices in the 1H dimension to the

mechanism in (C). Data for the free TCR are shown in black; stacked

colors indicate increasing concentrations of QL9-scLd.

The Journal of Immunology 3

by guest on April 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The QL9 peptide is also flexible, with exchange kinetics thatmatch those of the 2C CDR3b loop

In the 2C-QL9-Ld ternary complex, Phe7 of the QL9 peptide in-teracts with Gly97b and together with Asp8 forms the majority ofthe peptide contacts with the TCR (Fig. 2A). To complement theGly97b data, we examined Phe7 using a site-specifically 15N-labeledQL9 peptide, titrating unlabeled sc2C TCR into a solution of15N-labeled QL9–scLd. In the spectrum of the unliganded QL9–scLd

complex, two cross-peaks were observed for the amide nitrogenof Phe7, indicating the presence of two distinct peptide confor-mations (Fig. 3A). The detection of two conformations is con-sistent with the crystallographic structure of QL9-Ld (43), in whichthe two molecules in the asymmetric unit presented the peptidedifferently (Supplemental Fig. 2B). One peptide conformationwas close to that observed in the 2C-QL9-Ld ternary complex(24). Although the quality of the electron density was poorer,the second conformation would require complementary adjust-ments in CDR3b to avoid steric clashes and electrostatic repul-sion, as shown in Supplemental Fig. 2C (as noted above, due to thepotential influence of the Phe7 aromatic ring, the NMR experi-ments cannot clearly differentiate between Phe7 backbone andside-chain motion, although, as indicated by the crystallographicdata, both may be involved).Upon titration with the 2C TCR, both cross-peaks for Phe7

shifted, mimicking the behavior of Gly97 of CDR3b and sug-gesting that, as with CDR3b, Phe7 remains mobile in the TCR–pMHC complex (Fig. 3A). We thus performed a similar NMRline-shape analysis for the Phe7 data as was performed for Gly97b.We used the same two-state conformational exchange model asused for Gly97b, except that motion was located in the peptiderather than TCR (Fig. 3B). Intriguingly, this analysis yieldedvalues similar to those determined for Gly97b (Fig. 3C andTable I). The concordance between the data for Gly97b and Phe7

suggests that the two peptide conformations seen during titrationwith TCR reflect slow movement of the peptide and the CDR3bloop in the TCR–pMHC complex. As modeled in Fig. 3E, thiscould involve synchronous movement between the various CDR3band QL9 peptide conformations that have been observed crystal-lographically.A more complex spectrum consisting of at least five cross-peaks

was seen for the unliganded QL9–scLd complex when the nitrogenof Asp8 of the peptide was labeled (Fig. 3D), consistent with thepeptide sampling multiple conformations on a slow timescale.Upon titration with the TCR, however, the complexity was re-duced to two resonances of nearly equal volume. The complexityof the spectrum in the free QL9–scLd complex prohibited NMRline-shape analysis. However, the behavior is indicative of a rela-tively mobile peptide whose motions are reduced but not elimi-nated in the TCR–pMHC complex.

DiscussionConformational flexibility in TCR CDR loops has been linked toAg identification and engagement, with loop motions in the freeTCR permitting the optimization of structural and chemicalcomplementarity with target pMHC complexes. This has beenmost clearly demonstrated with the human TCR A6, for whichthe mobility of the hypervariable loops, and CDR3b in par-ticular, allows the receptor to engage a myriad of targets thatdiffer in the center of the peptide (9). Considered alongside thevariation in CDR loop conformation seen between the free andvarious bound structures of the 2C TCR, our observation that themajority of the CDR loops of the 2C TCR undergo a reduction inbackbone flexibility is consistent with a general role for TCR loopflexibility in facilitating Ag recognition.However, the observation of residual flexibility for the 2C

CDR3b loop in the TCR–pMHC complex was unanticipated.Particularly striking was our finding that at least two slowly ex-changing and essentially equally populated conformations existfor Gly97b of CDR3b. Although the presence of flexibility mightbe expected from a loop enriched in glycine, in this case, theobservation was puzzling, as in the 2C-QL9-Ld crystal structurethe majority of contacts to the QL9 peptide are formed by this

Table I. Rate and equilibrium constants for conformational exchangea

Constant Gly97 (2C CDR3b) Phe7 (QL9)

k2cf 0.19 (6 0.01) s21 0.30 (6 0.02) s21

kcf 0.21 (6 0.04) s21 0.43 (6 0.01) s21

Kcf 0.9 (6 0.2) 0.7 (6 0.1)k2cb 0.35 (6 0.05) s21 0.25 (6 0.03) s21

kcb 0.31 (6 0.01) s21 0.35 (6 0.01) s21

Kcb 1.1 (6 0.1) 0.7 (6 0.1)

aRate constants describe the rates of forward and reverse motion in the free andbound TCR and peptide as defined in Figs. 2C and 3B. The unitless equilibriumconstants Kcf and Kcb are the ratios of forward and reverse rate constants (e.g., Kcf =k2cf/kcf) and give the equilibrium distributions between the TCR and peptide con-formations in the free and bound states.

