Supporting Information - PNAS...2015/01/19 · Supporting Information Garstka et al....

Supporting InformationGarstka et al. 10.1073/pnas.1416543112SI Experimental ProceduresCell Lines and Antibodies. RMA (murine mutagenized Rauschervirus-induced T lymphoma) and TAP-deficient RMA-S cells (1)were maintained in RPMI1640 medium (Gibco), supplementedwith 8% FBS (Sigma) and penicillin–streptomycin (Gibco) at37 °C and 5% CO2.Mouse monoclonal Y3, specific for native H-2Kb molecules in

complex with β2m (2), and anti-exon8 antiserum, recognizing thecytoplasmic tail of all forms of H-2Kb molecules (3), were used.As secondary antibodies Alexa Fluor 647 goat anti-mouse IgG(Invitrogen) and IRDye 800CW goat anti-rabbit IgG (OdysseyInfrared Imaging Systems; LI-COR) were used.

Peptide Synthesis. Peptide FAPGNYPAL and its derivatives(Table S1) were synthesized (by solid-phase peptide synthesis,preloadedWang resin) on a SYRO II robot using Fmoc chemistrywith PyBop and Dipea as activator and base. Depending on theapplication, three types of modified peptides were synthesized:unlabeled peptides (for thermal stability and crystallizationpurposes), peptides coupled to biotin (for peptide binding in celllysates), and peptides coupled to the TAMRA fluorophore (forfluorescence polarization measurements). For biotin or TAMRAcoupling Gly in the peptide sequence was replaced with Lys(resulting in peptide FAPKNYPAL). For the synthesis, Fmoc-K(Mtt)-OH was used. In the fully protected peptide, the Mttgroup was selectively removed (using dichloromethane, 1%TFA, and 2% Triisopropylsilane), followed by coupling of biotinor TAMRA to the e-amino group of K using standard proce-dures. To promote binding of biotinylated peptides to bothH-2Kb and streptavidin e-amino caproic acid was introduced be-tween Lys and biotin.The following modifications of the FAPKNYPAL model

peptide were made: introduction of different amino acids, re-moval of terminal amino acids, extensions of the peptide, acet-ylation of peptide N terminus (using acetic anhydride), amidationof peptide C terminus (using a PAL-PEG-PS Wang resin), andremoval of one or both charges at the peptide C terminus (ex-change of carboxyl group with leucinol and isopentyl amine,respectively). For synthesis of C-terminal peptide alcohol, Fmoc-leucinol was preloaded on the resin prior Fmoc solid-phasepeptide synthesis (SPPS). For synthesis of C-terminal N-alkylamide, Boc-FAPK(ivDde)NYPA was synthesized using FmocSPPS. After removing the ivDde-protecting group from the lysineside chain and cleavage, the otherwise protected peptide wascoupled in solution to isopentyl amine. Finally, the remainingprotecting groups were removed to obtain a C-terminal alkylatedpeptide.Peptides were further purified by reversed-phase HPLC using

a water/acetonitrile gradient and their composition was con-firmed by mass spectrometry.

Detection of Peptide Binding in Lysates. RMA and RMA-S cellswere cultured at 26 °C or 37 °C for 16 h. One million cells percondition were lysed in 0.5% 3-[(3-cholamidopropyl)dimethy-lammonio]-1-propanesulfonate in PBS (containing protease in-hibitor mixture; Sigma). For detection of H-2Kb moleculesbound to endogenous peptides, immunoisolation with Y3 anti-body (specific for peptide H-2Kb) prebound to protein G beadswas performed for 1 h at 4 °C. Y3-reactive H-2Kb moleculeswere separated by SDS/PAGE, transferred to nitrocellulosemembranes, and detected by immunoblotting with anti-exon8antibodies.

For binding of exogenous peptides to H-2Kb molecules lysatesof RMA-S cells were incubated with 2 μM biotinylated peptidesfor 30 min at 4 °C, and associated H-2Kb molecules were isolatedby streptavidin beads, separated by SDS/PAGE, transferred tonitrocellulose membranes, and detected using anti-exon8 anti-bodies. Both exon8-specific bands were quantified (discussedbelow) and the amount of H-2Kb bound to biotinylated peptideswas expressed as percentage of total H-2Kb in cell lysates, assimilarly detected using exon8 antibody.

