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Peptide-N-Terminal Binding SiteComplex with an Empty ConservedHLA-A*0201/Octameric Tax Peptide The Structure and Stability of an

Biddison and Don C. WileyAmir R. Khan, Brian M. Baker, Partho Ghosh, William E.

http://www.jimmunol.org/content/164/12/6398doi: 10.4049/jimmunol.164.12.6398

2000; 164:6398-6405; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/164/12/6398.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Structure and Stability of an HLA-A*0201/OctamericTax Peptide Complex with an Empty ConservedPeptide-N-Terminal Binding Site1

Amir R. Khan,* Brian M. Baker,* Partho Ghosh, ‡ William E. Biddison,§ and Don C. Wiley2*†

The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV),from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higherresolution (1.8 Å) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminalresidue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace theterminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elementsat that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable,DTm 5 16°C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infectedindividuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a “closed conformation” of thepeptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does notfunctionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes. The Journal of Immunology,2000, 164: 6398–6405.

Short peptides (;9 residues) from proteins degraded in thecytoplasm of vertebrate cells are bound by class I MHCmolecules in the endoplasmic reticulum and transported to

the cell surface for recognition by the Ag-specific receptors (TCR)of T cells as part of the immune system’s surveillance for foreignAgs (1, 2). Any one class I molecule, of the many different allelesexpressed in the population, is capable of forming very stable com-plexes, with half-lives of tens of hours, with a large number ofdifferent short peptides. These long-lived MHC/peptide com-plexes, which can mark cells for destruction by CTL, appear to bekinetic traps for peptides. In vitro, the off-rates of peptides fromMHC molecules are very slow (3, 4), whereas peptide associationrates vary between experimental systems (5–9). The kinetics ofMHC assembly suggests a two step process involving a confor-mational change of the MHC molecule from a short-lived, recep-tive, “open” binding state to a long-lived, “closed” conformation(5, 10–13).

X-ray crystal structures of MHC molecules have revealed thestructure of the closed conformation with peptides bound (14–16).Some side chains of bound peptides (anchor residues) are held inpockets in the peptide-binding groove that are polymorphic in the

different MHC allelic products, providing a sequence-dependentelement to peptide binding (16–18). In class I MHC molecules thecharged N and C termini and main chain of the bound peptide areheld, through a network of hydrogen bonds and salt bridges, topolar residues conserved in all human and murine class I MHCmolecules (19–24). This network of conserved hydrogen bonds atboth termini of peptides provides an independent peptide se-quence-independent element to peptide binding. Because thesepeptide N- and C-terminal contacts to class I MHCmolecules areconserved for both all bound peptides and all class I alleles, whereasthe peptide side chain contacts to the polymorphic pockets of class Imolecules vary considerably with different peptides and differentMHC alleles, the conserved interactions at the peptide termini havebeen proposed to have an important role in forming the sharedproperty of long half-life peptide-bound conformations of class Imolecules (19).

The stability of class I MHC molecules, which are heterodimersof a polymorphic heavy chain (Hc)3 andb2-microglobulin (b2m),is strongly dependent on the presence of a bound peptide. In vitro,in the absence of bound peptides,b2m dissociates and Hc aggre-gates (17, 22, 25–29), whereas in vivo in the endoplasmic reticu-lum peptide-free class I molecules are stabilized by chaperoninsand the peptide transport and loading proteins, TAP and Tapasin(30, 31). Thermal denaturation studies of MHC/peptide complexesin which either the peptide N-terminal amino group or C-terminalcarboxylate group was substituted by a methyl group showed adecrease in theTm of the MHC/peptide of 22°C, indicating a de-crease in stability of;4.6 kcal/mol, whereas substituting both pep-tide anchor residues with alanine showed a decrease of only 5.5°Cor ;1.2 kcal/mol (28, 32). These thermodynamic data support thesuggestion that the conserved interactions between the N and C

*Department of Molecular and Cellular Biology and†Howard Hughes Medical In-stitute, Harvard University, Cambridge MA 02138;‡Department of Chemistry andBiochemistry, University of California at San Diego, La Jolla, CA 92093; and§Mo-lecular Immunology Section, Neuroimmunology Branch, National Institute of Neu-rological Disorders and Stroke, National Institutes of Health, Bethesda MD 20892