FIGURE 3. Phe7 of the QL9 peptide moves slowly between multiple

conformations in free and bound QL9-scLd. (A) HSQC data for Phe7 in

free QL9-scLd (blue spectrum) and in the sc2C-QL9-scLd complex (red

spectrum). (B) The least complex model consistent with the spectra for

Phe7. The model is identical to that used for Gly97b (Fig. 2C) except that

conformational exchange is located in the peptide. (C) Fit of HSQC slices

in the 1H dimension to the mechanism in (B). Data for free QL9-scLd are

shown in black; stacked colors indicate increasing concentrations of sc2C.

(D) HSQC data for Asp8 in free QL9-scLd (blue spectrum) and sc2C-QL9–

scLd complex (red spectrum). The data are consistent with peptide sam-

pling of multiple conformations in bound and free QL9-scLd. (E) Model

for synchronous conformational exchange of Phe7 and Gly97b in the 2C-

QL9-Ld complex. The right panel shows the conformations of CDR3b and

the peptide in the crystallographic structure of the 2C-QL9-Ld complex

(Protein Data Bank identification number 2OI9), whereas the left panel

shows the conformation of CDR3b in the structure of the free TCR and

the alternate conformation of the peptide in the structure of free QL9-Ld

(Protein Data Bank identification numbers 1TCR and 3ERY, respectively).

Red arrows indicate bonds that rotate significantly between the two con-

formations.

4 TCR SCANNING BY DYNAMIC COMPLEMENTARITY

by guest on April 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

region of CDR3b. Mutations in this region and substitutions tocontacted peptide residues have moderate to significant effects onrecognition (46, 47). How can a key region of the TCR bindingsite maintain substantial mobility within a TCR–pMHC complex?Insight was provided by the observations on the pMHC ligand,

which revealed that the region of the peptide that interfaces withCDR3b is also mobile, both in the free pMHC as well as thecomplex. This was particularly notable, as motion occurring withinpMHC complexes has not been widely considered in structuralimmunology. Remarkably, though, Phe7 of the peptide was found tomove with kinetics that closely match those of Gly97b. The datasuggest that in the TCR–pMHC complex, the CDR3b loop and thepeptide synchronously interconvert between compatible con-formations as modeled in Fig. 3E (although correlated intercon-version between structurally compatible conformational states asshown in Fig. 3E is the simplest interpretation of our data, ourexperiments cannot rule out the existence of other states, althoughgiven the proximity of Gly97b and Phe7, such states would be lessstable due to steric and electrostatic repulsion, as shown inSupplemental Fig. 2C).Intriguingly, our observations in the sc2C-QL9–scLd complex

are not wholly unique. Mobility in interfaces formed by flexibleproteins has been observed in other systems, most notably in com-plexes formed by proteins that engage multiple targets (30). Thisbehavior is believed to serve two purposes. First, it reduces theentropic penalty associated with fixing mobile regions. Second,if regions that interact possess complementary motions within theinterface, structural and chemical complementarity can be main-tained as the proteins move, preventing the loss of favorable contactsand/or the introduction of unfavorable ones. Our observations thushighlight a mechanism of reciprocal, dynamic complementaritybetween the receptor and ligand that enhances the stability of whatwould otherwise be a weak TCR–pMHC complex.It is further striking that the rates at which CDR3b and the

peptide move are similar in the free TCR and free pMHC. Wesuggest this is not coincidental: the fact that the peptide and CDR3b

move with similar rates, likely sampling compatible conformationsas shown in Fig. 3E, indicates that the two molecules are ideallypoised to interact from both a structural and dynamic perspective.As discussed above, ligands with mismatched motional propertiesare likely to be a poorer ligand for the receptor. The matching ofconformational exchange between receptor and ligand thus pro-vides a mechanism for facile engagement of QL9-Ld by the 2CTCR. Put more simply, as the dynamic 2C TCR scans for ligands,those that can match both its structural and motional properties arelikely to bind better.The influence of matched structural and motional properties on