Western Blot. Proteins were separated by a polyacrylamide gel[10% (vol/vol)] and transferred to a 0.45-μm nitrocellulosemembrane. Membranes were blocked with 5% milk in PBS for1 h and incubated with anti-exon8 antibody overnight at 4 °C inPBST with 2% milk. After six washes with PBST, membraneswere incubated with solution containing LI-COR Odyssey IRDye800CW antibody for 1 h at room temperature in the dark, fol-lowed by six washes with PBST. Membranes were scanned usinga LI-COR Odyssey Infrared Imaging System (Biosciences) andanalyzed using Odyssey LI-COR software package version 3.0.Data were represented as an average with the SD:

μ: average value μ= 1NPn

i=1xn

δ: SD δ=ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPn

i=1ðxi − μÞ2q

:

Protein Expression and Purification. The extracellular portion ofH-2Kb heavy chain and mouse β2 microglobulin were produced,purified, and refolded according to published protocols (4).Briefly, proteins were produced in Escherichia coli Bl21(DE3)pLysS from pET3a plasmid under T7 promoter. Protein ex-pression was induced by 0.4 mM isopropyl-beta-D-thiogalactopyr-anoside for 4 h before harvesting and lysis in 50 mM Tris·HCl(pH 8.0), 25% sucrose, and 1 mM EDTA. Inclusion bodies wereharvested by centrifugation and washed with 20 mM Tris(pH 8.0) and 100 mM NaCl. Next, inclusion bodies were dis-solved in 8 M urea and 20 mM Tris·HCl (pH 8.0) and insolublecontaminants were removed by centrifugation at 16,000 × g for2 min. The soluble fraction was used for refolding reactions ei-ther immediately or after storage at –80 °C. To refold H-2Kb

with β2 microglobulin in the presence of peptide, peptide wasdissolved in 20 mM Tris·HCl (pH 8.0) to a final concentration of8 mM. Refolding was performed with H-2Kb heavy chain, β2microglobulin, and peptide at a molar ratio of 1:1:8 in 4 M ureaand 20 mM Tris·HCl (pH 8.0) containing protease inhibitors(complete EDTA-free protease inhibitor mixture; Roche). Therefolding mixture was dialyzed (dialysis tubing MWCO500)against 10 mM potassium phosphate (pH 7.4) at 4 °C for 48 hwith an exchange of dialysis buffer after 24 h. Refolded com-plexes were purified using ion exchange (6 mL ResourceQ column) and size-exclusion chromatography (Superdex 20016/60 column) by an AKTA system (GE Healthcare Life Sci-ence). Properly folded complexes were concentrated usingAmicon Ultra-15 with 3-kDa cutoff (Merck Millipore) to a con-centration between 4.2 and 20 mg/mL, and aliquots were storedat –80 °C until further use.

Protein Unfolding and Aggregation. Thermal unfolding and ag-gregation of different H-2Kb–peptide complexes was determinedusing the Optim 1000 (Avacta Analytical). H-2Kb–peptidecomplexes were measured in 150 mM NaCl and 20 mM Tris·HCl(pH 7.5) buffer with protein concentrations of 0.2, 0.5, and1.0 mg/mL. Samples were heated using 1 °C step gradient with

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30-s temperature stabilization for each step. Tryptophans wereexcited at 266 and fluorescence was measured at a range from300 to 400 nm. Barycentric fluorescence was determined ac-cording to the equation:

BCMλ=P

IðλÞ× λPIðλÞ ;

where BCM λ is the barycentric mean fluorescence in nano-meters, I(λ) is the fluorescence intensity at a given wavelength,and λ is the wavelength in nanometers.The melting temperature (Tm) was calculated using bar-

ycentric fluorescence as a function of temperature according tothe equation

Tm =maxdBCMdT

�T�;

where max is the local maximum and dBCM=dTðTÞ is the firstderivative of barycentric fluorescence as a function of tempera-ture in nanometers per degree Celsius.The static light scattering signal at 473 nm was also recorded to

detect aggregate formation. Analysis was performed with OptimAnalysis Software v 2.0 (Avacta Analytical).

Fluorescence Polarization. Fluorescence polarization measure-ments were performed with refolded and purified H-2Kb–peptidecomplexes over a concentration range of 62.5 nM to 4 μM. Ex-periments were performed at 26 °C or 32 °C in 20 mM Tris·HCl(pH 7.5) and 150 mM NaCl after a 10-min preincubation ofH-2Kb–peptide complexes. TAMRA-labeled peptide was auto-matically injected at 2 nM final concentration and the reactionwas mixed and fluorescence polarization was recorded over time.Kinetic measurements were performed using PHERAstar (BMGLABTECH). Excitation and emission wavelengths were 555 and576 nm, respectively. Data were processed with GraphPad soft-ware using this user-defined equation:

FP=Bmax× ½A�½A�+ koff

�kon

×�1− expð−kon×½A�+koff Þt

�;

where FP is fluorescence polarization (milli-polarization), Bmaxis the FP signal when all of the labeled peptide is bound (milli-polarization), [A] is the concentration of H-2Kb complexeswith an unlabeled peptide (molar), kon is the association con-stant, on-rate [1/(mol × second)], koff is the dissociation constant,off-rate ð1=secondÞ, and t is time (seconds).Binding of the weak binders (Table S3) was fitted to a linear

regression equation of one phase association:

Kobs = kon × ½A�+ koff ;

where Kobs was estimated fitting the initial binding curves toexponential function

FP=FPo + �FPmax −FPo

�p�1− e−Kobs×t

�;

where FP is fluorescence polarization (milli-polarization), FP0 isthe initial signal (milli-polarization), FPmax is the maximal signal(milli-polarization), and t is time (seconds).

