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A loop structure allows TAPBPR to exert its dual function as MHC I chaperone and peptide editor

Adaptive immunity vitally depends on major histocompatibility complex class I (MHC I) molecules loaded with peptides. Selective loading of peptides onto MHC I, referred to as peptide editing, is catalyzed by tapasin and the tapasin-related TAPBPR. An important catalytic role has been ascribed to a structural feature in TAPBPR called the scoop loop, but the exact function of the scoop loop remains elusive. Here, using a reconstituted system of defined peptide-exchange components including human TAPBPR variants, we uncover a substantial contribution of the scoop loop to the stability of the MHC I-chaperone complex and to peptide editing. We reveal that the scoop loop of TAPBPR functions as an internal peptide surrogate in peptide-depleted environments stabilizing empty MHC I and impeding peptide rebinding. The scoop loop thereby acts as an additional selectivity filter in shaping the repertoire of presented peptide epitopes and the formation of a hierarchical immune response.


Introduction
Nucleated cells of higher vertebrates provide information about their health status by presenting a selection of endogenous peptides on MHC I molecules at the cell surface. By sampling these peptide-MHC I (pMHC I) complexes, CD8 + T lymphocytes are able to detect and eliminate infected or cancerous cells (Blum et al., 2013;Rock et al., 2016). In a process called peptide editing or proofreading, peptides derived from the cellular proteome are selected for their ability to form stable pMHC I complexes. This peptide editing is known to be catalyzed by the two homologous MHC I-specific chaperones tapasin (Tsn) and TAP-binding protein-related (TAPBPR) (Fleischmann et al., 2015;Hermann et al., 2015;Morozov et al., 2016;Tan et al., 2002;Thomas and Tampé, 2019;Wearsch and Cresswell, 2007;Wearsch et al., 2011). The selection of high-affinity MHC I-associated peptide epitopes is of pivotal importance not only for immunosurveillance by effector T lymphocytes, but also for priming of naïve T cells and T cell differentiation. As an integral constituent of the peptide-loading complex (PLC) in the endoplasmic reticulum (ER) membrane, the ER-restricted Tsn functions in a 'nanocompartment' characterized by a high concentration of diverse, optimal peptides. The peptides are shuttled into the ER by the heterodimeric ABC (ATPbinding cassette) transporter associated with antigen processing TAP1/2, the central component of the PLC (Abele and Tampé, 2018). In the ER, most peptides are further trimmed by the aminopeptidases ERAP1 and ERAP2 to an optimal length for binding in the MHC I groove (Evnouchidou and van Endert, 2019;Hammer et al., 2007). In contrast to Tsn, TAPBPR operates independently of the PLC and is also found in the peptide-depleted cis-Golgi network . Fundamental insights into the architecture and dynamic nature of the Tsn-containing PLC have come from a recent cryo-EM study of the fully-assembled human PLC (Blees et al., 2017), while the basic principles underlying catalyzed peptide editing have been elucidated by crystal structures of the TAPBPR-MHC I complex (Jiang et al., 2017;Thomas and Tampé, 2017a): TAPBPR stabilizes the peptide-binding groove in a widened conformation primarily through the MHC I a2-1 helix, distorts the floor of the binding groove, and shifts the position of b2-microglobulin (b2m). Furthermore, one of the two TAPBPR-MHC I complex structures revealed a remarkable structural feature in TAPBPR named the scoop loop (Thomas and Tampé, 2017a). In TAPBPR, this loop is significantly longer than the corresponding region in Tsn, which was not resolved in the X-ray structure of Tsn (Dong et al., 2009). Notably, the scoop loop of TAPBPR is located in the F-pocket region of the empty MHC I binding groove ( Figure 1A,B). By anchoring the C-terminal part of the peptide, the F pocket region is crucially involved in defining pMHC I stability (Abualrous et al., 2015;Hein et al., 2014). The scoop loop occupies a position that is incompatible with peptide binding and displaces or coordinates several key MHC I residues responsible for binding the C terminus of the peptide. We therefore proposed that the scoop loop can be regarded as a surrogate for the C terminus of the displaced peptide, stabilizing the inherently labile empty MHC I molecule (Thomas and Tampé, 2017a). At the same time, by occupying a region critical to peptide binding, the scoop loop might allow only highaffinity peptides to re-enter the MHC I binding groove after displacement of sub-optimal peptide. The proposed importance of the scoop loop for TAPBPR function has recently been scrutinized in a study by Ilca et al. investigating TAPBPR scoop-loop variants using immunopeptidomics and cellbased assays (Ilca et al., 2018). Ilca et al. found that a specific leucine residue in the scoop loop facilitates peptide displacement on MHC I allomorphs favoring hydrophobic peptide side chains in their F pocket. Here, we aimed to clarify the role of the scoop loop during TAPBPR-catalyzed peptide editing using in vitro interaction and peptide-exchange studies with defined, purified components. We demonstrate that the scoop loop is of critical importance for TAPBPR-mediated stabilization of empty MHC I clients in peptide-depleted environments and contributes to peptide quality control during editing by impeding released peptide to rebind in the MHC I groove. Collectively, our data support a crucial role for the TAPBPR scoop loop in establishing a hierarchical immune response. eLife digest Cells in the body keep the immune system informed about their health by showing it fragments of the proteins they have been making. They display these fragments, called peptides, on MHC molecules for passing immune cells to inspect. That way, if a cell becomes infected and starts to make virus proteins, or if it becomes damaged and starts to make abnormal proteins, the immune system can 'see' what is happening inside and trigger a response.
MHC molecules each have a groove that can hold one peptide for inspection. For the surveillance system to work, the cell needs to load a peptide into each groove before the MHC molecules reach the cell surface. Once the MHC molecules are on the cell surface, the peptides need to stay put; if they fall out, the immune system will not be able to detect them. The problem for the cell is that not all peptides fit tightly into the groove, so the cell needs to check each one before it goes out. It does this using a protein called TAPBPR.
TAPBPR has a finger-like structural feature called the "scoop loop", which fits into the end of the MHC groove while the molecule waits for a peptide. The experiments revealed that the scoop loop plays two important roles. The first is to keep the MHC molecule stable when it is empty, and the second is to hinder unsuitable peptides from binding. The scoop loop sticks into one side of the groove like a tiny hairpin, so that pushed-out, poorly fitting peptides cannot reattach. At the same time, it holds the MHC molecule steady until a better peptide comes along and only releases when the new peptide has slotted tightly into the groove.
Understanding how cells choose which peptides to show to the immune system is important for many diseases. If cells are unable to find a suitable peptide for a particular illness, it can stop the immune system from mounting a strong response. Further research into this quality control process could aid the design of new therapies for infectious diseases, autoimmune disorders and cancer.

