Downloaded by guest on October 1, 2021 immunity conformational the a into recruitment through MHC-I facilitates complex. by that edit- a mechanism affected peptide dynam- selection in also conformational in are groove involved groove. ics loop binding binding the MHC- tapasin toward MHC-I a The central ing steers the MHC-I. a antigen-loaded stabilizing plays I–linked of Tapasin PLC, reminiscent core. with the surround- conformation active structure, belt in catalytically layered protein role stable, a flexible (1.6 a has a environment forming PLC ing water modules The and editing simu- total). bilayer two (MD) PLC in lipid dynamics atoms the multimicrosec- explicit molecular million an of the all-atom in on using model lations dynamics scale atomistic time conformational an ond its report membrane-bound study com- large we and cryo-electron and this complex, low-resolution healthy of recent between a (cryo-EM) distinguish on microscopy to Based cells cells. that adaptive promised antigens T human processing the killer for of responsible allow peptide- cornerstone being a (MHC-I) system, is immune 2020) class-I 9, (PLC) March complex complex review for loading (received histocompatibility 2020 14, major July approved The and CT, Haven, New University, Yale Cresswell, Peter by Edited and J Forschungszentrum a Fisette Olivier MHC-I human complex the peptide-loading of dynamics and structure Atomistic ieo h ope n h rnin aueo h interac- the large the www.pnas.org/cgi/doi/10.1073/pnas.2004445117 of impres- of an nature represents light transient study In this the 1). components, and (Fig. its was complex between PLC study tions the the this of of in module size single PLC a full on the 9.9 for only obtained The (5). resolution (cryo-EM) map microscopy cryo-electron single-particle by high-affinity of selection editing). catalytic peptide (or and peptides process, antigenic dur- stabilization loading MHC-I the ER, ing the to (ER) molecular transport single antigen reticulum a machine: sev- into endoplasmic integrates functions antigen-processing the assembly MHC-I eral macromolecular to large Anchored requires this 5). MHC-I membrane, (PLC) for (4, complex affinity 1) peptide-loading high (Fig. the a machinery, find have sophisticated to that products a degradation peptides cytosolic few of the pool vast the Triaging receptors cell T apoptosis. their via induce complexes and (pMHC-I) peptide–MHC-I the exposed. are in both mutated, or CD8 mutation, exposed; viral Patrolling cancer-causing either are peptides, or “self” “nonself” virus and the contents: self a from cellular by peptides compromised mirror only cells therefore at cells, MHC-I exposed healthy from by resulting In Peptides surface acids) proteins. the cell amino intracellular for 12 the of are, to antigens degradation (8 These peptides the (1–3). short by cells part, cells T for most of killer proteins surface by (MHC-I) the class-I recognition at complex exposed histocompatibility are major antigens whereby anism T simulations dynamics molecular hoeia hmsr,Rh nvriyBcu,D470Bcu,Germany; Bochum, D-44780 Bochum, University Ruhr Chemistry, Theoretical h oeua tutr ftePCwsol eetyresolved recently only was PLC the of structure molecular The value. immunogenic no or little have sequences peptide Most d ua dpieimn ytmrle nasgaigmech- signaling a on the relies pathogens, system intracellular immune and adaptive cancer human against us protect o hsc eatet Heinrich-Heine-Universit Department, Physics | ,wihcudb mrvdt 5.8 to improved be could which A, ˚ MHC-I a + | etd-odn complex peptide-loading ypoye eettitdclsb scanning by cells tainted detect lymphocytes T lc,D545J D-52425 ulich, ¨ unrF Schr F. Gunnar , | rti dynamics protein lc,Germany; ulich, ¨ oder ¨ | b,c,d tD at ¨ n asV Sch V. Lars and , c J lc etefrSrcua ilg JSrc) oshnsetu J Forschungszentrum (JuStruct), Biology Structural for Centre ulich ¨ sedr,D425D D-40225 usseldorf, ¨ yfocusing by A ˚ afer ¨ sedr,Germany usseldorf, ¨ b a,1 nttt fBooia nomto rcsig(B-:Srcua Biochemistry), Structural (IBI-7: Processing Information Biological of Institute is ulse uut1,2020. 11, August published First 1 the under Published Submission.y Direct PNAS a is article This interest.y competing no paper.y declare O.F., the authors wrote research; L.V.S. The and performed O.F. O.F. and data; research; analyzed designed L.V.S. and L.V.S. G.F.S., and O.F. contributions: Author dual sur- the core and rigid proteins, a accessory form of belt MHC-I flexible and a Tsn by PLC. rounded the in subunits full biological to the them pin- relates in and functions. approach events observed dynamic of biology we origin dynam- the structural what points protein computational to compared systems This truncated and complex. these removed in were parts ics large complex even or the subunits, of domains, the individual of where on models alternative PLC unfold simulated the complex also the We solvated scale. of time motions a microsecond the into (MD) which dynamics embedded in molecular simulations, all-atom was to subjected structure and bilayer atomic lipid model This atomistic 2). an cryo-EM obtain informa- the (Fig. to structural components integrated PLC high-resolution We individual with on level. tion complex atomic the the of at density PLC the PLC of ics the hindered be rendering can 19), they (18, since mechanisms these inoperative. health evasion Understanding human immune to MHC-I. or by crucial sub- selection, antigen-loaded is of peptide of components events recruitment of the various release process com- complex, the the the the stable MHC-I, us of in a loaded inform roles of place optimally not the formation take the assembly, does pic- that in as cryo-EM averaged events such by dynamic static, plex, provided many A the one workings. about the required inner PLC like its the structure ture of elucidate low-resolution picture fully atomic-level a to the provide such not However, does achievement. sive owo orsodnemyb drse.Eal Lars.Schaefer@ruhr-uni- Email: addressed. be may correspondence bochum.de. whom To foraatv mueresponse. immune pro- adaptive results our underpinnings of The molecular the atoms). into insights million unprecedented vide (1.6 membrane environment a water in and simulations dynamics molecular microsecond ebita l-tmmdlo h ua H- L by PLC MHC-I human the of 9.9- model recent dynamic all-atom the and combining an size built unknown. its remained PLC We to the of Due details atomic-level cells peptides. the infections nature, in nonself viral apoptosis expose inducing against contents that by protect cellular mutations which mirror cancer-causing cells, to and T surface cell patrolling the for at complexes presented peptide–MHC-I are pep- Stable degradation antigenic molecules. load MHC-I cytosolic to onto reticulum tides translocates endoplasmic the to (PLC) products complex peptide- (MHC-I) loading class-I complex histocompatibility major The Significance u eut ihih h eta oeo h w aai (Tsn) tapasin two the of role central the highlight results Our dynam- conformational and structure the present we Here, y PNAS NSlicense.y PNAS | uut2,2020 25, August eouincy-Mdniywith density cryo-EM resolution A ˚ | lc,D545J D-52425 ulich, ¨ o.117 vol. | o 34 no. lc,Germany; ulich, ¨ | 20597–20606