X-Ray Crystallization. Crystals of H-2Kb in complex with peptideswere grown using the sitting drop method at 277 K or 293 K.Purified protein (>5 mg/mL) was mixed with reservoir solu-tion containing sodium/potassium phosphate buffer (1.8–2.0 M,pH 6.5) and 2-methyl-2,4-pentanediol (0.5–2%) at a protein:reservoir solution ratio of 1:1 and 1:2. Crystals were generally

obtained within 2 wk and were harvested and incubated in res-ervoir solution supplemented with 20% glycerol before vitrifi-cation in liquid nitrogen at 100 K.Diffraction data were collected at the Swiss Light Source,

Villigen, Switzerland, at beamline X06DA (H-2Kb in complexwith peptide FAPGNYPAL) and at the European SynchrotronRadiation Facility, Grenoble, France at beamlines ID23-I(H-2Kb in complex with peptide FAPGNYPAF and FAPGNY-PAW) and ID23-II (H-2Kb in complex with FAPGNWPAL andFAPGN¥PAL).For each MHC–peptide complex, X-ray diffraction data were

collected and then integrated with Mosflm (CCP4 1994) or XDS(5) and scaled using Scala (6, 7) as implemented in the CCP4suite (8). An initial solution for the dataset was obtained bymolecular replacement with Phaser (9–11). A crystal structure ofH-2Kb in complex with an ovalbumin peptide (PDB ID code3P9L) (12) and a structure of H-2Kb bound to a peptide from theSendai virus nucleoprotein (PDB ID code 2VAB) (13) were usedas search models for molecular replacement. The initial so-lution was rigid-body refined with Refmac (14) and rebuiltmanually with Coot (15), followed by replacement of the nec-essary residues within the peptide with the correct amino acids.PDB_REDO (16) was used to rebuild the model and to optimizerefinement parameters for Refmac (14). Simple translation, li-bration, and screw-rotation (TLS) (17) models of one group perchain (or H-2Kb and the peptide combined) were refined, to-gether with individual, isotropic B-factors. For H-2Kb in a com-plex with FAPGNWPAL, TLS plus a single overall B-factorwas used.Several rounds of manual rebuilding in Coot (15), guided by

validation reports from WHAT_CHECK (18) and MolProbity(19) and followed by refinement in Refmac, were used to com-plete the models. During this process, a helical fragment wasdiscovered in the electron density of H-2Kb in a complex withFAPGN¥PAL, which was modeled as unknown residues. Anadditional model was created by taking the 1.7-Å resolutionstructure of H-2Kb/VSV8 (PDB ID code 1KPV) from thePDB_REDO databank (20) and following the procedure as de-scribed above. In addition, occupancies of the two Tyr116 con-formations were refined using Refmac.To assess the effect of peptide binding on the dimensions of

the peptide-binding groove, the coordinates ESU from Refmacbased on the maximum likelihood approach were determinedand used for the calculations of distances between two relevantamino acids.

Error model : sðA−BÞ2 = sðAÞ2 + sðBÞ2;

where sðA−BÞ= ffiffiffi2p × sðAÞ:Model ensembles were created using ensemble refinement in

Phenix using default settings (21). The structure models wereanalyzed in YASARA (22).Data collection and refinement statistics are provided in Table

S4. Graphics were generated with PyMOL (PyMOL MolecularGraphics System; Schrödinger) and CCP4MG (23).

Mass Spectrometry Analysis.Sample preparation. RMA cells were used as sources of H-2Kb

molecules. Cells (1010) were expanded in roller bottles usingDMEM supplemented with 8% FCS, penicillin/streptomycin,and L-glutamine. Cells were cultured at 26 °C or 37 °C for 72 h.Cells were washed twice with ice-cold PBS and stored as a pelletat −80 °C until use.The Y3 hybridoma cell line was expanded in roller bottles

to obtain Y3 (anti–H-2Kb–peptide complex) antibody usingDMEM supplemented with 1% of ultra-low IgG FBS (Gibco),glutamine, β-mercaptoethanol (2.3 mg/mL), sodium selenite



(4.5 × 10−4 mg/mL), and penicillin/steptomycin. Y3 hybridomasupernatant was first concentrated using an artificial kidney, andY3 antibodies were purified using an ABx column (BakerbondABx, 7269−00; J. T. Baker) by HPLC. Antibodies were elutedby an ammoniumsulfate gradient. Y3-containing fractions werepooled and concentrated with a 100-KDa-cutoff filter and washedthoroughly with PBS.Y3 antibody was used to produce an immunoaffinity column