Design of TAPBPR scoop-loop variants
To investigate the function of the scoop loop, we prepared two human TAPBPR variants: TAPBPR Tsn-SL , in which the TAPBPR scoop loop was replaced with the corresponding shorter loop of Tsn, and TAPBPR DSL , in which the original scoop loop was essentially deleted by replacing it with three glycine residues to preserve proper folding of the MHC I chaperone ( Figure 1C). The ERlumenal domains of wildtype (wt) TAPBPR and the variants, each harboring a C-terminal histidine tag, were expressed in insect cells and purified from the cell culture supernatant via immobilizedmetal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). As MHC I chaperone clients, we chose mouse H2-D b and human HLA-A*02:01, which are known to interact with TAPBPR Ilca et al., 2019;Morozov et al., 2016). HLA-A*02:01, the major MHC I allomorph in the Caucasian population and found in more than 50% of the global population, presents a diverse spectrum of immunodominant autoimmune, viral, and tumor epitopes and is therefore medically highly relevant (Boucherma et al., 2013). The MHC I allomorphs were expressed in E. coli as inclusion bodies and refolded in the presence of b2m and fluorescently-labeled or photo-cleavable peptide (Rodenko et al., 2006). The highly pure TAPBPR variants and pMHC I complexes eluted as monodisperse samples at expected size during SEC ( Figure 1D-F).