receptor/ligand selectivity is diagrammed schematically in Fig. 4.Fig. 4A illustrates our observations with sc2C and QL9–scLd: theTCR and pMHC move between compatible conformations atsimilar rates and retain their mobility in the TCR–pMHC com-plex. Due to the structural complementarity between the differentreceptor and ligand configurations, there are two ways in whichthe complex can form. Fig. 4B illustrates formation of a complexfrom molecules possessing mismatched motional properties andfor which motion is restricted in the TCR–pMHC complex. Com-pared to the case in Fig. 4A, the resulting complex is of weakerthermodynamic stability. This results from the need for structuraladjustments to optimize complementarity, as well as a greater en-tropic penalty due to the restriction of motion in the complex. Thereduced stability of the complex results in both weaker binding andfaster dissociation.Should we expect these observations to be unique to 2C and

QL9-Ld? A number of studies have demonstrated mobility of bothpeptides in MHC binding grooves and TCR CDR loops (5, 9, 17,21, 48–50), and other studies have directly linked crystallo-graphically observed conformational changes to TCR and pMHCmotion (5, 9). Moreover, conformational melding, or the obser-vation of conformational changes occurring in both pMHC andTCR upon binding, has been observed in multiple cases. We thuspropose that the matching of structural fluctuations between re-ceptor and ligand is one element of the process through which

FIGURE 4. Matched TCR and pMHC conformational exchange promotes facile peptide binding. (A) In their free states, the TCR (blue curve) and

pMHC (green curve) sample structurally complementary conformations with similar kinetics, as shown by the superposition of the TCR and pMHC curves.

Either set of compatible conformations can bind, as shown by the two orange binding curves. Continued TCR and pMHC conformational exchange in the

TCR–pMHC complex results in two distinct yet kinetically linked bound-state conformations. This scheme mirrors our observations on recognition of

QL9-scLd by sc2C. (B) Structural fluctuations between incompatible TCR and peptide conformations results in a weaker and more rapidly dissociating

complex. In the example shown, the free TCR and pMHC sample noncomplementary conformations with different kinetics, with one set of conformations

too divergent to permit binding (high DEconf on the left). The lack of fluctuations in the complex, reflecting the freezing out of TCR and pMHC motions,

increases the entropic penalty for binding and lowers structural complementarity, weakening the thermodynamic and kinetic stability of the complex (lower

DGbind and a correspondingly lower barrier for dissociation).

The Journal of Immunology 5

by guest on April 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

TCRs scan for and identify the most compatible ligands from amyriad of potential targets. As a range of structural changes andmotions over different timescales have been observed for TCRsand pMHC complexes (4, 9, 10, 17–19, 51), this strategy wouldcertainly be used to varied extents with different interactions. Forexample, more rigid TCRs may rely less on matched motions.Further, flexibility in a free receptor does not necessarily requireretained motion in the complex, as entropic penalties associatedwith reducing motions could be offset by energetic gains else-where in an interface. Nonetheless, the strategy identified in thisstudy is generalizable and readily superimposable onto other mech-anisms that influence how TCRs discriminate among ligands forthose that can bind sufficient well to enable T cell signaling.

AcknowledgmentsWe thank Kristina Davis of the Notre Dame Center for Research Computing

for assistance in generating Fig. 4.

DisclosuresThe authors have no financial conflicts of interest.

References1. Armstrong, K. M., K. H. Piepenbrink, and B. M. Baker. 2008. Conformational

changes and flexibility in T-cell receptor recognition of peptide-MHC com-plexes. Biochem. J. 415: 183–196.

2. Garcia, K. C., M. Degano, L. R. Pease, M. Huang, P. A. Peterson, L. Teyton, andI. A. Wilson. 1998. Structural basis of plasticity in T cell receptor recognition ofa self peptide-MHC antigen. Science 279: 1166–1172.

3. Ding, Y. H., B. M. Baker, D. N. Garboczi, W. E. Biddison, and D. C. Wiley.1999. Four A6-TCR/peptide/HLA-A2 structures that generate very differentT cell signals are nearly identical. Immunity 11: 45–56.