(Y3–Prot-A Sepharose 2.5 mg/mL). The Y3 antibody was co-valently bound to Prot-A Sepharose beads using dimethylpime-limidate. The columns were stored in PBS pH 8.0 and 0.02%NaN3 at 4 °C.Isolation of the H-2Kb–associated peptidome. Extraction of peptidesassociated to MHC class I molecules was performed as describedelsewhere (24, 25). Briefly, pellets from RMA cells were lysed in50 mM Tris·HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, and0.5% Zwittergent 3-12 and supplemented with complete pro-tease inhibitor (Sigma-Aldrich) to a final concentration of 0.1 ×109 cells/mL.After 2-h lysis at 4 °C, preparation was centrifuged for 10 min

at 1,450 × g at 4 °C. Supernatant was transferred to a new tubeand centrifuged for 45 min at 39,000 × g and 4 °C, preclearedwith CL4B beads, and subjected to the antibody Y3 immuno-affinity column at a flow rate of 2.5 mL/min. After washing,bound H-2Kb–peptide complexes were eluted from the columnand dissociated with 10% acetic acid. Peptides were separatedfrom the H-2Kb molecules via passage through a 10-kDa-cutoffmembrane (macrosep centrifuge devices; Pall). The filtrate waslyophilized, and peptides were further purified by solid phaseextraction (C18 Oasis, 100-μL bed volume; Waters). Peptideswere eluted from the C18 Oasis column with 500 μL 50/50/0.1water/acetonitrile (ACN)/formic acid (vol/vol/vol) and againlyophilized.Peptide separation. Peptide pools were fractionated using a SCXcolumn (320-μm inner diameter, 15 cm, polysulfoethyl A 3 μm,Poly LC) run at 4 μL/min. Gradients were run for 10 min at100% solvent A (0.1% TFA in H2O), after which a linear gra-dient started to reach 100% solvent B (250 mM KCl and 35%ACN/0.1% TFA) over 15 min, followed by 100% solvent C(500 mM KCl and 35% ACN/0.1% TFA) over the next 15 min.The gradient remained at 100% solvent C for 5 min and thenswitched again to 100% solvent A. Twenty 4-μL fractions were

collected in vials prefilled with 100 μL 95/3/0.1(vol/vol/vol)H2O/ACN/TFA.Liquid chromatography-MS/MS analysis. The dissolved fractions wereanalyzed by online nano-HPLC-MS using a conventional Agilent1100 gradient HPLC system (Agilent), as described (26), coupledto a Q-Exactive mass spectrometer (Thermo). Fractions wereinjected onto a homemade pre-column (100 μm × 15 mm;Reprosil-Pur C18-AQ 3 μm; Dr. Maisch) and eluted via a home-made analytical nano-HPLC column (15 cm × 50 μm; Reprosil-Pur C18-AQ 3 μm). The gradient was run from 0 to 50% solventB [10/90/0.1 H2O/ACN/TFA (vol/vol/vol)] over 30 min. Thenano-HPLC column was drawn to a tip of ∼5 μm and acted asthe electrospray needle of the MS source. The Fourier transformmass spectrometer (FTMS) was operated in data-dependentmode, automatically switching between MS and MS/MS acqui-sition. Full scan mass spectra were acquired in a Fourier transformion cyclotron resonance mass spectrometer with a resolution of25,000 at a target value of 5 million. The two most intense ions werethen isolated for accurate mass measurements by a selected ion-monitoring scan in Fourier transform ion cyclotron resonance witha resolution of 50,000 at a target accumulation value of 50,000.Selected ions were fragmented in the linear ion trap using collision-induced dissociation at a target value of 10,000. The Q-Exactivemass spectrometer was operated in top10 mode. Parameters wereresolution 70,000 at an AGC target value of 3 million, maximum filltime of 100 ms (full scan), and resolution 35,000 at an AGC targetvalue of 1 million/maximum fill time of 128 ms for MS/MS at anintensity threshold of 800,000. Apex trigger was set to 1–5 s andallowed charges were 1, 2, and 3. In a postanalysis process, raw datawere converted to peak lists using Proteome Discoverer 1.4.

Data Analysis. The tandem mass spectra were matched against theIPI mouse database (version 3.87) using theMascot search engine(version 2.2.04; Matrix Science) with a precursor mass toleranceof 2 ppm, with methionine oxidation and cysteinylation, asa variable modification and a product ion tolerance of 0.5 Da (forthe FTMS data) and 10 ppm and 20 mmu (for the Q-Exactivedata). Scaffold software, version 4, was subsequently used toprocess the Mascot output files and generate spectrum reports.Duplicates were removed, and peptides that had a best Mascotion score of ≥35 and were 8–14 aa long were selected for theproduction of Dataset S1.