Scoop-loop variants have reduced chaperone activity towards peptidefree MHC I
During peptide exchange, MHC I molecules go through a peptide-free high-energy intermediate state after peptide release and before re-entry of a new peptide. A hallmark of peptide editors like TAPBPR is their ability to recognize and chaperone this intermediate until it is located in a peptiderich environment where a high-affinity peptide ligand can enter the MHC I binding groove (Thomas and Tampé, 2019;Thomas and Tampé, 2017b). To scrutinize the role of the scoop loop in chaperoning empty MHC I, we tested the ability of our TAPBPR variants to stabilize peptide-free H2-D b . Hence, H2-D b (10 mM) loaded with a photo-cleavable peptide was incubated with TAPBPR (3 mM) under UV exposure. Subsequent SEC analysis revealed that both TAPBPR Tsn-SL and TAPBPR DSL are, in principle, competent to form complexes with MHC I (Figure 2A). However, in comparison to TAPBPR wt ( Figure 2A,B), the amount of H2-D b complex detected for TAPBPR Tsn-SL and TAPBPR DSL during SEC was reduced by around 40% and 90%, respectively ( Figure 2C). After reanalysis of the MHC I chaperone complexes by SEC, the mutant complexes were mostly dissociated, indicating kinetic instability ( 10 mM) loaded with a photo-cleavable peptide (RGPGRAFJ*TI, J* denotes photocleavable amino acid) was irradiated with UV light in the presence of TAPBPR wt (3 mM, red), TAPBPR Tsn-SL (blue), or TAPBPR DSL (yellow) and subsequently analyzed by SEC. The different elution volumes of the first main peak, marked by dashed lines, already hint at different complex stabilities. (B) Deconvolution of size-exclusion chromatogram from TAPBPR wt complex formation (experiment independent of the sample shown in (A)). The experimental chromatogram (red) was deconvoluted using three Gaussian functions (gray) that can be ascribed to the TAPBPR-H2-D b complex (1.06 mL), free TAPBPR (1.12 mL), and free H2-D b (1.20 mL). The sum of the three Gaussians is shown as dotted curve. The residual plot depicted beneath the main panel shows the difference between the experimental data and the sum. (C) Stability of complexes formed by TAPBPR wt , TAPBPR Tsn-SL , and TAPBPR DSL , respectively, as judged by the area of the complex peak obtained by deconvolution. Data represent mean ± SD (n = 2).
The online version of this article includes the following figure supplement(s) for figure 2: Scoop-loop variants retain their function in catalyzing peptide dissociation from MHC I After investigating the chaperone activity of the TAPBPR scoop-loop mutants, we tested their ability to displace MHC I-bound peptide. To this end, we employed an in-vitro peptide exchange assay similar to the one previously described for measuring the activity of Tsn (Fleischmann et al., 2015;Chen and Bouvier, 2007). Dissociation of medium-affinity fluorescent peptide from refolded and purified p*MHC I (p* denotes fluorescently-labeled peptide) was monitored by fluorescence polarization after addition of a 1000-fold molar excess of unlabeled high-affinity competitor peptide in the absence or presence of TAPBPR ( Figure 3A). The large molar excess of unlabeled competitor peptide ensures that once a fluorescent peptide dissociates, it does not rebind, but is replaced by an unlabeled competitor-peptide molecule. The observed rate constant is thus solely determined by the dissociation rate constant of the fluorescent peptide. The condition of this assay mimics the environment of the