Received for publication January 28, 2000. Accepted for publication March 30, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 A.R.K. is supported by a Human Frontiers Long Term Fellowship. B.M.B. is sup-ported by a fellowship from the Cancer Research Institute. D.C.W. is an investigatorof the Howard Hughes Medical Institute.2 Address correspondence and reprint requests to Dr. Don C. Wiley, Howard HughesMedical Institute, Department of Molecular and Cellular Biology, Harvard Univer-sity, 7 Divinity Avenue, Cambridge, MA 02138. E-mail address: wiley@xta10.harvard.edu

3 Abbreviations used in this paper: Hc, heavy chain;b2m, b2-microglobulin; Tm,melting temperature; HTLV-1, human T cell lymphotrophic virus-1; HAM/TSP,HTLV-I-associated myelopathy/tropical spastic paraparesis; CD, circular dichroism.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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termini of bound peptides may dominate in the formation and sta-bilization of peptide/MHC complexes.

In this paper we have studied the effect of removing the N-terminal amino acid of an antigenic peptide (Tax9) on the stabilityand structure of its interaction with HLA-A2, and on its recogni-tion by T cells as an HLA-A2/Tax8 complex. Tax9 is the dominantantigenic peptide inducing cytotoxic T cells in human T cell lym-photrophic virus-1 (HTLV-1)-infected individuals with the neuraldegenerative disorder HAM/TSP (33). The x-ray structure of theHLA-A2 complex with Tax9 (34) was previously refined to 2.5-Åresolution, whereas the complexes of HLA-A2/Tax9 with two hu-man abTCRs, A6 and B7, were determined at 2.6-Å resolution(35–37). Although HLA-A2/Tax8 activates cytotoxic T cells inHTLV-1-infected individuals (38), we find that it does not cross-react functionally with HLA-A2/Tax9-specific T cells in cell lysisassays and has a low affinity for a HLA-A2/Tax9-specific TCR.The stability of the HLA-A2/Tax8 complex is markedly reduced,as expected for loss of the conserved interactions at the N-terminalpeptide-binding site, but the structure of the complex is remarkablysimilar with water molecules substituting for some of the peptideinteractions in the binding site.

Materials and MethodsProtein purification and crystallization

The extracellular region of the HLA-A2 Hc andb2m were expressed sep-arately inEscherichia colias inclusion bodies (39). The inclusion bodieswere refolded together in the presence of excess Tax8 (LFGYPVYV) orTax9 (LLFGYPVYV) peptide. Briefly, milligram amounts ofb2m, Hc, andpeptide were injected into 500 ml of a refolding buffer (100 mM Tris, 400mM arginine-HCl, 2 mM NaEDTA, 0.5 mM oxidized glutathione, and 5mM reduced glutathione, pH 8.3). The final concentrations of the constit-uents of the complexes were 2mM b2m, 1 mM Hc, and 50mM peptide(Tax9). A significantly larger excess of Tax8 peptide (100–150mM) wasrequired to produce that complex in sufficient quantity for biochemicalstudies, and the resulting yield was very low (;5%) compared with typicalyields with Tax9 (20–25%). The ternary complexes were purified by ion-exchange and gel filtration chromatography as described previously (34).

Crystals of Tax9-A2 were obtained by vapor diffusion from hangingdrops containing equal volumes of protein (5 mg/ml; 25 mM MES, pH 6.5)and 13–20% polyethylene glycol (PEG) 6000 (25 mM MES, pH 6.5).Tax8-A2 crystals were obtained by seeding with Tax9-A2 crystals. A 3-mlsolution of seed crystals was incubated with a 3-ml solution containing 3mg/ml Tax8-A2 and 400mM Tax8 peptide in 25 mM MES (pH 6.4) and0.1% NaN3. The drop was equilibrated against 13% PEG 6000, 25 mMMES, and 0.1% NaN3 at 18°C.

Data collection and structural refinement

Crystals of Tax8-A2 were transferred to a 20% glycerol solution in steps of4–10% and flash-cooled in a stream of cryo-cooled nitrogen gas. Data werecollected on a Mar345 detector (Mar Research, Hamburg, Germany)mounted on an Elliot GX-13 x-ray generator (GEC Avionics, London,U.K.). The structure of Tax8-A2 was refined by using the previously de-

termined structure of Tax9-A2 as the starting model (Protein Data Bankcode 1hhk; Ref. 34). A subset (10%) of the reflection data were flagged(R-free) and excluded from refinement protocols. As the crystals ofTax8-A2 and Tax9-A2 were isomorphous, the same set of flagged reflec-tions previously used for monitoring Tax9-A2 refinement was used in theR-free data set for Tax8-A2. Because the structure of Tax9-A2 was pre-viously refined to only 2.5-Å resolution (without waters), the data wereextended to 1.8 Å and the model refined to that resolution with the inclu-sion of water molecules.