BIOPHYSICS AND COMPUTATIONAL BIOLOGY Fig. 1. Structural overview of the MHC-I PLC. The PLC can be divided into three parts, ER-luminal, TM, and cytosolic. The ER-luminal part consists of two editing modules, M1 and M2, each of which contains one Tsn, ERp57, Crt, and MHC-I. MHC-I proteins (1–3, 6, 7) are heterodimers formed of a variable α heavy chain (αHC) and an invariant, light β2 microglobulin (β2m). αHC comprises three soluble domains, two of which, α1 and α2, form the peptide-binding groove (PBG). MHC-Is have an N-linked branched glycan that reflects their loading status: A terminal glucose allows recognition and binding by Crt and acts as a signal that MHC-I should be recruited to the PLC for peptide editing; antigen-loaded MHC-Is that exit the ER are deglucosylated. Newly synthesized “empty” MHC-I proteins (eMHC-I) are unstable; in the PLC, Tsn acts as an MHC-I . Tsn (8–10) is the central component of the editing modules. It has two ER-luminal domains: N-terminal TN and C-terminal TC. M1 and M2 are organized around a pseudosymmetry axis at the interface between the two Tsns. Tsn forms a complex with MHC-I and accelerates the off rate of low-affinity MHC-I–bound peptides to perform peptide editing (9, 11–13). Tsn is also disulfide bonded to ERp57 (14), a four-domain protein playing a structural role. Crt (15, 16) consists of three soluble domains, a globular lectin domain with a binding site for the monoglucosylated branch of the N-linked MHC-I glycan, a flexible P domain that extends over the PBG and contacts ERp57, and a calcium-sensing C domain with an extended α-helix that contacts Tsn. The transporter associated with (TAP) (17) is the main component of both the TM and cytosolic parts of the PLC. TAP shuttles peptides from the cytosol to the ER, providing the PLC with its substrate for antigen processing. TAP is a heterodimer of TAP-1 and TAP-2, each of which has an N-terminal four-helix TM domain, TMD0, that provides a docking site for the TM helix of Tsn. A TM helix also anchors MHC-I to the ER membrane.

editing modules formed by Tsn are essential for global com- Results and Discussion plex stability. MHC-I binding-groove width is modulated by the ERp57 and Crt Form a Flexible Belt around Tsn•MHC-I. To study environment of the PLC. The MHC-I N-linked glycan modulates the structural dynamics of the human PLC at the atomic level, the conformational dynamics of a solvent-exposed Tsn loop puta- all-atom MD simulations of the entire PLC, embedded in a lipid tively involved in peptide editing [called the “scoop loop” (20) by bilayer and solvated by water and ions (Fig. 2), were carried out. some authors]. Finally, the calreticulin (Crt) C domain constrains We acquired five 1-µs trajectories of this 1.6-million-atom system. Tsn motions to facilitate the docking of MHC-I proteins to the To the best of our knowledge, this represents an unprecedented complex, which is the last step of PLC assembly and the onset of time scale for such a large membrane-bound protein complex. antigen processing. First, as a quality check, the structural integrity of the individual

AB

Fig. 2. Atomistic model of the MHC-I peptide-loading complex. (A) The 1.6-million-atom molecular dynamics simulation system contains the complete human MHC-I PLC (75.939 atoms), embedded in a POPC lipid bilayer (101.036 atoms) and solvated by explicit water with Na+ and Cl− ions. (Only a small slab of water is shown for clarity.) (B) The PLC model was built by fitting atomistic subunit structures to the cryo-EM density for a single editing module (EMD-3906) (5) and then duplicating this module and fitting to the cryo-EM data (EMD-3094) (5) for the pseudosymmetric assembly (cryo-EM density for a single module shown as a surface).