PLC, where optimal, high-affinity peptides abound. For the mouse MHC I allomorph H2-D b , TAPBPR wt and the scoop-loop variants accelerated the uncatalyzed peptide release (2.53 ± 0.37 Â 10 À3 s À1 ) to a similar extent. The TAPBPR DSL mutant lacking the entire scoop loop exhibited slightly reduced activity (7.68 ± 1.17 Â 10 À3 s À1 ) compared to the wt protein (10.41 ± 0.54 Â 10 À3 s À1 ), whereas TAPBPR Tsn-SL was slightly more active (12.64 ± 1.03 Â 10 À3 s À1 ) ( Figure 3B,C). When we performed the experiment at a much lower TAPBPR concentration (75 nM), the TAPBPRs retained their activity, and the gradual activity differences between the variants remained ( Figure 3-figure supplement 1). This suggests that TAPBPR wt and the scoop-loop mutants have similar affinities for H2-D b . TAPBPR wt was even able to catalyze displacement of a high-affinity peptide from H2-D b , although the catalytic effect was considerably smaller (1.8-fold acceleration) than for H2-D b loaded with the medium-affinity peptide (4.1-fold acceleration) (Figure 3-figure supplement 2A,B). In a second set of experiments, we analyzed peptide dissociation from the human MHC I allomorph HLA-A*02:01. Similar to the experiments with H2-D b , in a peptide-rich environment (1000-fold molar excess of peptide), the highest catalytic activity towards HLA-A*02:01 was observed for TAPBPR Tsn-SL , followed by TAPBPR wt and TAPBPR DSL ; yet, the differences in activity between the three TAPBPRs were more pronounced, and the acceleration of the uncatalyzed peptide dissociation from HLA-A*02:01 (1.90 ± 0.04 Â 10 À3 s À1 ) by TAPBPR Tsn-SL (26.31 ± 2.59 Â 10 À3 s À1 ) and TAPBPR wt (15.79 ± 0.71 Â 10 À3 s À1 ) was significantly higher than for H2-D b , while the activity of TAPBPR DSL (8.52 ± 1.18 Â 10 À3 s À1 ) remained almost the same ( Figure 3D,E).
The validity of our peptide exchange assay was confirmed by two interface mutants of TAPBPR wt , TN3-Ala and TN6. The TN3 (E72K) and TN6 (E185K, R187E, Q189S, Q261S) mutants were initially described for Tsn to significantly reduce or abolish MHC I binding (Dong et al., 2009). The impact of the TN6 mutations on MHC I interaction was later confirmed for TAPBPR (Morozov et al., 2016). According to the TAPBPR-MHC I crystal structures (Jiang et al., 2017;Thomas and Tampé, 2017a), the residue in TAPBPR (E105) corresponding to the mutated residue in Tsn-TN3 forms a hydrogen bond with the swung-out Y84 of the MHC heavy chain, which is involved in coordinating the C-terminus of the peptide in liganded MHC. We reasoned that a mutation to Ala instead of Lys might increase the mutational effect and therefore generated the TN3-Ala mutant. Two of the mutated residues in TN6 (R210 and Q212) are part of the jack hairpin of TAPBPR and form several interactions with MHC I heavy-chain residues, while Q275 lies in the interface with the a2-1 helix and the b8 sheet in the floor of the MHC I binding groove. Consequently, TN3-Ala and TN6 displayed drastically reduced activity towards H2-D b in our peptide-exchange experiment, with peptide dissociation rate constants close to the value of the uncatalyzed reaction ( Figure 3C,F). In summary, the results of our exchange assays demonstrate that under peptide-rich condition, the tested TAPBPR variants differ gradually in their displacement activity in an allomorph-dependent manner. But even the TAPBPR DSL mutant lacking the scoop loop is still able to substantially accelerate peptide dissociation from MHC I.