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2. Jones B, Janeway CA, Jr (1981) Cooperative interaction of B lymphocytes with anti-gen-specific helper T lymphocytes is MHC restricted. Nature 292(5823):547–549.

3. Smith RN, Amsden A, Sudilovsky O, Coleman N, Margolies R (1986) The alloantibodyresponse in the allogeneically pregnant rat. IV. Analysis of the alloantibody specific-ities with monoclonal antibodies. J Immunol 136(11):4063–4069.

4. Rodenko B, et al. (2006) Generation of peptide-MHC class I complexes throughUV-mediated ligand exchange. Nat Protoc 1(3):1120–1132.

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7. Evans PR (2011) An introduction to data reduction: Space-group determination,scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr 67(Pt 4):282–292.

8. Collaborative Computational Project, Number 4 (1994) The CCP4 suite: Programs forprotein crystallography. Acta Crystallogr D Biol Crystallogr 50(Pt 5):760–763.

9. McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ (2005) Likelihood-enhanced fasttranslation functions. Acta Crystallogr D Biol Crystallogr 61(Pt 4):458–464.

10. Read RJ (2001) Pushing the boundaries of molecular replacement with maximumlikelihood. Acta Crystallogr D Biol Crystallogr 57(Pt 10):1373–1382.

11. Storoni LC, McCoy AJ, Read RJ (2004) Likelihood-enhanced fast rotation functions.Acta Crystallogr D Biol Crystallogr 60(Pt 3):432–438.

12. Denton AE, et al. (2011) Affinity thresholds for naive CD8+ CTL activation by peptidesand engineered influenza A viruses. J Immunol 187(11):5733–5744.

13. Fremont DH, Matsumura M, Stura EA, Peterson PA, Wilson IA (1992) Crystal structures oftwo viral peptides in complex with murine MHC class I H-2Kb. Science 257(5072):919–927.

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15. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot.Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501.

16. Joosten RP, Joosten K, Cohen SX, Vriend G, Perrakis A (2011) Automatic rebuildingand optimization of crystallographic structures in the Protein Data Bank. Bio-informatics 27(24):3392–3398.

17. Schomaker V, Trueblood KN (1968) On the rigid-body motion of molecules in crystals.Acta Crystallogr B 24(1):63–76.

18. Hooft RW, Vriend G, Sander C, Abola EE (1996) Errors in protein structures. Nature381(6580):272.

19. Davis IW, et al. (2007) MolProbity: All-atom contacts and structure validation forproteins and nucleic acids. Nucleic Acids Res 35(web server issue):W375-83.

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26. Meiring MS, et al. (2002) In vitro effect of a thrombin inhibition peptide selected byphage display technology. Thromb Res 107(6):365–371.



C

20 30 40 50 60

0.2

0.5

1.0

Con

c. (

mg/

ml)

Temperature ( C)o

Melting Temperature

B Unfolding

dBC

M/dT

(nm/ C

)

o

BC

M (

nm)

354

352

350

348

346

FAPGNYPAL0.2 mg/ml

354

352

350

348

346

354

352

350

348

346

0.20

0.15

0.10

0.05

020 30 40 50 60

20 30 40 50 60

20 30 40 50 60

0.20

0.15

0.10

0.05

0

0.20

0.15

0.10

0.05

0

Temperature ( C)o

FAPGNYPAL0.5 mg/ml

FAPGNYPAL1.0 mg/ml

A Aggregation

0.2 mg/ml0.5 mg/ml1.0 mg/ml

20 30 40 50 60 70 80 90

473

nm

sca

tter

(10

)4

2

1

3

4

5

0

Temperature ( C)o

Fig. S1. (Continued)



UnfoldingD

Fig. S1. H-2Kb–peptide complexes unfolding undergoes in two steps. (A–C) The H-2Kb–FAPGNYPAL complex at different concentrations (0.2, 0.5, and 1.0 mg/mL) was used for the optimization of the measurements of temperature-induced unfolding and aggregation using the Optim apparatus. (A) Aggregationrelates to protein concentration; 473-nm light scattering intensity was used to measure temperature-induced aggregation of H-2Kb–FAPGNYPAL complexes.Aggregation will induce light scattering. Protein concentrations used were 0.2, 0.5, and 1.0 mg/mL. (B) Thermal unfolding by Trp fluorescence. Thermal de-naturation of H-2Kb–FAPGNYPAL complexes measured by barycentric mean fluorescence (BCM, in black). Fit to first derivate of BCM (in red) shows meltingtemperature, Tm, as a maximum. (C) Quantification of two independent experiments from B represented as average ± SD. (D) Thermal denaturation of H-2K

b

complexed to different peptides measured by BCM. BCM (in black) plotted together with its fit to the first derivative (in red) that allows to determine meltingtemperature, Tm. Note that the different H-2Kb peptide complexes all show one fluorescence alteration around 26 °C and a second at higher and variabletemperatures dependent on the associated peptide.