The starting model of Tax8-A2, stripped of water molecules and thepeptide was initially subjected to rigid-body fitting using the program CNS(40). Electron density maps clearly indicated the position of Tax8 withinthe peptide-binding groove of HLA-A2. The structure was further refinedby multiple cycles of energy minimization and model building using theprograms CNS and O (40, 41). All reflections between 50 Å and the res-olution limits of the data sets (2.14 Å for Tax8-A2, 1.8 Å for Tax9-A2)were used during refinement and electron density map calculations (TableI). Water molecules were gradually introduced during the course of modelbuilding and were selected from.3s peaks in difference (Fo 2 Fc) elec-tron density maps within 2.5–3.6 Å of hydrogen bond donors or acceptors.

During the course of refinement, it was observed that the N-terminalleucine residue (Leu2) of Tax8 has a backbone conformation that is distinctfrom that residue in Tax9, resulting in a net shift of 1.0 Å in the positionof the a-amino group of Tax8. Simulated annealing omit maps, in whichthe peptide and surrounding water molecules were removed, confirmed thatthe N terminus was both well positioned and ordered in both moleculesof the asymmetric unit. The conformation of Leu2 precludes formation ofa salt bridge with Glu63 of the B pocket; in the Tax9 structure, the identicalamide nitrogen hydrogen bonds to Glu63. To further test the validity of theTax8 model, one further cycle of positional refinement was performed in

Table II. Refinement statistics

Tax8 Tax9

Resolution (Å) 50-2.15 50-1.8Rfree (%)a 25.41 (2.26-2.2 Å)

28.7%25.02 (1.86-1.8 Å)

30.7%Rcryst (%)a 19.18 (2.26-2.2 Å)

21.6%19.77 (1.86-1.8 Å)

24.9%rms deviations from

idealityBonds (Å) 0.01 0.03Angles (°) 1.6 2.4Dihedrals (°) 25.3 26.2Improper (°) 0.9 1.6

Luzzati e.s.d. (Å)b 0.23 0.21Protein atoms 6306 6322Water molecules 496 607Ramachandran mapc

Most favored (%) 89.3 93.2Disallowed (%) 0 0

a Rcryst (Rfree) 5 (ShuFo 2 Fcu/(SFo). Rfreecalculated using 10% of the reflections,randomly generated and excluded from refinement protocols.

b Estimated coordinate accuracy of the structures from the method of Luzzati (51).c Analyzed using the program PROCHECK (52).

Table I. Crystallographic dataa

Tax8-A2 Tax9-A2

Space group P1 P1Molecules in AU 2 2Cell parameter a 5 50.34 Å,b 5 62.86 Å,c 5 74.84 Å a 5 50.56 Å,b 5 63.79 Å,c 5 75.08 Å

a 5 82.01°,b 5 76.1°,g 5 77.96° a 5 81.58°,b 5 75.66°,g 5 77.38°Total reflections 60,478 137,168Rmerge(%) 4.7 4.2Resolution (Å) 50-2.15 50-1.80Unique reflections 38,048 72,184Completeness (%) 78.0 85.3F/sF . 3.0 (2.26-2.15 Å) 38.3% (1.94-1.80 Å) 59.9%

(2.26-2.15 Å) 33.2% (1.94-1.80 Å) 45.4%

a Rmerge5 ShkljuIhklj 2 ,Ihklj.uShklj,Ihkl. 3 100%.

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which thea-amino group was manually re-fitted by adjustment of torsionangles to within 3.2 Å of the side-chain of Glu63 (Oe1). Following crys-tallographically restrained energy minimization in the CNS program, it wasagain observed that the N terminus shifted away, to a position 3.6 Å fromGlu63 (Oe1), but to within 3.29 Å of the hydroxyl group of Tyr159. Thestatistics from the final cycle of refinement are shown in Table II. Coor-dinates and structure factors have been submitted to the Protein Data Bank(A2-Tax8 and A2-Tax9 PDB codes 1DUY and 1DUZ, respectively).