The scoop loop acts as an internal peptide competitor
In the TAPBPR-MHC I crystal structure, the scoop loop binds in the F pocket region of the MHC binding groove and appears to act as a surrogate for the peptide C terminus (Thomas and Tampé, 2017a). This notion is corroborated by our SEC analyses, which show that the scoop loop stabilizes peptide-free MHC I. We therefore wondered if the scoop loop impedes rebinding of displaced peptide and functions 'in cis' as a tethered, internal peptide competitor in the F pocket with extremely high effective concentration. To test this hypothesis, we modified the peptide exchange assay for H2-D b and HLA-A*02:01 by adding in a first step only TAPBPR without competitor peptide, which allowed us to monitor the change in free and bound fluorescent peptide under the influence of peptide rebinding in the presence of TAPBPR ( Figure 4A). This condition mimics the physiological environment TAPBPR is operating in, where optimal replacement peptides are scarce. Strikingly, after addition of the different TAPBPRs to H2-D b loaded with fluorescent peptide, the polarization changes, which correspond to the changes in the ratio of free to bound peptide, diverged dramatically ( Figure 4B). Peptide dissociation was most pronounced for TAPBPR wt with the native scoop loop, reaching~60% peptide release, whereas only~12% of the peptide population was released from H2-D b by TAPBPR Tsn-SL , and almost no decrease in polarization was caused by TAPBPR DSL . Similar to our original peptide exchange assay (Figure 3), differences between the two MHC I allomorphs were observed: In comparison to H2-D b , TAPBPR Tsn-SL -induced peptide dissociation from HLA-A*02:01 was significantly stronger, approaching the level of peptide release induced by TAPBPR wt (Figure 4-figure supplement 1A). Peptide release was also peptide-dependent, as H2-   Figure 4 continued on next page D b loaded with a high-affinity peptide led to a significantly smaller decline in bound peptide (Figure 3-figure supplement 2C). After addition of competitor peptide (2 nd step), the observed dissociation rate constants were in the same range as the values determined for the one-step experiment. Moreover, the level of released peptide after TAPBPR addition was titratable and reached saturation at 3 mM TAPBPR ( Figure 4C-E, Figure 4-figure supplement 1B). Under the given conditions, TAPBPR wt was able to dissociate 70% (H2-D b ) and 80% (HLA-A*02:01) of total MHC I-associated peptide, respectively ( Figure 4C, Figure 4-figure supplement 1B). These results suggest that the scoop loop interferes with re-binding of displaced peptide. It can only be completely dislodged from the MHC I binding pocket by a high-affinity peptide. The scoop loop thus acts as a crucial selectivity filter during peptide editing on MHC I.