20598 | www.pnas.org/cgi/doi/10.1073/pnas.2004445117 Fisette et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 iet tal. et Fisette PLC. entire the for and individually components i.3. Fig. C- single the a and by membrane TN the to N-terminal anchored the is Tsn latter domains, the of TC; two part terminal ER-luminal of The composed peptides. cytosolic is of pep- pool from antigens vast and immunodominant the binding of selection MHC-I the i.e., for editing, tide conformation productive a relaxation adopt the slow over exhibit were stable typically times overall convergence systems equilibrium. toward Long is large C simulations. the since PLC MD shows expected The the also complex. of 3 course whole Fig. subunits, the individual for of that to and Tsn by editing. formed peptide core, to dedicated PLC and rigid that MHC-I more proteins a accessory of stabilize belt and flexible encircle a form ERp57 and 5.3 Crt and that C 6.8 mean of consid- and distributions are values broad ERp57 with and flexible more Crt erably whereas proteins, structured highly (α MHC-I [β that microglobulin shows 3 Fig. C simulations. of devi- root-mean-square (RMSD) the of ation terms in assessed was components h eea rhtcueo h L lostetoTn to Tsns two the allows PLC the of architecture general The opposed as PLC, overall the of stability global the verify To C α mdfo h trigsrcue acltdfrER-luminal for calculated structure, starting the from rmsd 2 ] n s xii o mdvle yia for typical values rmsd low exhibit Tsn and m]) α ,rsetvl.Ti uprstenotion the supports This respectively. A, ˚ tm rmtesatn tutr fthe of structure starting the from atoms ev hi [α chain heavy α HC α rmsd rmsd ], β 2 ftePC() s Netbihsa nefc ihteMHC- the with by interface formed an domain establishes binding-groove TN Tsn first I structure (5). low-resolution and PLC the 21) the in of (20, Tsn for structures feature experimentally TAPBPR•MHC-I a observed in interfaces, separate present two also through MHC-I contacts Tsn cau- been these between had proximity the which 2,400 of components. basis module, two of the on editing area (5) opposite suggested surface tiously the buried in mean first ERp57 a the a with (SD) and and 200 interface (TN) (a an domain domains in N-terminal ERp57 fourth Tsn and the Con- 1). involve 5, Tsn cal- (Fig. tacts First, ERp57 was found. disulphide-conjugated components the were between with PLC interfaces interacts area interaction other main surface and Five buried domains culated. these Tsn the map soluble To simulations, the PLC. our the in of components contacts exten- other establishes (23). with Tsn contacts repertoire roles, functional sive antigen key its these with optimize keeping stabilizes that In rapidly editing that to peptide MHC-I chaperone for catalyst enables a a assembles, and MHC-I, PLC Components. peptide-deficient the PLC which Individual around Bridges Tsn experiments (22). stoichiometry pulldown TAP:Tsn with 1:2 and a a consistent suggest (5) form are that structure to results cryo-EM PLC, These two the human binding. of with the MHC-I assembly for in scaffold least the stable at that required, plausible is seems Tsn-ERp57s mem- the it Crt•MHC-I of proximity Therefore, of close very brane. dissociation the of and because apo-PLC the association and the hamper stable. could is angle tilt reference the PLC, the full in the contrast, of In simulations center. complex’s the from outward that shows a all 4 to Fig. in sys- leads structure. initial Crt this the and from of Tsn-ERp57, PLC deviation MHC-I, pronounced simulations one single-module MD of a removal 500-ns scaf- The of tem. five PLC model stable performed computational a and a of (∆M2) formation built the we in fold, modules editing PLC. two the the Stabilize Modules Editing Dual of features specific on these and of complex. stability effects the global the truncated the assessed we on and answer, modifications an components provide PLC To removed core. or struc- stable the which a for of of essential are formation question contacts the intercomponent to and raises elements compared tural This MHC-I changes (10). with conformational Tsn complex significant MHC-I–free in show exhibits TAPBPR 21) Tsn protein (20, structures that homologous while (13) the PLC, the of work of outside previous flexibility interdomain in large reported We α-helix. ofrainldnmc r ecie nmr eali later a in Tsn detail on more glycan in subsection. MHC-I described and the are MHC-I of dynamics effects conformational dynamic our between site; form In stretch glycan-binding loop Crt polysaccharide proteins. the the of the loaded apex with properly the contacts in which at terminus residues location C the that peptide simulations, MHC- at the the with bound of directly pocket is F or the selection groove with peptide binding either in I interacting role a by play 25) 11– to 24, Tsn suspected (20, is the loop and This glycan loop. MHC-I 20 N86-linked the involves MHC-I. MHC-I and TC Tsn of (13), interface ours binding including specific study, the computational predicted no that noting worth is between lodged 2,600 2; neetnl,teNtria nefc ewe s and Tsn between interface N-terminal the Interestingly, napeiu opttoa td,(3,w hwdthat showed we (13), study, computational previous a In it vivo, in happen to were change conformational a such If 2smltos h dtn ouelasincreasingly leans module editing the simulations, ∆M2 ± A ˚ 500 2 eddntosreaycnatbtenTnand Tsn between contact any observe not did We . A α ˚ PNAS 3 2 ,wieteTnCtria oan(C is (TC) domain C-terminal Tsn the while ), (α HC | and ) uut2,2020 25, August β 2 Fg ,3 1,800 3; 5, (Fig. m α oivsiaeterl of role the investigate To 0 1 | epciey,resulting respectively), , and o.117 vol. s sacnrlhub central a is Tsn α 2 | in o 34 no. ± α HC 500 Fg 5, (Fig. | A ˚ 2 20599 .It ). ±

BIOPHYSICS AND COMPUTATIONAL BIOLOGY entation through the action of the C domain; this hypothesis is addressed in a later subsection. Finally, the Tsn proteins in both editing modules contact each other to form a large Tsn•Tsn interface (Fig. 5, 5; 1,900 ± 300 A˚ 2) that involves their N233-linked N-acetyl-D-glucosamine . As discussed above, paired Tsns are essential to sta- bilize the PLC. This interface is strictly TN•TN, with the TC domains tilted away from the complex center so that a central cavity is formed that can act as a reservoir for TAP-translocated peptides (5). Tsn variants were used by Dong et al. (10) to map residues and features important for MHC-I association. The TN3 variant is a single-point E72K substitution that reduces Tsn peptide-loading activity to 58% of that of the wild-type (WT) protein. In our sim- ulations, the E72 side chain is lodged between the MHC-I α1

Fig. 4. Effects of ∆M2 on PLC structural stability. To measure the tilt of the editing module, θ is defined as the angle between the centers of geometry of the two soluble Tsn domains (TN and TC). ∆θ is the deviation from the ini- tial orientation; it is reported for the reference system (full PLC, Ref.) (Top) and for ∆M2 (Middle). (Bottom) The starting structure of ∆M2 and the structure with tilted editing module obtained after 500 ns of MD simulation.

Tsn also contacts Crt (Fig. 5, 4). The C-terminal Crt domain (C domain) is known to fluctuate between disordered and α- helical states as a function of calcium concentration (26). In our simulations, the highly structured C domain contacts Tsn TC at a location directly opposite the interface with MHC-I (700 ± 200 A˚ 2 mean buried surface area). In previous work (13), we commented on the high flexibility between TN and TC and the conformational selection enforced by MHC-I binding. Crt could Fig. 5. Interfaces between ER-luminal Tsn domains and other PLC compo- facilitate MHC-I binding by constraining TC to a specific ori- nents. TM regions are not shown.