Fig. S2. Binding of peptides with modified anchor residues to H-2Kb. (A and B) kon and koff values of 10 peptides with charged anchor residues or alteredsecondary anchors. The FP measurements were all performed at 26 °C. Shown are average values of at least two independent experiments ± SD. ND, nobinding resolved at the highest concentration of H-2Kb (4 μM). (C) Representation of peptide modifications applied: N-terminal acetylation, C- terminalamidation, and exchange of C-terminal leucine to leucinol or isopeptylamine.



Fig. S3. Peptides with extended anchor residue side chains fill corresponding pockets in the peptide-binding groove of H-2Kb. (A) Structure of H-2Kb boundwith FAPGNYPAL, heavy chain in cyan, β2 microglobulin in orange, and peptide in yellow. (B) Top view on peptide-binding groove with bound FAPGNYPAL,ribbon representation of H-2Kb, and stick of peptide. (C and D) The diiodo-tyrosine anchor residue in peptide FAPGN¥PAL widens the peptide-binding groove.Width of the peptide-binding groove was represented as four distances, Lys66 (Nζ atom) to Tyr159 (OH atom), Asn70 (Cγ atom) to Arf155 (Cδ atom), Ser73 (Cβatom) to GLu152 (Cδ atom), and Asp77 (Cγ atom) to Lys146 (Ce atom) with graphical representation (C) and quantification with error from Refmac based on themaximum likelihood approach (D).



Fig. S4. Increase in the size of the C-terminal residue of the peptide decreases flexibility of Tyr116 of H-2Kb peptide-binding groove. (A) Quantification ofdistances Tyr116–Gln114 (distance represented in Fig. 4A by blue line), Tyr116–Tyr123 (orange line), and Tyr116–Ile124 (green line) on the basis of ensemblerefinement indicates decreased flexibility of the F pocket. (B) The calculated torsion angles of gatekeeper amino acid Tyr116 (in Fig. 4B) were plotted for thedifferent H-2Kb complexes and peptides, as indicated, illustrating the restrained mobility for expanded anchor residues in the F pocket of H-2Kb.



Table S1. Peptides used in this study and descriptions of their modifications

No. Sequence Modification

1 FAPGNYPAL2 FAPKNYPAL Biotin attachment to K, TAMRA attachment to K3 FAPGNYPAW4 FAPKNYPAW Biotin attachment to K, TAMRA attachment to K5 FAPGNYPAF6 FAPKNYPAF Biotin attachment to K, TAMRA attachment to K7 FAPGNYPAA8 FAPKNYPAA Biotin attachment to K, TAMRA attachment to K9 FAPGNWPAL10 FAPKNWPAL Biotin attachment to K, TAMRA attachment to K11 FAPGNAPAL12 FAPKNAPAL Biotin attachment to K, TAMRA attachment to K13 FAPGN¥PAL14 FAPKN¥PAL Biotin attachment to K, TAMRA attachment to K15 FAPGNWPAW16 FAPKNWPAW Biotin attachment to K, TAMRA attachment to K17 FAPKNAPAA Biotin attachment to K, TAMRA attachment to K18 FAPKNLPAL Biotin attachment to K19 FAPKNFPAL Biotin attachment to K20 FAPKNYPAD TAMRA attachment to K21 FAPKNYPAR TAMRA attachment to K22 FAPKNDPAL TAMRA attachment to K23 FAPKNRPAL TAMRA attachment to K24 FAPKAAAAL TAMRA attachment to K25 FAAKAAAAL TAMRA attachment to K26 FAAKAYAAA TAMRA attachment to K27 AAPKAAAAL TAMRA attachment to K28 AAPKAYAAA TAMRA attachment to K29 AAAKAYAAL TAMRA attachment to K30 -APKNYPAL TAMRA attachment to K31 FAPKNYPA- TAMRA attachment to K32 *FAPKNYPAL *Acetylation, TAMRA attachment to K33 FAPKNYPAL# # Amidation, TAMRA attachment to K34 *FAPKNYPAL# *Acetylation, # amidation, TAMRA attachment to K35 FAPKNYPA& & Leucinol, TAMRA attachment to K36 FAPKNYPA^ ^ Isopentyl amine, TAMRA attachment to K37 AAAFAPGNYPAL TAMRA attachment to K38 HGEFAPGNYPAL TAMRA attachment to K39 FAPGNYPALWSY TAMRA attachment to K40 FAPGNYPALAAA TAMRA attachment to K

Peptides used are derivatives of FAPGNYPAL, modified at anchor positions, termini, and/or in length (in-dicated in bold). For the attachment of either biotin or TAMRA, glycine in the peptide was substituted to lysine(resulting in peptide FAPKNYPAL) and the corresponding label was attached to the side chain of Lys.