Circular dichroism (CD)

The thermal stability of peptide-MHC complexes was monitored by CDspectroscopy using a Jasco J-710 instrument (Jasco International, Tokyo,Japan) equipped with a Peltier temperature regulator. Solution conditionswere 20 mM phosphate and 75 mM NaCl (pH 7.4). Protein concentrationswere ;0.15 mg/ml. The spectrum between 250 and 190 nm indicated apredominantlyb-sheet conformation. Temperature denaturation was mon-itored at the minimum of 218 nm between 10 and 90°C using a gradient of1°C/min. Scans were repeated twice with fresh protein and the data pointsaveraged. Rather than constraining the data to a two-state unfolding model,the data in the transition region were simply fit to a nine-order polynomialequation, and the apparentTm was determined from the maximum of thefirst derivative of the fitted curve.

Binding measurements

The 2G4 T cell clone has a TCR (A6-TCR) specific for Tax9 complexedwith HLA-A2, and the interaction of this receptor with a number of alteredpeptides complexed with HLA-A2 has been previously investigated (37,

42). The binding of Tax8-A2 to the A6-TCR was investigated here usingan equilibrium BIAcore assay as described previously (37). Briefly, re-combinant A6-TCR with a single free thiol at the C terminus of theb-chainwas coupled to a CM5 sensor chip using standard thiol coupling. Multipleconcentrations of Tax8-A2 were injected at a flow rate of 10ml/min, andthe responses at equilibrium (;400 s after injection) were determined. Thetemperature of the sample was maintained at 4°C, and Tax8-A2 dilutionswere made from a highly concentrated stock immediately before injectionto minimize dissociation of the Tax8 peptide from HLA-A2. The responsesfrom injections over a mock surface were subtracted from the data, and allinjections were repeated twice. Equilibrium responses were fit against theconcentration of injected Tax8-A2, assuming a single-site binding model.Solution conditions were 10 mM HEPES, 150 mM NaCl, 3 mM EDTA,and 0.005% polysorbate-20 (pH 7.4) at 25°C.

T cell assays

T cell-mediated cytotoxicity was quantitated by a time-resolved fluoromet-ric assay using HLA-A2-transfected cells as targets as previously described(36). Effector cells were the A6-TCR-expressing CD81 T cell clone 2G4that recognizes the Tax11–19 peptide presented by HLA-A2 and was iso-lated from a patient with HAM/TSP (43).

For the antagonism assay, HLA-A2-transfected Hmy2.C1R cells weretreated with 100mg/ml mitomycin C (Sigma, St. Louis, MO) for 2 h at37°C, washed three times with PBS, and then pulsed with 1000 nM ofcandidate antagonist peptides for 2 h at 37°C. The cells were washed, and1 3 105 cells were incubated with 13 105 2G4 T cells for 1 h at 37°C. Taxpeptide was added at concentrations of 0.1–10 nM, and the cells wereincubated for 48 h. Supernatants were collected and assayed for IFN-gcontent as previously described (36).

ResultsPeptide residues P2-P9 of the Tax8-A2 and Tax9-A2 complexesbind very similarly

The structures of Tax8-A2 and Tax9-A2 were refined to reason-able agreement between observed and calculated structure factors,as well as good stereochemistry and geometry (Table II). Althoughdata extending to 2.15 Å were used in the refinement of Tax8-A2,the data were incomplete in the highest resolution shell (Table I).The number of observed reflections corresponds to a 100% com-plete data set to 2.30-Å resolution. The overall structure of theTax8-A2 complex is identical to Tax9-A2 and is shown in Fig. 1.The quality of the structures permits a detailed discussion of thepeptide-binding groove and its bound solvent.

The conformation and position of Tax8 within the peptide-bind-ing groove of HLA-A2 is identical to the structure observed inTax9-A2 (Fig. 2). Hydrogen bonds and hydrophobic contacts are

FIGURE 1. Molecular structure of Tax8-A2. The overall architecture isidentical with the Tax9-A2 structure.Upper, The various domains are dif-ferentiated by color. The position of the peptide (red) and three watermolecules that occupy the A site of the groove are shown. The transmem-brane anchor would extend down from the green (a3) domain of Hc intothe lipid bilayer.Lower, View of Tax8 within the MHC groove. The viewis looking down from the “top” of theupper panel. The images werecreated with MOLSCRIPT (53).