20600 | www.pnas.org/cgi/doi/10.1073/pnas.2004445117 Fisette et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 iet tal. et Fisette MHC-I and to TN shown Tsn been Only have (10). trajectories. loop MHC-I that with from association α D12 Tsn for and 11–20 Tsn important E11 the be MHC-I residues of partner between and interaction lodged E72 suggested is a loop. also E72 is (A) which Y84, pocket. contacts F the of vicinity 6. Fig. and differently (27) environment alone. PLC Tsn MHC-I the by peptide- than by of free affected groove is binding for MHC-I the deficient that obtained the found We in results its (13). dynamics Tsn•MHC-I of previous MHC-I terms to in compared PLC plasticity we MHC-I fluctuations, affects structural environment Dynamics. PLC MHC-I the Dampens PLC ligands. The peptide with and competes or Y84, groove Tsn of binding how the understand vicinity to up studied the opens be in should Tsn effects interact several synergistic Thus, to their residues activity. together WT loop the come of involves 41% elements only which has (10), D12, and loop variant E11 only using TN4 performed the was study edit- fragments, this peptide While the Tsn (25). of the process drivers two and as ing proposed Y84 the recently between were but Interactions loop interactions scale. 11–20 6B), stable time (Fig. establish 100-ns and the simulation proximity on typical close in a stay substantially of residues fluctuates course distance the E72–Y84 pep- over antigen The the terminus. with C bonds hydrogen tide essential form to known is and B A 1 and α 2 α neatosbtenTnE2adteMCIbniggov nthe in groove binding MHC-I the and E72 Tsn between Interactions .E2itrcswt H- 8,which Y84, MHC-I with interacts E72 6A). (Fig. helices 2 7 oY4dsac iesre ntwo in series time distance Y84 to E72 (B) clarity. for shown are et oivsiaewhether investigate to Next, α 1 and α 2 and n roei h eino h etd emns possibly terminus, C peptide the of region bind- the the its with in than directly shorter groove interact much can ing is also it loop results homolog, 11–20 Our TAPBPR Tsn study. 22–35 the further in although deserves that rearrangement that show are hypothesis structural these a a Whether is much induce TN contacts. a that establish at interactions can albeit specific regions two glycan, show the clearly the simulations that MD of the have Nevertheless, absence also scale. and time would the slower glycan that in motion the observed a between accelerate been hindrance simply might steric loop possible the loop; the and a the glycan about MHC-I of the speculate For motions of cautiously presence simulations. the only our between can scale, relationship in we time sampled millisecond reason, may incompletely to this loop, be micro- 11–20 might the Tsn on which the dynamics as simula- slow such MD exhibit regions, all-atom flexible with reached Highly be tions. can that times simulation terminus. C dis- their at the peptides from loaded MHC-I the with and seen in loop, interacts located be the of is can apex which the as at Y84, L18, groove, the between trajectories, the 8B) (Fig. two on tance region In down F-pocket 8C). (Fig. closes the groove loop toward binding pushed MHC-I steric is the to of loop due the the inaccessible and of becomes and presence hindrance, region the MHC-I this In between however, 8 D). fluctuates (Fig. glycan, it domain glycan, glycan-binding Crt the the of differs. absence loop the the 8A). by (Fig. In explored removed is space it conformational when the and However, MHC-I on present is glycan the per- this and of model simulations PLC MD system. our 500-ns understand from additional To glycan five loop. formed MHC-I 11–20 the dynamics, its removed conformational Tsn of we impact vicinity might interaction the this in how the Tsn of with Dynamics tacts Conformational Loop. the 11–20 Modulates Tsn high- Glycan MHC-I a The with efficiently peptide- the loaded to be close d peptide. groove can affinity near binding bond conformation empty disulfide an loaded its a that with by shown (28) constrained has protein width MHC-I groove empty binding an helices of flanking recently structure both The that determined simultaneously. by peptides so incoming itself groove with loading interact the can facilitate of also opening the may conforma- chaperoning limiting and the optimize loading to may peptide closer This during groove MHC-I. peptide-loaded binding of the tion keeps PLC full the 7B). Fig. in bar observed, median the still d (below the is is of conformation distribution half open about distance the the however, Although PLC, broadened. the of environment widened is conformation confor- open (16.4 closed the and but observed, open still two PL are these mations (15.7 the complex, wider Tsn•MHC-I to much the similar In other the one and conformations: state groove two binding the between however, fluctuates MHC-I, (PD) peptide-deficient an In with the stable, of very mass and packed (d tightly distance is average region groove this the binding MHC-I, binds that (PL) the pocket peptide-loaded modulates of F In Tsn the terminus. addition, near C In groove antigen binding 7A). the (Fig. of PLC also width full the is the (13), in Tsn•MHC-I here of seen simulations previous in observed we the in h eylresz ftessessmltdhr iisthe limits here simulated systems the of size large very The when fluctuations high similarly exhibits loop 11–20 Tsn The chaperone, and editor peptide a as vitro in active is Tsn While especially fluctuations, its lowering by MHC-I chaperones Tsn )aditreit itne r esppltd nthe In populated. less are distances intermediate and A) ˚ α 1 and α α 2–1 2 h 8-ikdMCIgya salse con- establishes glycan MHC-I N86-linked The eie ftebniggov.Ti fet which effect, This groove. binding the of helices PNAS ei rgetadteopst einin region opposite the and fragment helix F F ausaecoe otoese nMHC-I in seen those to closer are values f13.3 of 7B) Fig. , | uut2,2020 25, August α 1 ei ftebniggov and groove binding the of helix ewe h etr of centers the between A ˚ | )admr populated. more and A) ˚ o.117 vol. | o 34 no. ∆ Glycan | 20601 α PL 1 F .

BIOPHYSICS AND COMPUTATIONAL BIOLOGY A to the integration of these MHC-I•Crt dimers into the PLC is unknown, as is the role of Crt, if any, in the assembled complex. To gain insights into the effects of MHC-I and Crt binding on the structural dynamics of the PLC, two modified PLC systems were built and subjected to five 500-ns MD simulations: one where MHC-I has been removed (∆MHC) and one where both MHC- I and the C domain of Crt have been removed (∆MHC-∆CrtC). It should be stressed that these two systems do not correspond to in vivo situations: Since Crt delivers MHC-I to the PLC, it would not be expected for Crt to be present in the PLC while MHC-I is absent. However, these theoretical models enable testing hypotheses that are relevant to the PLC and its assembly. Previous work (13) showed that, in the absence of MHC-I, Tsn exhibits a significant degree of rotational flexibility between its two soluble domains, TN and TC. In the Tsn•MHC-I complex, this degree of freedom becomes essentially frozen. Since, in the PLC, the Crt C domain contacts TC (Fig. 5, 4) at a location oppo- B site the TC•α3 interface (Fig. 5, 3), we posited that Crt could extend the long α-helix of its C domain to constrain TC to a con- formation that is conducive to MHC-I binding (Fig. 9A). To test this hypothesis, we computed TC Cα rmsd using the TN domain as a reference frame (Fig. 9B). Using the TN domain (instead of the complete protein) for structural superposition yields a measure of the TN/TC interdomain flexibility. Fig. 9B shows that this flexibility increases slightly in the absence of MHC-I. Removal of the C domain, however, leads to a more drastic increase and a broadened distribution (Fig. 9B, ∆MHC-∆CrtC). The Crt C domain thus limits TN/TC interdomain fluctuations and favors a Tsn conformation similar to the one adopted in the