Discussion
Tsn and TAPBPR are MHC I-dedicated chaperones, which facilitate loading and selective exchange of antigenic peptides and thereby generate stable pMHC I complexes that shape a hierarchical immune response. The molecular underpinnings of their chaperone and peptide proofreading activities have only recently been uncovered by crystal structures of the TAPBPR-MHC I complex (Jiang et al., 2017;Thomas and Tampé, 2017a). Notably, one of the X-ray structures resolved a loop structure, termed the scoop loop, that is wedged into the F-pocket region of the empty MHC I binding groove and has been postulated to play an important role during peptide exchange (Thomas and Tampé, 2017a). Here, we show that the TAPBPR scoop loop is indeed critically important in chaperoning intrinsically unstable empty MHC I clients in a peptide-depleted environment. This is illustrated by the reduced chaperone activity of TAPBPR Tsn-SL , which harbors the shorter Tsn scoop loop, and by the dramatically reduced lifetime of the TAPBPR DSL complex. In a peptide-rich, PLC-like environment, emulated by our one-step displacement experiments, the TAPBPR Tsn-SL mutant displays the highest activity, while TAPBPR DSL retains the ability to displace peptide. The latter observation appears to be in contrast to the study by Ilca et al. which found that TAPBPR with a mutated, but full-length scoop loop loses its ability to effectively mediate peptide dissociation (Ilca et al., 2018). In addition to stabilizing the chaperone-MHC I complex, we demonstrate that the TAPBPR scoop loop acts as an internal peptide competitor, and thus, as a selectivity filter in the discrimination between low-and high-affinity peptides. Although a direct competition appears to be the most obvious explanation for the effect on peptide rebinding, we cannot exclude that the scoop loop exerts its influence on peptide rebinding by an allosteric mechanism. The peptide-filtering activity seems to be allomorph-dependent for TAPBPR Tsn-SL . Our current interpretation of this allomorph specificity is that the Tsn scoop loop interacts more strongly with the F-pocket region of HLA-A*02:01 and is therefore able to impede peptide rebinding more efficiently than in the case of H2-D b . In contrast, TAPBPR wt shows a strong peptide release activity towards both MHC I allomorphs.
Based on the new insights, we propose the following model of TAPBPR-catalyzed peptide optimization on MHC I ( Figure 5): The large concave surface formed by the N-terminal domain of TAPBPR mediates its initial encounter with a suboptimally-loaded MHC I, assisted by the C-terminal domain of TAPBPR, which contacts the a3 domain of the MHC I heavy chain and b2m. TAPBPR facilitates the release of low-to medium-affinity peptides primarily by widening the peptide-binding groove through the MHC I a2-1-helix, fastening the peptide-coordinating Tyr84, distorting the floor of the binding groove, and shifting the position of b2m (Jiang et al., 2017;Thomas and Tampé, 2017a). This remodeling is made possible by the intrinsic plasticity of MHC I molecules Garstka et al., 2011;McShan et al., 2019;Natarajan et al., 2018;Thomas and Tampé, 2017b;van Hateren et al., 2017;van Hateren et al., 2015;Wieczorek et al., 2017), and it appears to be induced primarily by structural elements of TAPBPR that lie outside the scoop loop. As a result, the TAPBPR DSL mutant lacking the scoop loop is still able to catalyze peptide displacement. Once the suboptimal peptide has been released, the scoop loop occupies the position of the peptide C terminus in the F-pocket region. The scoop loop thereby contributes to the stabilization of the peptidedeficient binding groove. Our two-step peptide exchange -mimicking a peptide-depleted environment -demonstrates that the scoop loop functions at the same time as a peptide selectivity filter by impeding re-binding of the replaced peptide, either through direct competition with the C terminus of the incoming replacement peptide or through an allosteric mechanism. Hence, the scoop loop contributes to the significant affinity decrease of incoming peptides for the MHC I groove in the presence of TAPBPR (McShan et al., 2018). Assuming a mode of direct competition, the replacement peptide would dock in the MHC I groove first with its N terminus, before it competes with the TAPBPR scoop loop over the F pocket region (Hafstrand et al., 2019;Thomas and Tampé, 2017a). Negative allosteric coupling between different parts of the MHC I molecule might play a role in the final release of TAPBPR . The shorter scoop loop in Tsn suggests that its selective pressure on the replacement peptide is weaker than in TAPBPR. Indeed, our fluorescence polarization and SEC analyses show that the tapasin scoop loop in TAPBPR Tsn-SL is less efficient in preventing re-binding of dissociated peptide.
Physiologically, these observations might be explained by the fact that Tsn functions within the PLC, a 'nanocompartment' characterized by an abundant and diverse supply of optimal peptides, reaching a bulk concentration of up to 16 mM before the TAP transporter is arrested by trans-inhibition (Grossmann et al., 2014). Moreover, Tsn is supported by other PLC chaperones in stabilizing empty MHC I clients. In contrast, TAPBPR operates as a single MHC I-dedicated chaperone outside the PLC in environments where the concentration of high-affinity peptides is drastically lower and  Figure 5. Proposed mechanistic functions of the scoop loop in catalyzed peptide proofreading. MHC I molecules bound to low-affinity peptide are recognized by the peptide editor (TAPBPR) (step 1). The editor lowers the peptide affinity of the suboptimally-loaded MHC I and induces dissociation of the low-to medium-affinity peptide (step 2). The scoop loop, which inserts into the F-pocket region of the peptide-binding groove, crucially contributes to the stabilization of the empty MHC I. In the absence of suitable peptides, empty MHC I clients are thereby held in a stable state until they can be loaded with an optimal epitope, for example in the PLC. Re-binding of the low-affinity peptide (step 3) is impeded by the scoop loop, through direct competition and/or via allosteric means. Only high-affinity peptides are able to compete with the editor over key regions of the peptidebinding groove (step 4) to eventually displace the scoop loop and the editor from the MHC I (step 5). The displaced editor is now ready for a new round of peptide selection, and the stable pMHC I complex is licensed to travel via the Golgi apparatus to the cell surface.
MHC I clients have to be stabilized in a peptide-receptive state for extended periods of time. Longterm stabilization of suboptimally-loaded or empty MHC I by TAPBPR also allows the major ER/cis-Golgi glycoprotein folding sensor UGGT1 (UDP-glucose:glycoprotein glucosyltransferase 1) to reglucosylate the MHC I molecule in order to feed it back into the calnexin/calreticulin cycle and/or allow recruitment of the MHC I to the PLC Thomas and Tampé, 2019). In conclusion, the evidence provided by our study indicates that the scoop loop is evolutionarily finetuned to enable Tsn and TAPBPR to accomplish their dual function as chaperone and proofreader in the specific subcellular location they operate in. By serving both as a stabilizing element and as selectivity filter in TAPBPR, the scoop loop influences peptide editing and impacts the repertoire of MHC I-associated epitopes presented on the cell surface.