Table S2. Refolding efficiency of H-2Kb peptide complexes usedin this study

Peptide Refolding efficiency, %

FAPGNYPAL 4.2FAPGNWPAL 4.1FAPGNYPAW 2.3FAPGN¥PAL 3.5FAPGNYPAF 3.7FAPGNWPAW 2.4FAPGNYPAA 2.6FAPGNAPAL 2.8

Refolding efficiency represented as a percentage of the isolated andproperly folded H-2Kb–peptide complex related to input free heavy chain(from inclusion bodies). The introduction of nonclassical anchor residues didnot alter refolding efficiencies in vitro. ¥, diiodo-Tyr.



Table S3. Kinetic values of peptides binding to H-2Kb

Peptide name Temperature, °C kon ×103, M−1 × s−1 koff ×10

−3, s−1 Kd, nM

AnchorsSize

FAPKNYPAL 26 31.0 ± 0.3 0.5 ± 0.1 16 ± 232 25.6 ± 0.1 2.7 ± 0.0 108 ± 2

FAPKNYPAA 26 27.7 ± 0.2 0.5 ± 0.1 18 ± 332 14.4 ± 0.0 2.1 ± 0.0 144 ± 4

FAPKNYPAF 26 25.1 ± 0.2 3.0 ± 0.1 119 ± 432 25.1 ± 0.3 3.1 ± 0.1 123 ± 4

FAPKNYPAW 26 33.1 ± 0.3 0.5 ± 0.1 17 ± 332 22.8 ± 0.2 3.5 ± 0.0 152 ± 4

FAPKN¥PAL 26 16.3 ± 3.6 21.4 ± 4.8 1,311 ± 41332 ND ND —

FAPKNWPAL 26 57.2 ± 0.4 1.9 ± 0.1 33 ± 132 40.4 ± 0.2 1.3 ± 0.0 31 ± 2

FAPKNAPAL 26 ND ND —32 ND ND —

FAPKNWPAW 26 ND ND —32 ND ND —

FAPKNAPAA 26 ND ND —32 ND ND —

ChargeFAPKNRPAL 26 54.4 ± 0.9 29.5 ± 0.5 542 ± 13

32 51.1 ± 0.8 18.2 ± 0.4 355 ± 6FAPKNYPAR 26 ND ND —

32 ND ND —FAPKNDPAL 26 ND ND —

32 ND ND —FAPKNYPAD 26 ND ND —

32 ND ND —Anchors only

FAPKAAAAL 26 30.7 ± 0.3 8.5 ± 0.1 279 ± 532 28.7 ± 4.0 16.9 ± 9.6 588 ± 8

FAAKAAAAL 26 ND ND —32 ND ND —

FAAKAYAAA 26 ND ND —32 ND ND —

AAPKAAAAL 26 44.2 ± 2.2 13.7 ± 0.6 310 ± 2132 13.7 ± 2.1 30.8 ± 4.8 2,254 ± 34

AAPKAYAAA 26 34.1 ± 3.8 31.3 ± 5.7 917 ± 19632 9.9 ± 1.6 25.9 ± 4.0 2,610 ± 411

AAAKAYAAL 26 38.4 ± 0.4 6.7 ± 0.1 1,74 ± 332 13.9 ± 0.3 16.7 ± 0.8 1,201 ± 28

TerminiMissing

-APKNYPAL 26 3.0 ± 0.5 7.4 ± 0.8 2,442 ± 47432 ND ND —

FAPKNYPA- 26 ND ND —32 ND ND —

Removed charge*FAPKNYPAL 26 4.3 ± 0.6 15.0 ± 1.3 3,478 ± 576

32 ND ND —FAPKNYPAL# 26 37.8 ± 0.3 0.3 ± 0.1 8 ± 2

32 14.6 ± 0.3 7.8 ± 0.3 531 ± 10*FAPKNYPAL# 26 ND ND —

32 ND ND —FAPKNYPA& 26 29.7 ± 0.2 1.2 ± 0.1 39 ± 2

32 5.9 ± 2.4 20.6 ± 5.6 3,482 ± 1,440FAPKNYPA^ 26 15.2 ± 4.9 34.2 ± 4.5 2,242 ± 786

32 13.0 ± 2.4 43.9 ± 3.5 3,388 ± 702Extended

AAAFAPKNYPAL 26 3.2 ± 0.5 11.1 ± 0.7 3,454 ± 64732 5.5 ± 2.6 14.5 ± 3.4 2,788 ± 783

HGEFAPKNYPAL 26 3.7 ± 0.2 10.6 ± 0.4 2,865 ± 19632 ND ND —



Table S3. Cont.