A

Fig. 7. MHC-I dynamics in the PLC. (A) Effects of Tsn and PLC binding on RMSF along the αHC sequence. SD is shown for the PLC system. (B) Effects of peptide, Tsn, and PLC binding on binding-groove width dF, measured as the distance between the centers of mass of Cα atoms in residues 75 to 85 (α1) and 138 to 150 (α2–1). B

competing with ligands for binding to MHC-I during the peptide editing process. These results are fully compatible with the partial glycan model presented with the cryo-EM PLC structure (5). How- ever, since the cryo-EM model included only the few sugar units nearest MHC-I N86 and Crt W319, the effects of the complete, branched glycan could not be inferred in that study. Tsn L18 CD was identified in another work (24) as a lever that mediates peptide dissociation. Recent evidence (29) suggests instead that the 11–20 loop might act as peptide surrogate, stabilizing the empty binding groove and competing with peptides at the bind- ing step. However, both published TAPBPR•MHC-I structures (20, 21) show poor electron density in this region, in line with the pronounced conformational dynamics observed in our MD simulations. Since the Tsn and TAPBPR loops are of different lengths (11–20 in Tsn vs. 22–35 in TAPBPR), they may also have different modes of action. Clearly, more studies are needed to determine precisely the possible role of this region.

The Crt C Domain Facilitates MHC-I Binding to Tsn through Confor- Fig. 8. Tsn 11–20 loop dynamics. (A) Effects of glycan removal on RMSF mational Selection. Upon assembly, TAP and two Tsn-ERp57 along the TN sequence. SD is shown for the reference system. (B) TN L18 to form an apo-PLC ready to bind suboptimally-loaded MHC-I. Crt αHC Y84 distance time series. (C) Contact between the 11–20 loop and the recruit MHC-I whose glycan is glucosylated, and deliver them to MHC-I binding groove in a simulation of the full PLC. (D) Typical 11–20 loop the PLC (30). However, the precise sequence of events leading conformation in a simulation of the ∆Glycan system.

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Fig. D B AC h vrg ln fteTC the of plane average The ) rvossmltoso reTn(3 hwdta h average the that showed (13) Tsn free of simulations Previous β settoward -sheet nuneo h r oano s Ccnomtoa dynamics. conformational TC Tsn on domain C Crt the of Influence β n measured and 9C) (Fig. MHC-I contacts that -sheet θ ∆MHC), β ewe hspaeadisiiiloinaini the in orientation initial its and plane this between settwr where toward -sheet θ fthe of α mdo CuigT C TN using TC of rmsd α β θ 3 sett t nta retto,vee rmthe from viewed orientation, initial its to -sheet neetnl,i h bec fMHC- of absence the in Interestingly, . so average on is β setrgo htcnat H- (residues MHC-I contacts that region -sheet α 3 and β 2 ol e hrfr,the Therefore, be. would m β +9 α 2 n locnatdb the by contacted also and m o tutrlsuperposition. structural for ◦ rmisiiilvalue, initial its from 16 ◦ hsobser- This . −7 ◦ lxwt H- hw wsigo h -adCtria oul domains, com- soluble in C-terminal to and TAPBPR us N- Tsn, the allowed free of of 21) twisting to structures (20, shows Compared MHC-I MHC-I two with interface. with of plex Tsn•MHC-I complex publication subcomplex the in recent this TAPBPR refine The homolog of Tsn (13). structure the simulations a MD obtained from low-resolution we a provides Previously, PLC the of approximation. structure is cryo-EM Tsn-ERp57 the and but MHC-I by available, formed not subcomplex active catalytically the of ture Interface. Tsn•MHC-I the Modeling for Methods way and the Materials up repre- opens and work direction present this in The work. step future first questions. key these a sents answer successfully synergistic be to can in applied approaches, simulations, experimental dynamics with molecular combination that TC that of groove? Tsn exits expect binding dual MHC-I two We the the toward do the diffusion and peptide between reservoir, steer cavity peptide TAP, a atop as act cavity handle. domains, the to For peptide MHC-I. does is to easier instance, binding investigation and are membrane closer the that through warrants translocation the systems that of question smaller context Another the on now, neglected focusing Until have PLC, Tsn•MHC-I interfaces? of protein–protein and studies affinity various most the the influence MHC-I groove of the binding stability of MHC-I there- the influence high-affinity in questions a the of antigen is presence of the one would number How first status: A peptide-loading The open. studied. different remain be of fore could number that the systems limiting TC demanding, and computationally TN Tsn by the apo-PLC of the orientations to domains. relative binding MD the MHC-I the constraining facilitates mechanism Finally, selection CrtC glycan. conformational edit- involving nearby a explicitly peptide not that this far in suggest of so simulations involved effects have likely loop the that region considered of of a studies dynamics Experimental loop, conformational peptide- ing. 11–20 the a Tsn influence in N86-linked to the stabilized found MHC-I is was the that glycan groove Furthermore, binding conformation. tighter receptive intermodule. a result- and dynamics, in MHC-I intra- ing modulates both environment protein–protein 5), complex five (Fig. This found Tsn Tsn•MHC-I We involving bridg- together. two in interfaces Tsn components that of the role PLC Crt crucial by the ing and highlight formed also ERp57 results scaffold Our by dimers. central formed can- belt the PLC circular single-module encloses the A assemble modules. editing not two ER-luminal requires 10-protein PLC a to membrane cytosolic ER a macro- from assembly. the extends of interac- system their variety the through PLC, of the the part affinity of to case low the due the In tions. often, also just and, but involved not size, molecules large challenging system such of are of because complexes simulations protein time dynamics microsecond membrane-bound full the molecular on the All-atom dynamics of structural model scale. its atomic-resolution study an and present PLC we work, this In of Conclusions orientation precise the to related not is TC the on domain C Crt 9B, (Fig. to domain compared C the of of removal ∆MHC- Further dissociation 32). (31, subsequent researchers to groove the signal binding the peptide-loading in from a transduction role with consistent a MHC-I, antigen-loaded play “spring- TC could Such binding. loading” provided MHC-I energy, upon store fluctuations, could thermal Tsn by that hypothesis the fuels vation l-tmM iuain fti .-ilo-tmsse are system 1.6-million-atom this of simulations MD All-atom MHC-I human the of stability the that shown have We β -sheet. rC osnthv infiatadtoa impact additional significant a have not does CrtC) ∆ H,sgetn httesaiiigefc fthe of effect stabilizing the that suggesting MHC, PNAS | uut2,2020 25, August naoi-eouineprmna struc- experimental atomic-resolution An | α o.117 vol. 3 spooe yother by proposed as , | o 34 no. | 20603