4. Reiser, J. B., C. Gregoire, C. Darnault, T. Mosser, A. Guimezanes,A. M. Schmitt-Verhulst, J. C. Fontecilla-Camps, G. Mazza, B. Malissen, andD. Housset. 2002. A T cell receptor CDR3beta loop undergoes conforma-tional changes of unprecedented magnitude upon binding to a peptide/MHCclass I complex. Immunity 16: 345–354.

5. Borbulevych, O. Y., K. H. Piepenbrink, B. E. Gloor, D. R. Scott, R. F. Sommese,D. K. Cole, A. K. Sewell, and B. M. Baker. 2009. T cell receptor cross-reactivitydirected by antigen-dependent tuning of peptide-MHC molecular flexibility.Immunity 31: 885–896.

6. Gagnon, S. J., O. Y. Borbulevych, R. L. Davis-Harrison, R. V. Turner,M. Damirjian, A. Wojnarowicz, W. E. Biddison, and B. M. Baker. 2006. T cellreceptor recognition via cooperative conformational plasticity. J. Mol. Biol. 363:228–243.

7. Armstrong, K. M., F. K. Insaidoo, and B. M. Baker. 2008. Thermodynamics ofT-cell receptor-peptide/MHC interactions: progress and opportunities. J. Mol.Recognit. 21: 275–287.

8. Boehr, D. D., R. Nussinov, and P. E. Wright. 2009. The role of dynamicconformational ensembles in biomolecular recognition. Nat. Chem. Biol. 5: 789–796.

9. Scott, D. R., O. Y. Borbulevych, K. H. Piepenbrink, S. A. Corcelli, andB. M. Baker. 2011. Disparate degrees of hypervariable loop flexibility controlT-cell receptor cross-reactivity, specificity, and binding mechanism. J. Mol. Biol.414: 385–400.

10. Borbulevych, O. Y., S. M. Santhanagopolan, M. Hossain, and B. M. Baker. 2011.TCRs used in cancer gene therapy cross-react with MART-1/Melan-A tumorantigens via distinct mechanisms. J. Immunol. 187: 2453–2463.

11. Chen, J.-L., G. Stewart-Jones, G. Bossi, N. M. Lissin, L. Wooldridge,E. M. L. Choi, G. Held, P. R. Dunbar, R. M. Esnouf, M. Sami, et al. 2005.Structural and kinetic basis for heightened immunogenicity of T cell vaccines.J. Exp. Med. 201: 1243–1255.

12. Wu, L. C., D. S. Tuot, D. S. Lyons, K. C. Garcia, and M. M. Davis. 2002. Two-step binding mechanism for T-cell receptor recognition of peptide MHC. Nature418: 552–556.

13. Holler, P. D., and D. M. Kranz. 2004. T cell receptors: affinities, cross-reactivities, and a conformer model. Mol. Immunol. 40: 1027–1031.

14. Boniface, J. J., Z. Reich, D. S. Lyons, and M. M. Davis. 1999. Thermodynamicsof T cell receptor binding to peptide-MHC: evidence for a general mechanism ofmolecular scanning. Proc. Natl. Acad. Sci. USA 96: 11446–11451.

15. Baker, B. M., S. J. Gagnon, W. E. Biddison, and D. C. Wiley. 2000. Conversionof a T cell antagonist into an agonist by repairing a defect in the TCR/peptide/MHC interface: implications for TCR signaling. Immunity 13: 475–484.

16. Borbulevych, O. Y., K. H. Piepenbrink, and B. M. Baker. 2011. Conformationalmelding permits a conserved binding geometry in TCR recognition of foreignand self molecular mimics. J. Immunol. 186: 2950–2958.

17. Hare, B. J., D. F. Wyss, M. S. Osburne, P. S. Kern, E. L. Reinherz, andG. Wagner. 1999. Structure, specificity and CDR mobility of a class II restrictedsingle-chain T-cell receptor. Nat. Struct. Biol. 6: 574–581.

18. Pohlmann, T., R. A. Bockmann, H. Grubm€uller, B. Uchanska-Ziegler, A. Ziegler,and U. Alexiev. 2004. Differential peptide dynamics is linked to major histo-compatibility complex polymorphism. J. Biol. Chem. 279: 28197–28201.

19. Hawse, W. F., M. M. Champion, M. V. Joyce, L. M. Hellman, M. Hossain,V. Ryan, B. G. Pierce, Z. Weng, and B. M. Baker. 2012. Cutting edge: Evidencefor a dynamically driven T cell signaling mechanism. J. Immunol. 188: 5819–5823.