FIGURE 2. Least-squares superimposition of Tax8-A2 and Tax9-A2.Only the peptides and conserved water molecules are shown. Tax9 is col-ored green to distinguish the molecule from Tax8. Superimposition wasperformed using only the backbone atoms of the Hc (1100 atoms), givinga root-mean-square deviation of 0.33 Å. The image was created withMOLSCRIPT (53).

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preserved from Phe3 to Val9 of Tax8 and the corresponding MHCbinding pocket, indicating that loss of the position 1 Leu (P1) doesnot induce global shifts or conformational changes in the peptideor MHC. Following alignment of the HLA Hc, the root-mean-square difference in the coordinates of the common atoms of Tax8and Tax9 was 0.36 Å. However, thea-amino group of Leu2 (Tax8)is positioned about 1 Å from the equivalent P2 amide nitrogen ofTax9 (seeMaterials and Methods), making a hydrogen bond withthe hydroxyl of Tyr159 rather than Glu63 as in Tax9 (compare Fig.3, a andb). The distance between the side chain of Glu63 and theP2 amino group of Tax8 is 3.9 Å, which may still permit a favor-able electrostatic interaction.

Two water molecules occupy the N-terminal peptide residuebinding pocket in the Tax8 complex

In the Tax8 complex two water molecules, Wat-1 and Wat-2, par-tially fill the space occupied by the P1 peptide residue, Leu1, in theTax9 complex (Fig. 3). Wat-1 provides a bridge from the N ter-minus of Tax8, via hydrogen bonds, to the carboxylate group ofGlu63 (Fig. 3). Wat-2 is in the position that is occupied by thea-amino group of Leu1 in Tax9 (Fig. 3,a andb). This water pro-vides a bridge from the hydroxyl group of Tyr7, in the b-sheetforming the floor of the peptide-binding groove, via hydrogen

bonds to Tyr171 in a-helix forming one side of the binding site(Fig. 3). In the Tax9 complex, the same hydrogen bonded bridgebetween elements of secondary structure is made through theamino group at Leu1 of the bound peptide (Fig. 3b). Wat-2 is alsowithin hydrogen bonding distance of Wat-1 (distance of 2.82 Å;Fig. 3a) and a third water (Wat-3) found in both the Tax8 and Tax9complexes (Fig. 3).

The hydrogen bonds in the peptide N-terminal region of thebinding groove for the Tax8-A2 and Tax9-A2 structures are sum-marized in Table III. Although in the Tax8 complex there is a lossof three hydrogen bonds that went directly from the bound peptideto the MHC molecule relative to Tax9 complex, the shift in thenitrogen position of Tax8 and the addition of two water moleculescreates a network of hydrogen bonds that both satisfies all of thehydrogen bonding acceptors and donors of the conserved MHCresidues and cross-links together the same secondary structure el-ements as in the Tax9 complex.

The absence of the P1 peptide residue apparently causes somesmall conformational changes in HLA-A2. The indole ring ofTrp167 and the side chain of Glu163 are about 1 Å closer to thespace that would be occupied by Leu1 of Tax9 (data not shown).Despite these observed structural differences at the binding site forthe peptide N terminus, the side chain of the P2 peptide residue,

FIGURE 3. Hydrogen bonding inter-actions at the N-terminal anchor region.a, Tax8 is represented as a ball-and-stickmodel. The peptide bond vectors aregreen, and atom color corresponds toatom type. The A2 side chains and sec-ondary structure elements are light blue;water molecules are magenta. The pep-tide is truncated at Phe3. b, Hydrogenbonding interactions at the N-terminal re-gion of Tax9. The color scheme is thesame as ina and a similar orientation isshown. The images were created withMOLSCRIPT (53).

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Leu2, occupies the identical hydrophobic pocket in both Tax8 andTax9 complexes, forming contacts with Met45 and Phe9 in the Bpocket (16).