BIOPHYSICS AND COMPUTATIONAL BIOLOGY allowing the peptide editor to lodge its C-terminal domain between MHC-I system by rigid body superposition using the cryo-EM density of the full PLC α3 and β2m. A similar binding mode is observed for Tsn in the structure of (EMD-3905) (5); while the resolution of the density in that region is low, TAP the PLC. anisotropy and the protruding nucleotide-binding domains allowed unam- Starting from the structure of the Tsn-ERp57 conjugate (Protein Data biguous placement. Harmonic position restraints on all Cα atoms were then Bank [PDB] ID 3F8U, chains A and B, which contains only soluble domains) applied in the MD simulations to preserve the structural integrity of the TAP (10), we removed the 269–271 linker between TN and TC and fitted TN- homology model. Since we do not study TAP dynamics in the present work, ERp57 and TC to their homologs from the TAPBPR•MHC-I complex (20) this is sufficient for our purposes. separately. The linker was then rebuilt using Modeller (version 9.19) (33), The Tsn and MHC-I TM helices were modeled as ideal α-helices and posi- as were the unresolved Tsn loops. Human MHC-I protein HLA B*4402 (PDB tioned in the plane of the membrane underneath the termini of the soluble ID 1M6O, which contains only soluble domains) (34) was then superposed domains they connect to, yielding a topology similar to what has been onto the mouse MHC-I protein from the structure of the TAPBPR•MHC- reported (5). Previously, we proposed the structure and binding mode of the I complex. This allele was chosen as it is known to be PLC dependent TMD0 domain from TAP1 (44). Given the position of the core TAP domains and has been extensively studied both in experiments and in simulations. and Tsn TM helix, the only location TMD0 can occupy in the membrane with- Tsn and MHC-I side chains with at least one heavy atom within 2.5 A˚ of out steric clashes is between the Tsn and MHC-I TM helices (Fig. 1). At this another heavy atom from the binding partner were rebuilt with Modeller to position, TMD0 is close enough to the core TAP domains for the flexible remove steric clashes, followed by 100 steps of steepest-descent (SD) energy linker to connect them. TMD0 and the Tsn TM helix were then oriented minimization. to reproduce the published binding mode (44). Since there is no available Finally, MD simulations allowed the subcomplex to relax. The structure structure of the TMD0 domain from TAP2, we used the one from TAP1 for was solvated in a periodic rhombododecahedral box (10 A˚ minimal distance both editing modules; since both TMD0s are four-helix bundles and share between protein surface and box edge) with 0.15 M Na+Cl−. The result- the same binding mode (44), this should have little impact on overall PLC ing system was subjected to 5 ns of MD simulation with position restraints behavior. Membrane insertion depth was determined using the Orientation on all Cα atoms. This was followed by unrestrained MD simulation in the of Proteins in Membranes (OPM) Positioning of Proteins in Membrane (PPM) isothermal-isobaric (NpT) ensemble; protein coordinates after 45 ns were (45) server independently for the core TAP domains, the MHC-I TM helix, used in the next step. and the subcomplex formed by TMD0 and the Tsn TM helix. The three flex- ible linkers were built, connecting TMD0 to the core TAP domains, the Tsn Fitting to the Cryo-EM Density. The two ER-luminal editing modules are TM helix to the Tsn soluble domains, and the MHC-I TM helix to the MHC- composed of the soluble regions of Tsn-ERp57, MHC-I, and Crt. We first I soluble domains. The structure was finally energy minimized for 100 SD superposed the above-described Tsn-ERp57•MHC-I subcomplex to the cryo- steps. EM density for a single editing module (EMD-3906) (5) as a rigid body using Chimera (version 1.12) (35). This approximate positioning was then refined MD Simulation System Setup. A preequilibrated 128-lipid 1-palmitoyl-2- using DireX (version 0.7) (36). We performed 300 optimization steps using oleoylphosphatidylcholine (POPC) bilayer was overlaid onto the PLC structure a deformable elastic network (DEN) with the recommended default param- at the previously determined insertion depth. The membrane normal and the eters; the DEN was applied separately to each polypeptide chain, with no long axis of the PLC lie in the z dimension. To accommodate the full PLC, the restraint between the chains. Additional harmonic distance restraints were bilayer was replicated fourfold in the x and y dimensions and the protein com- applied to conserve hydrogen bonds in the starting structure. Hydrogen plex was centered on the resulting 2,048-lipid bilayer. The water on both sides bonds were determined using the FindHBond (37) Chimera program, with of the lipid bilayer was then extended in the z dimension by replicating a 216- donor–acceptor constraints relaxed by 0.4 A˚ and 20◦. molecule preequilibrated water box as needed to accommodate the full PLC To complete the positioning of the editing modules, we took the Crt par- and a 20-A˚ buffer on both sides. The membed (46) GROMACS