Materials and methods
Key resources

DNA constructs
The DNA constructs of human b2m, the ectodomain of mouse H2-D b , and TAPBPR wt were identical to the ones previously described (Thomas and Tampé, 2017a), except for position 97 in TAPBPR wt , which contained the native cysteine. The TAPBPR scoop loop mutants TAPBPR Tsn-SL and TAPBPR DSL were generated by overlap extension PCR, the TN3-Ala and TN6 mutants were generated by sitedirected mutagenesis. The TN3-Ala and TN6 mutants harbored the same mutations that were described for the corresponding mutants of Tsn (Dong et al., 2009), except that in TN3-Ala E105 was mutated to alanine. TAPBPR Tsn-SL , TAPBPR DSL , TN3-Ala, and TN6 all contained the C97A mutation. Human HLA-A*02:01 (amino acids 1-278) was cloned into pET-28 (Novagen, Merck Millipore, Darmstadt, Germany) and ended in a C-terminal His 6 -tag preceded by a linker (sequence: HE). The amino acid numbering of TAPBPR is based on the mature protein as defined by N-terminal sequencing (Zhang and Henzel, 2004).

Protein expression
Human b2m and the ectodomains of mouse H2-D b and human HLA-A*02:01 were expressed as inclusion bodies in Escherichia coli BL21(DE3) as described before (Rodenko et al., 2006;Thomas and Tampé, 2017a). TAPBPR proteins were expressed in Spodoptera frugiperda (Sf21 or Sf9) insect cells according to standard protocols for the Bac-to-Bac system (Thermo Fisher Scientific, Waltham, MA). A high-titer recombinant baculovirus stock was used to infect the insect cells at a density of 1.5-2.0 Â 10 6 cells/mL, which were cultivated in Sf-900 III SFM medium (Thermo Fisher Scientific) at 28˚C. The cell culture medium containing secreted TAPBPR was harvested 72 hr after infection.
Refolding and purification of MHC I allomorphs H2-D b and HLA-A*02:01 were refolded from inclusion bodies by rapid dilution in the presence of purified b2m and peptide according to established protocols (Rodenko et al., 2006). Refolded MHC I complexes were purified by SEC (Superdex 200 Increase 10/300, GE Healthcare) in 1xHBS and concentrated by ultrafiltration (Amicon Ultra, Merck Millipore).

Purification of TAPBPR proteins
TAPBPR proteins were purified from the insect cell culture medium by IMAC according to a protocol published earlier (Thomas and Tampé, 2017a), polished by SEC (Superdex 200 Increase 10/300, GE Healthcare) in 1xHBS, and concentrated by ultrafiltration (Amicon Ultra, Merck Millipore).

Peptide exchange
Dissociation of fluorescently labeled peptide from MHC I was monitored at 23˚C in 1xHBS by fluorescence polarization (Fluorolog-3 spectrofluorometer, Horiba Jobin Yvon, Bensheim, Germany) with l ex/em of 530/560 nm. One-step and two-step dissociation assays were carried out with 300 nM MHC I loaded with TAMRA-labeled peptide, 1 mM TAPBPR, and 300 mM competitor peptide. Dissociation rate constants were determined in GraphPad Prism using a one-phase exponential decay regression.

MHC I-chaperone complex formation
In the presence of purified TAPBPR (3 mM), photo-P18-I10-loaded H2-D b (10 mM) was irradiated with UV light (36 nm, 185 mW/cm 2 , 120 s) on ice and afterwards incubated for 10 min at room temperature. Samples were subsequently centrifuged at 10,000xg for 10 min and analyzed by analytical SEC on a Superdex 75 (3.2/300) column (GE Healthcare). SEC runs were conducted in 1xHBS and monitored by absorbance at 280 nm. Chromatograms were deconvoluted into three Gaussian functions using the program Fityk 1.3.1 (Wojdyr, 2010). The amount of complex was assessed by the area of the complex peak.

TAPBPR-MHC I complex stability
Purified peptide-deficient TAPBPR wt -H2-D b , TAPBPR Tsn-SL -H2-D b , and TAPBPR DSL -H2-D b complexes were analyzed via analytical SEC either on a Superdex 75 (3.2/300) or a Superdex 200 (3.2/300) column (GE Healthcare) at a flow rate of 0.075 mL/min. A separate sample of purified TAPBPR wt -H2-D b complex was incubated with a 100-fold molar excess of high-affinity peptide prior to re-analysis by SEC.