20. Scott, D. R., C. F. Vardeman, 2nd, S. A. Corcelli, and B. M. Baker. 2012.Limitations of time-resolved fluorescence suggested by molecular simulations:assessing the dynamics of T cell receptor binding loops. Biophys. J. 103: 2532–2540.

21. Fabian, H., H. Huser, D. Narzi, R. Misselwitz, B. Loll, A. Ziegler,R. A. Bockmann, B. Uchanska-Ziegler, and D. Naumann. 2008. HLA-B27subtypes differentially associated with disease exhibit conformational dif-ferences in solution. J. Mol. Biol. 376: 798–810.

22. Kleckner, I. R., and M. P. Foster. 2011. An introduction to NMR-basedapproaches for measuring protein dynamics. Biochim. Biophys. Acta 1814:942–968.

23. Garcia, K. C., M. D. Tallquist, L. R. Pease, A. Brunmark, C. A. Scott,M. Degano, E. A. Stura, P. A. Peterson, I. A. Wilson, and L. Teyton. 1997.Alphabeta T cell receptor interactions with syngeneic and allogeneic ligands:affinity measurements and crystallization. Proc. Natl. Acad. Sci. USA 94: 13838–13843.

24. Colf, L. A., A. J. Bankovich, N. A. Hanick, N. A. Bowerman, L. L. Jones,D. M. Kranz, and K. C. Garcia. 2007. How a single T cell receptor recognizesboth self and foreign MHC. Cell 129: 135–146.

25. Lee, P. U. Y., H. R. O. Churchill, M. Daniels, S. C. Jameson, and D. M. Kranz.2000. Role of 2CT cell receptor residues in the binding of self- and allo-majorhistocompatibility complexes. J. Exp. Med. 191: 1355–1364.

26. Sykulev, Y., A. Brunmark, M. Jackson, R. J. Cohen, P. A. Peterson, andH. N. Eisen. 1994. Kinetics and affinity of reactions between an antigen-specificT cell receptor and peptide-MHC complexes. Immunity 1: 15–22.

27. Schlueter, C. J., T. C. Manning, B. A. Schodin, and D. M. Kranz. 1996. A residuein the center of peptide QL9 affects binding to both Ld and the T cell receptor.J. Immunol. 157: 4478–4485.

28. Jones, L. L., S. E. Brophy, A. J. Bankovich, L. A. Colf, N. A. Hanick,K. C. Garcia, and D. M. Kranz. 2006. Engineering and characterization ofa stabilized alpha1/alpha2 module of the class I major histocompatibility com-plex product Ld. J. Biol. Chem. 281: 25734–25744.

29. Garcia, K. C., M. Degano, R. L. Stanfield, A. Brunmark, M. R. Jackson,P. A. Peterson, L. Teyton, and I. A. Wilson. 1996. An alphabeta T cell receptorstructure at 2.5 A and its orientation in the TCR-MHC complex. Science 274:209–219.

30. Tompa, P., and M. Fuxreiter. 2008. Fuzzy complexes: polymorphism andstructural disorder in protein-protein interactions. Trends Biochem. Sci. 33: 2–8.

31. Varani, L., A. J. Bankovich, C. W. Liu, L. A. Colf, L. L. Jones, D. M. Kranz,J. D. Puglisi, and K. C. Garcia. 2007. Solution mapping of T cell receptordocking footprints on peptide-MHC. Proc. Natl. Acad. Sci. USA 104: 13080–13085.

32. Shusta, E. V., M. C. Kieke, E. Parke, D. M. Kranz, and K. D. Wittrup. 1999.Yeast polypeptide fusion surface display levels predict thermal stability andsoluble secretion efficiency. J. Mol. Biol. 292: 949–956.

33. Marley, J., M. Lu, and C. Bracken. 2001. A method for efficient isotopic labelingof recombinant proteins. J. Biomol. NMR 20: 71–75.

34. Vranken, W. F., W. Boucher, T. J. Stevens, R. H. Fogh, A. Pajon, M. Llinas,E. L. Ulrich, J. L. Markley, J. Ionides, and E. D. Laue. 2005. The CCPN datamodel for NMR spectroscopy: development of a software pipeline. Proteins 59:687–696.

35. Bodenhausen, G., and D. J. Ruben. 1980. Natural abundance nitrogen-15 NMRby enhanced heteronuclear spectroscopy. Chem. Phys. Lett. 69: 185–189.