Peptide name Temperature, °C kon ×103, M−1 × s−1 koff ×10

−3, s−1 Kd, nM

FAPKNYPALWSY 26 ND ND —32 ND ND —

FAPKNYPALAAA 26 4.7 ± 1.4 17.6 ± 2.8 3,716 ± 1,26532 10.7 ± 2.4 13.6 ± 3.2 1,268 ± 284

kon, koff, and Kd values ± SD from at least two independent experiments. ND, no binding resolved at thehighest concentration of H-2Kb (4 μM). Modifications: ¥, di-iodo-Tyr; *Acetylation, # amidation, & leucinol, and^ Isopentylamine.

Table S4. X-ray data collection and refinement statistics

Protein MHC I: H-2Kb 4PG9 MHC I: H2Kb 4PGB MHC I: H2Kb 4PGC MHC I: H2Kb 4PGD MHC I: H2Kb 4PGE

Peptide FAPGNYPAL FAPGNWPAL FAPGN¥PAL FAPGNYPAF FAPGNYPAWData collectionSpace group P21212 P21 P21 P21212 P21212Cell dimensions

a, b, c, Å 87.72, 136.53, 45.20 65.55, 89.26, 88.46 66.18, 90.30, 89.37 88.05, 137.03, 45.18 87.93, 136.67, 45.23α, β, γ, ° 90, 90, 90 90, 110.67, 90 90, 111.58, 90 90, 90, 90 90, 90, 90

Resolution, Å(high-resolution shell)

40–2.40 (2.53–2.40) 45–2.80 (2.95–2.80) 51–2.30 (2.42–2.30) 46–2.70 (2.85–2.70) 44–2.00 (2.11–2.00)

Rmerge, % 12.7 (48.8) 6.8 (38.6) 7.7 (52.9) 13.9 (55.0) 10.6 (62.3)Average I/σI 8.3 (2.6) 10.4 (2.3) 12.0 (2.2) 10.6 (3.8) 9.1 (2.3)Multiplicity 3.8 (3.8) 2.5 (2.5) 3.5 (3.5) 3.8 (3.9) 3.8 (3.9)Completeness (%) 98.3 (98.9) 98.0 (95.8) 99.5 (99.4) 99.7 (99.6) 99.7 (99.6)RefinementResolution, Å 39–2.40 45–2.80 51–2.30 43–2.70 42–2.00No. of reflections 20,335 21,992 41,159 14,832 35,766Rwork/Rfree 21.0/25.0 25.8/28.1 21.2/24.3 21.0/25.9 20.1/23.5No. of atoms

Protein 3,149 6,328 6,388 3,123 3,161Water 52 0 57 15 104Other 60 0 36 6 52

B-factors, Å2

All 27 51 40 30 28MHC 27 51 40 31 28β2m 26 51 36 26 27Peptide 25 78 62 23 24Other 31 N/A 49 28 33

ValidationrmsZ/rmsd†

Bond lengths 0.245/0.005 0.197/0.004 0.319/0.007 0.219/0.004 0.252/0.005Bond angles 0.443/1.010 0.358/0.859 0.507/1.171 0.399/0.926 0.459/1.053

Ramachandran plotFavored,‡ % 97 97 98 98 99Outliers,‡ % 0.0 0.4 0.3 0.0 0.0

Clash score (percentile)‡ 2.06 (100) 2.26 (100) 1.28 (100) 0.82 (100) 0.63 (100)MolProbity score (percentile)‡ 1.16 (100) 1.28 (100) 0.98 (100) 0.78 (100) 0.84 (100)

Single crystals were used to collect data for each of the structures. ¥, diiodo-Tyr; N/A, not applicable.†As given by Refmac for all bonds and angles.‡As given by MolProbity.



Table S5. Interactions of peptide with H-2Kb

No. Peptide Total contacts Hydrogen bondsHydrophobicinteractions π–π bonds Cation–π bonds

1 FAPGNYPAL 76 15 (4) 54 2 52 FAPGN¥PAL 61 9 (1) 44 2 63 FAPGNWPAL 85 11 (0) 61 7 64 FAPGNYPAF 101 15 (3) 69 12 55 FAPGNYPAW 103 20 (9) 68 9 6

Water-mediated bonds are given in parentheses. Contacts between peptide atoms and H-2Kb residues in the respective crystalstructures: total contacts, hydrogen bonds, hydrophobic, π–π, and cation–π interaction for FAPGNYPAL, FAPGN¥PAL, FAPGNWPAL,FAPGNYPAF, and FAPGNYPAW. ¥, diiodo-Tyr.

Other Supporting Information Files

Dataset S1 (XLSX)

http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416543112/-/DCSupplemental/pnas.1416543112.sd01.xlsxwww.pnas.org/cgi/content/short/1416543112

Supporting Information - PNAS...2015/01/19 · Supporting Information Garstka et al....

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