Solvent molecules in the peptide-binding sites of both Tax8 andTax9 complexes

In the middle portion of the peptide, three buried water moleculesform part of the interface between Tax8 and Hc (Wat-4, Wat-5,Wat-6; Fig. 4). These waters are also present in the structure ofTax9-A2 refined here, but were not visible in the previously pub-lished, lower resolution (2.5 Å) structure (34). The carbonyl oxy-gen of Tyr5 is linked via Wat-4 to thee-amino group of Arg97. Thecarbonyl oxygen of Val7 is linked via Wat-6 to the carboxylateoxygen of Asp77. These water-mediated, hydrogen bond bridgesare formed with the part of the peptide that is central to recognitionby TCRs (35, 37). Upon binding of both the A6- and B7-TCRs, aconformational change takes place in the Tax9 peptide such thatthe side chain of Pro6 becomes buried in the pocket occupied bytwo of these water molecules (35), and the Val7 side chain projectsoutward toward the TCR. Consequently, two water molecules,Wat-4 and Wat-5, must be displaced upon TCR binding. Anotherwater molecule, Wat-7, bridges the C-terminal carboxylate of thepeptide via hydrogen bonds to the side chain of Thr80 (Og1) inboth the Tax8-A2 and Tax9-A2 structures.

Thermal stability of the HLA-A2/Tax8 complex

The thermal denaturation curves of Tax8-A2 and Tax9-A2 weredetermined by monitoring the loss of secondary structure using

CD. The mid-point of the transitions were 49°C for Tax8-A2 and65°C for Tax9-A2 (Fig. 5), indicating a significant destabilizationof the peptide-A2 complex following loss of the N-terminal resi-due of the peptide. A second transition observed in the denatur-ation curves beginning near 80°C corresponds to the unfolding ofb2m (27, 32). Consequently, the early loss of secondary structureobserved for Tax8-A2 is attributed to loss of the peptide and un-folding of the Hc, as previously shown (27, 32). Due to linkagebetween peptide binding and stable folding of the Hc/b2mheterodimer, it follows that Tax8 binds the A2molecule with amuch lower affinity than Tax9 (28, 44). These results are consistentwith peptide-binding studies of the murine class I MHC H2-Kd, inwhich deletion of the N-terminal residue of a nonomer peptidesignificantly reduced the stability of the MHC/peptide complex (45).

TCR binding and T cell activation

Binding affinity of Tax8-A2 to the A6-TCR, specific for HLA-A2/Tax9, was measured using an equilibrium plasmon resonance ex-periment. The A6-TCR clone, isolated from a patient with theHTLV-1-associated autoimmune disorder HAM/TSP, is a class I-restricted TCR specific for Tax9 bound to HLA-A2 (43). Equilib-rium binding experiments (Fig. 6A) revealed that the association ofthe A6-TCR with Tax8-A2 is about 16-fold weaker (KD 5 15 62 mM) relative to the full-length Tax9 peptide-MHC complex (KD

5 0.9 6 0.1 mM; Ref. 37). Although we attempted to minimizesuch contributions during the experiment, it is possible that thisresult is influenced slightly by dissociation of Tax8 from the MHC(seeMaterials and Methods).

The biological activity of the Tax8 peptide was measured in aspecific lysis (cytotoxicity) assay (Fig. 6B). The data show that,compared with the wild-type Tax9 peptide, Tax8 has a very limitedability to activate A6-TCR-bearing T cells (barely detectable lysisat a million-fold increase in peptide concentration). In addition, noTCR antagonism (vs Tax9) was detected with Tax8 peptide (Fig.6C). However, Tax8 could sensitize targets cells for lysis by someTax-specific CTL (38).

Table III. Hydrogen bonds in the peptide N-terminal region of thebinding groovea

Peptide Water Heavy Chain

Tax9-A2 structureLeu1 (N) Tyr7 (OH) (2.82 Å)Leu1 (N) Tyr171 (OH) (2.84 Å)Leu1 (O) Tyr159 (OH) (2.71 Å)Leu2 (N) Glu63 (OE1) (2.91 Å)Leu2 (O) Lys66 (NZ) (3.02 Å)

Tax8-A2 structureLeu2 (N) Wat-1 (3.04 Å)

Wat-1 Glue63 (3.02 Å)Leu2 (N) Tyr159 (OH) (3.29 Å)Leu2 (O) Lys66 (NZ) (3.32 Å)

Wat-2 Tyr7 (OH) (2.53 Å)Wat-2 Tyr171 (OH) (2.72 Å)

a Only hydrogen bonds differing between the Tax8-A2 and Tax9-A2 structures arelisted.

FIGURE 4. Water-mediated hydrogen bonds between Tax8 and the MHCgroove. Water molecules (magenta) are represented as spheres, and the sidechains of A2 are green. The image was created with MOLSCRIPT (53).