36. Kay, L. E., M. Ikura, R. Tschudin, and A. Bax. 2011. Three-dimensional triple-resonance Nmr-spectroscopy of isotopically enriched proteins. 1990 J. Magn.Reson. 213: 423–441.

37. Clubb, R. T., V. Thanabal, and G. Wagner. 1992. A new 3D HN(CA)HA ex-periment for obtaining fingerprint HN-Halpha peaks in 15N- and 13C-labeledproteins. J. Biomol. NMR 2: 203–210.

38. Grzesiek, S., and A. D. Bax. 1992. Improved 3D Triple-Resonance NMRTechniques Applied to a 31 kDa Protein. J. Magn. Reson. 96: 432–440.

39. Wittekind, M., and L. Mueller. 1993. HNCACB, a High-Sensitivity 3D NMRExperiment to Correlate Amide-Proton and Nitrogen Resonances with theAlpha- and Beta-Carbon Resonances in Proteins. J. Magn. Reson. B. 101: 201–205.

40. Greenwood, A. I., M. J. Rogals, S. De, K. P. Lu, E. L. Kovrigin, andL. K. Nicholson. 2011. Complete determination of the Pin1 catalytic domainthermodynamic cycle by NMR lineshape analysis. J. Biomol. NMR 51: 21–34.

41. Kovrigin, E. L. 2012. NMR line shapes and multi-state binding equilibria. J.Biomol. NMR 53: 257–270.

42. McConnell, H. M. 1958. Reaction rates by nuclear magnetic resonance. J. Chem.Phys. 28: 430–431.

43. Jones, L. L., L. A. Colf, A. J. Bankovich, J. D. Stone, Y.-G. Gao, C. M. Chan,R. H. Huang, K. C. Garcia, and D. M. Kranz. 2008. Different thermodynamicbinding mechanisms and peptide fine specificities associated with a panel ofstructurally similar high-affinity T cell receptors. Biochemistry 47: 12398–12408.

44. Mittag, T., B. Schaffhausen, and U. L. G€unther. 2004. Tracing kinetic inter-mediates during ligand binding. J. Am. Chem. Soc. 126: 9017–9023.

45. De, S., A. I. Greenwood, M. J. Rogals, E. L. Kovrigin, K. P. Lu, andL. K. Nicholson. 2012. Complete thermodynamic and kinetic characterization ofthe isomer-specific interaction between Pin1-WW domain and the amyloid

6 TCR SCANNING BY DYNAMIC COMPLEMENTARITY

by guest on April 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

precursor protein cytoplasmic tail phosphorylated at Thr668. Biochemistry 51:8583–8596.

46. Bowerman, N. A., L. A. Colf, K. C. Garcia, and D. M. Kranz. 2009. Differentstrategies adopted by K(b) and L(d) to generate T cell specificity directed againsttheir respective bound peptides. J. Biol. Chem. 284: 32551–32561.

47. Manning, T. C., C. J. Schlueter, T. C. Brodnicki, E. A. Parke, J. A. Speir,K. C. Garcia, L. Teyton, I. A. Wilson, and D. M. Kranz. 1998. Alanine scanningmutagenesis of an alphabeta T cell receptor: mapping the energy of antigenrecognition. Immunity 8: 413–425.

48. Baker, B. M., D. R. Scott, S. J. Blevins, and W. F. Hawse. 2012. Structural anddynamic control of T-cell receptor specificity, cross-reactivity, and bindingmechanism. Immunol. Rev. 250: 10–31.

49. Insaidoo, F. K., O. Y. Borbulevych, M. Hossain, S. M. Santhanagopolan,T. K. Baxter, and B. M. Baker. 2011. Loss of T cell antigen recognition arisingfrom changes in peptide and major histocompatibility complex protein flexi-bility: implications for vaccine design. J. Biol. Chem. 286: 40163–40173.

50. Fabian, H., B. Loll, H. Huser, D. Naumann, B. Uchanska-Ziegler, and A. Ziegler.2011. Influence of inflammation-related changes on conformational character-istics of HLA-B27 subtypes as detected by IR spectroscopy. FEBS J. 278: 1713–1727.

51. Deng, L., R. J. Langley, P. H. Brown, G. Xu, L. Teng, Q. Wang, M. I. Gonzales,G. G. Callender, M. I. Nishimura, S. L. Topalian, and R. A. Mariuzza. 2007.Structural basis for the recognition of mutant self by a tumor-specific, MHCclass II-restricted T cell receptor. Nat. Immunol. 8: 398–408.

The Journal of Immunology 7

by guest on April 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from