program was tial model from the published PLC structure (PDB ID 6ENY) (5). This model then used to embed the protein structure in this orthorhombic lipid/water is based on structures of the Crt lectin domain (PDB ID 3O0W) (38), the box. Membed proceeded over 1,000 steps of simulation and considered five tip of the Crt P domain (PDB ID 1HHN) (39), and the homologous region TM fragments: TAP core, the two MHC-I TM helices, and the two subcomplexes of the P domain (PDB ID 1JHN) (40); the C domain is modeled as formed by TMD0 and its bound Tsn TM helix. After embedding was complete, an α-helix. We filled in missing side chains and missing regions of the P all waters whose oxygen was closer than 4.0 A˚ to lipid or protein heavy atoms domain with Modeller and superposed the resulting model onto the cryo- were removed, thus avoiding trapping waters inside the membrane or in small EM density as described above. We then performed another round of DireX cavities inside or between protein subunits. Na+Cl− ions were then added to optimization (300 steps), keeping Tsn-ERp57 and MHC-I fixed and using a a 0.15-M concentration by replacing randomly chosen water molecules. A 100 DEN as described above. The complete editing module was duplicated, and SD steps energy minimization was then performed. Finally, 50 ns of MD simu- the two modules were fitted, as rigid bodies, to the cryo-EM density for the lation was performed in the constant temperature and lateral area ensemble pseudosymmetric assembly (EMD-3904) (5). (frozen xy plane, pressure coupling applied only in the z dimension) with posi- Finally, the MHC-I N-linked glycan was modeled to bind the Crt lectin tion restraints on all PLC heavy atoms. This allowed the box size to stabilize domain. The glycan sequence is N86-GlcNAc2-Man-(Man3-Glu, Man-[Man2, and the water and lipids to equilibrate around the complex. The final system 3 Man2]), where GlcNAc is N-acetyl-D-glucosamine, Man is D-mannose and (Fig. 2) contains 1.6 million atoms in an approximately 240 × 240 ×300 A˚ box. Glu is D-glucose; this corresponds to a typical glucosylated high-mannose All deletion systems were built using a similar protocol. The vacuum cre- N-glycan. Topology and initial coordinates were generated with doGlycans ated by the removal of selected PLC regions was filled by water molecules, (source code date: 17 August 2017) (41). The four sugars of the Man3- except in the TM regions. This was followed by 50 ns of MD simulation in Glu moiety were then individually repositioned to reproduce the binding the constant temperature and lateral area ensemble with position restraints mode observed in the structure of the Crt lectin domain in complex with on all PLC heavy atoms. a tetrasaccharide (38). Monte Carlo (MC) sampling was then used to find a combination of dihedral angles that would allow the GlcNAc2-Man moiety MD Simulations. Simulations were carried out with GROMACS (version 5.1.2) to link N86 and Man3-Glu with no steric clashes. Finally, another dihedral (47). The Amber99SB*-ILDNP protein force field (48–53), the united-atom MC search was used to orient the Man-(Man2, Man2) moiety to avoid steric Berger lipid force field (54), the GLYCAM06h carbohydrate force field (55), clashes. and the TIP3P water model (56) were used. The SETTLE (57) and LINCS (58) algorithms were applied to constrain the internal degrees of freedom of Modeling Transmembrane and Cytosolic PLC Components. The transmem- water molecules and the bonds in other molecules, respectively. To allow brane (TM) and cytosolic parts of the PLC comprise TAP and the single TM for a 4-fs integration time step, we used virtual site hydrogens (59) for pro- helices of Tsn and MHC-I. While these are not the focus of the present work, teins. Since GLYCAM does not support virtual sites, we used hydrogen mass they were included in our simulations to properly position and anchor the repartitioning (HMR) (60) instead to slow down vibrations involving hydro- editing modules in the membrane. gen atoms in the carbohydrate moieties. The Berger lipids intrinsically allow While a cryo-EM structure of TAP in complex with the ICP47 viral inhibitor for a 4-fs integration time step since they contain no explicit hydrogens. has been resolved (42), this structure shows TAP in an arrested conforma- Short-range nonbonded Coulomb and Lennard-Jones 6-12 interactions were tion with its two nucleotide-binding domains separated. In the absence of treated with a Verlet buffered pair list (61) with smoothly shifted poten- an atomic-resolution structure of TAP that could provide suitable starting tials. Long-range Coulomb interactions were treated by the particle mesh coordinates for our all-atom MD simulations, we used Modeller to generate Ewald (PME) method (62). Analytical dispersion corrections were applied a homology model based on the heterodimeric ABC exporter TM287/288 in for energy and pressure to compensate for the truncation of the Lennard- the apo state (PDB ID 4Q4H) (43). The TAP model was then added to the Jones interactions. The thermodynamic ensemble was NpT unless otherwise