FIGURE 5. CD thermal denaturation profiles of Tax8-A2 and Tax9-A2.The apparentTm values given by the inflection points are indicated by soliddiamonds. They-axis for both data sets was normalized for illustrativepurposes and does not indicate percent completion of the unfolding tran-sition (seeMaterials and Methods).

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DiscussionStructure and stability of Tax8/HLA-A2

Until now, all the structures of class I MHC molecules withbound peptides determined by x-ray crystallography, whetherthe peptides were 8-mer, 9-mer, or 10-mer, have had a peptideresidue in the P1 binding site making the conserved array ofhydrogen bonds to nonpolymorphic MHC residues (Fig. 3b). Inthe human class I molecule HLA-B35, a short peptide was ac-commodated by stretching out a kink usually found in longerpeptides (46), and in murine class I molecules the shape of thebottom of the binding site is conducive to the binding of shorterpeptides (21, 47). The Tax8/Tax9 pair of peptides were studiedhere, because it seemed likely that the 8-mer would bind justlike the 9-mer, except with the P1 site empty. This offered theopportunity to see whether the loss of the conserved hydrogenbonds, which both held bound peptides in the site and tetheredthe secondary structures of the binding site together, would af-

fect the conformation of the binding site. In particular, wehoped to see whether the binding site might adopt a partially“open state” as proposed to exist from the slow binding anddissociation kinetics of peptides.

In the Tax8 complex, as a result of no peptide residue occupyingthe P1 binding site, we observed a loss of three hydrogen bondsdirectly from the bound peptide to the MHC molecule, relative tothe Tax9 complex. However, the shift in the primary amine posi-tion of Tax8, and the addition of two water molecules, created anew network of hydrogen bonds that satisfies all of the hydrogenbonding acceptors and donors of the conserved MHC residues(Fig. 3). Furthermore, the new network of hydrogen bonds in theTax8 complex cross-links together the same secondary structureelements as in the Tax9 complex. Thus, although the structureindicates that the Tax8 peptide might be expected to bind moreweakly than Tax9, due to the loss of the three conserved hydrogenbonds from peptide to MHC, the restoration of a hydrogen bondingnetwork using water molecules apparently serves to stabilize the“closed conformation” of the MHC binding site.

In previous studies with a different peptide, the influenza virusmatrix peptide (GILGFVFTL), the N-terminal amino group of thepeptide was replaced, synthetically, by a methyl group (32). Themodified N terminus prevented the formation of two of the hydro-gen bonds that group normally makes with conserved residues ofthe MHC molecule (28). This loss of interactions resulted in adecrease of the thermal denaturation temperature of the peptide/HLA-A2 complex of 21°C, corresponding to aDDG° value ofabout 4.6 kcal/mol (28, 32). A structure of the complex revealedthat the substituted methyl group rotated away from the hydrogenbonding groups of the MHC molecule and was replaced by a watermolecule occupying the position vacated by the peptide N termi-nus (28) and located identically to water Wat-2 observed in theTax8 complex (Fig. 3a). The bound water molecule formed a sim-ilar set of hydrogen bonds to the conserved MHC residues as thefull-length 9-residue matrix and Tax peptides, but did not provideany bridging hydrogen bonds to the modified peptide. Apparentlythe loss of these two hydrogen bonds to the N-terminally modifiedpeptide resulted in the large (;4.6 kcal/mol) decrease in stabilityof the peptide/MHC complex.

The thermal denaturation temperature of the Tax8 complexwas observed in this paper to be 16°C lower than that of theTax9 complex. This difference also presumably results partlyfrom the loss of direct hydrogen bonds between the peptide andMHC molecule. Although the bridging water molecules Wat-1and Wat-2 form a network of hydrogen bonds, the new hydro-gen bonds do not directly link the peptide to the MHC molecule(Fig. 3a). Thus, as in the earlier study of the modified matrixpeptide/MHC complexes, eliminating the conserved hydrogenbonds from the peptide N terminus to the MHC molecule in theTax8 complex also very substantially decreased the stability ofthe MHC molecule. In both cases, despite drastic changes in thestability of the MHC molecules, when hydrogen bonding inter-actions with the peptide were removed, the conformation of thepeptide-binding site of the destabilized molecules was notchanged. Instead, in both cases, water molecules were observedto bind and created networks of hydrogen bonds that apparentlymaintained the binding site structure. This suggests the possi-bility that even an empty binding site, devoid of peptide, whichis quite unstable relative to peptide/MHC complexes (27),might maintain this closed conformation as the result of bindingwater molecules at its ends. “Closed-empty” and “closed-full”binding sites may look similar and dominate at equilibrium,with “open-empty” and “open-full” states being more transientstates separated from the closed states by the high free energy