20604 | www.pnas.org/cgi/doi/10.1073/pnas.2004445117 Fisette et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 1 .P ilas .A e,A .Prel .MCuky .Elot piiainof Optimization Elliott, T. McCluskey, Fleischmann J. G. Purcell, 12. W. A. Peh, A. C. MHC Williams, into Insights P. A. Reinisch, M. 11. K. Cresswell, P. Peaper, R. D Wearsch, A. P. Dong, G. 10. 2 .S atr .Ji,R .Lohrt .H,P rswl,Dnmc fmjrhisto- major of Dynamics Cresswell, P. Ha, T. Leonhardt, M. R. Jain, A. Panter, S. M. 22. Jiang J. 21. Tamp R. Thomas, C. 20. in Lessons F evasion: F. immune Liaci, Viral M. Wiertz, A. J. H. Praest, J. P. E. 19. Luteijn, D. R. Weijer, de van L. M. the 18. of system Tamp chaperone R. Trowitzsch, calnexin/calreticulin S. The 17. lectins: Beyond Williams, B. D. and calreticulin 16. for Roles Cresswell. P. Spies, T. Ortmann, B. Lehner, J. P. disulfide- Sadasivan, stable B. a 15. form ERp57 and Tapasin Cresswell, P. Wearsch, A. P. Peaper, R. D. 14. Wingberm S. Fisette, O. 13. iet tal. et Fisette glycan MHC-I the in mannose closest the kJ·mol from C5 (50,000 W319 and Crt the nitrogen between chain harmonic added was one main module simulations, editing MD each our for in restraint established distance stably also is (44) structure position stabi- these to without cryo-EM, performed in then resolution C restraints. were simulation low trajectories Crt a of Production has on ns lize. which restraints posi- 100 interface, position Crt•ERp57 with Second, the with out rotations. performed carried side-chain was was protocol through C simulation generation stabilize box all of system to on the ns the by restraints to 50 preceded of tion addition First, were end above). (in simulations the described these protocol at initial of performed equilibration random All Coordinates equilibration two-step ps. 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Momburg, F. 8. antigen I MHC Ayres the M. in C. editing peptide 7. and plasticity Protein Hateren, van A. Elliott, T. 6. Blees A. 5. .K .Rc,E et,J efe,Peetyusl!b H ls n H ls II class MHC and I class MHC Tamp by R. yourself! Hulpke, Present Neefjes, S. J. 4. Reits, processing. E. antigen Rock, of L. Pathways K. Cresswell, 3. P. Wearsch, A. P. Blum, of S. understanding J. systems 2. a Towards Bakke, O. Paul, P. Jongsma, M. L. M. Neefjes, J. 1. oesr hta neato iia oteoeosre ntecryo-EM the in observed one the to similar interaction an that ensure To (see 1,000-ns to 500- five system, each For fppielaigadediting. and loading peptide of complex. I tapasin–MHC the in editing tapasin. on complex. loading dependent is cargo peptide I (2002). class MHC the oxidoreductase thiol tapasin-ERp57 the of structure the heterodimer. from loading peptide I class selection. peptide for mechanism reticulum. endoplasmic the in molecules (2002). I 217–233 class MHC by binding flexibility. global MHC I class of tuning pathway. processing (2017). 525–528 optblt ope ls soito ihtehmnppielaigcomplex. peptide-loading human Chem. the with Biol. association I class complex compatibility presentation. antigen in editing peptide viral by inhibition TAP of mechanisms and proteins. complex evasion peptide-loading immune I class MHC the presentation. antigen I class MHC Biol. Mol. TAP. reticulum. with endoplasmic molecules I class MHC of interaction the in Immunity tapasin, glycoprotein, novel a complex. peptide-loading I (2005). class MHC the within dimer linked immunity. adaptive molecules. Immunol. presentation. antigen II class MHC (2011). and I class MHC rsa tutr faTPP-H- ope eel h ehns of mechanism the reveals complex TAPBPR-MHC-I a of structure Crystal al., et tutr ftehmnMCIppielaigcomplex. peptide-loading MHC-I human the of Structure al., et 4149 (2018). 4481–4495 430, (2013). 443–473 31, 0–1 (1996). 103–114 5, rnsImmunol. 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W. 34. Tamp R. Thomas, C. Hennig, F. Sagert, L. 29. Anjanappa R. 28. Wingberm S. Fisette, O. 27. Boelt G. S. 26. Hafstrand I. 25. Ilca Tudor F. 24. Tamp R. Thomas, C. 23. optn ieo h C uecmue ueMCa ebi Super- Leibniz Tamp at Robert SuperMUC thank discussions. We Supercomputer (http://www.lrz.de). GCS Center the computing on providing time by Supercomputing project computing for this funding Center for (http://www.gauss-centre.eu Gauss ) e.V. (GCS) the 2033–390677874– acknowledge gratefully Strategy–EXC We Excellence RESOLV. Germany’s under gemeinschaft are data study ACKNOWLEDGMENTS. All Tsn. this and complex; MHC-I whole stable article. the more this visu- for the in the for than included adjusting range, case rather by the RMSF subunit done especially large better specific is is that a subunit for fluctuations. to given scale leads compute a the Crt to inside of fluctuations frame each of domain reference for alization P computed the flexible was as the structure used Since average and An chain factors. protein B for used pro- typically incorporates and glycans and C subunits tein protein all contains in format, PDB structure, The (https://www.modelarchive.org/doi/10.5452/ma-whom2). are Availability. maximum) Data and (minimum envelopes average. series running the series time alongside Time running shown ns; with points. 10 time, data equilibration over all including averages trajectories encompass entire whiskers from for shown while extend are bar, boxes median plots; Proper- a box/distribution equilibrium. with as the shown toward are relaxation ties allow to trajectory production Analysis. itcmaiiiycmlxcasImolecules. I class complex histocompatibility plasticity. calreticulin. chaperone ER the lacking cells P e evr eore o oiinn fpoen nmembranes. in proteins of positioning for Res. Resources server: web PPM lock-switch. ionic conserved (2012). conformation. inward-facing its in transporter ABC transporter. TAP the of analysis GROMACS. for Model. polymers Inf. carbohydrate Chem. J. and glycolipids, glycoproteins, of simulations folding. protein of control U.S.A. Chem. survey. crystal a from information improvements. side-chain analysis. and recognition. cell T and repertoire, (2003). peptide 679–691 structure, 198, I class alters type Bioinformatics editor. peptide and chaperone I MHC as function dual selection. peptide of mechanisms reveal molecules groove. binding MHCI the (2017). of 408 dynamics structural the influences Proteomics tapasin. by (2019). editing 5060 and loading peptide I class lever. leucine Immunol. α 30D7 (2012). D370–D376 40, MF(nagtos o yooi opnns trdi h column the in stored components, cytosolic for angstroms) (in RMSF 1333 (2001). 3133–3138 98, 81–82 (2010). 38612–38620 285, sebyadatgnpeetn ucino H ls oeue in molecules I class MHC of function antigen-presenting and Assembly al., et rpriswr optdatrdsadn h rt10n feach of ns 100 first the discarding after computed were Properties tal. et sebyo h H etd-odn ope eemndb a by determined complex peptide-loading I MHC the of Assembly al., et olcn–ol o rprn abhdaesrcue o atomistic for structures carbohydrate preparing for doGlycans–Tools al., et c.Rep. Sci. –5(2019). 9–15 58, eetrfnto fMCImlclsi eemndb protein by determined is molecules I MHC of function Selector al., et tal. et tal. et apn h a2)idcdsrcua hnei calreticulin. in change structural induced Ca(2+) the Mapping al., et 3–4 (2016). 138–148 142, ucsiecytlsrcuesasossgettebssfrMHC for basis the suggest snapshots structure crystal Successive al., et h tutr fclei,a Rcaeoeivle nquality in involved chaperone ER an calnexin, of structure The al., et ABRmdae etd iscainfo H ls sn a using I class MHC from dissociation peptide mediates TAPBPR al., et .Cmu.Chem. Comput. 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