FIGURE 6. T-cell receptor binding and T cell activation.A, Equilib-rium binding of Tax8-A2 to the A6-TCR performed using a BIAcoreassay as described.B, Sensitization of HLA-A2 target cells by Tax9 (Œ)and Tax8 (M) to lysis by the A6-TCR bearing CTL clone 2G4 (E:T52.5:1). A control peptide (influenza M1 57– 68) that lacks biologicalactivity with the A6-TCR is shown as a reference (f). C, Tax8 does notantagonize A6-bearing T-cells for IFN-g production by Tax9. Tax-P6A,a known antagonist, is a positive control (37), and M1 58 – 66 is anegative control.

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barriers responsible for the slow association and dissociationkinetics. It is these transient states that enzyme-like moleculesmust stabilize in vivo to accelerate peptide loading.

TCR binding and activation

HTLV-1 Tax8 peptide-specific HLA-A2-restricted CTL cell lineshave been generated from PBL of patients with HTLV-1-associ-ated neurological disease (38). The x-ray structure determined hereof the Tax8/HLA-A2 complex establishes the P1 peptide pocket isempty, so that the TCR of Tax8-specific CTL must bind to a pep-tide/MHC complex with no P1 peptide side chain.

The x-ray structures of two TCR specific for Tax9/HLA-A2have been determined complexed with Tax9/HLA-A2 (35–37). Inboth cases the major contacts between TCR and peptide are withpeptide residues P4 to P8, but the TCR does cover and make oneatomic contact to the P1 sidechain (Leu1). If we model a TCRinteraction with Tax8/HLA-A2 by superimposing the MHC/pep-tide coordinates from the Tax8/MHC structure with the Tax9/MHC/TCR structure, then the solvent accessible surface area ofthe MHC/peptide buried by the TCR would be decreased by 194Å2 (20%) as the result of deleting P1. However, a Tax8-specificTCR might fill in this cavity with either a different CDR1a se-quence or conformation.

In published studies, it is the kinetic off-rate of the TCR from theMHC/ligand complex that most often correlates with the nature ofthe T cell response (48). The kinetic off-rate of A6-TCR withTax9/HLA-A2 is 0.093 s21 (37, 42), in the range of a typicalagonist. The kinetic off-rate of the A6-TCR with Tax8/HLA-A2was too fast to measure by surface plasmon resonance, consistentwith the extremely low T cell response observed (Fig. 6,B andC).

The affinity for a Tax9 specificabTCR ectodomain for theTax8/HLA-A2 complex was measured as 16-fold lower than itsaffinity for Tax9/HLA-A2 (Fig. 6A). This affinity difference is atleast qualitatively consistent with the modeling that indicates apotential cavity in the interface. A 16-fold magnitude of affinitydifference in other TCR/peptide/MHC complexes has been ob-served to be sufficient to alter T cell responses dramatically (37,42, 49, 50). Qualitatively consistent with this expectation, we ob-served markedly reduced (.106 reduction) activity in Tax8-in-duced cell lysis of a Tax9-specific T cell line (Fig. 6B) and noantagonism of Tax9 activity at 1mM Tax8 peptide concentration(Fig. 6C). Quantitatively, however, the absolute affinity,KD 515 6 2 mM, of A6-TCR for the Tax8/HLA-A2 ligand, is in therange that often either signals as a partial agonist or an antagonist(37, 42), rather than as a null as observed here. This comparisonsuggests that the Tax8/HLA-A2 complex may have unusual prop-erties as a cell surface ligand for a T cell. Apparently the decreasedstability of the Tax8/MHC complex (Tm 5 49°C), coupled with the16-fold decrease in TCR affinity, combine somehow to decreasethe effectiveness of the Tax8/MHC complex as a ligand in cellularassays (since neither alone prevents either partial agonist or an-tagonist responses).

AcknowledgmentsWe thank R. Crouse, A. Haykov, and C. Garnett for technical support.

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