Effects of HLA single chain trimer design on peptide presentation and stability

Abstract

MHC class I "single-chain trimer" molecules, coupling MHC heavy chain, β₂-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation.

Keywords: HLA single-chain trimers; X-ray crystallography; human class I leukocyte antigens; pan-anti-class I antibodies; peptide presentation.

Figure 1

SCT design, structure, and peptide binding. (A) Native pHLA and SCT design schematics: the HLA heavy chain α1/α2/α3 and β₂m domains are shown in blue. The linkers incorporated into the SCT design, shown in purple, link the C-terminus of the peptide to the N-terminus of β₂m and the C-terminus of β₂m to the N-terminus of the HLA heavy chain. The peptide binding pockets, A and F, for the N- and C-terminal peptide residues, respectively are highlighted. While native HLA contains a transmembrane domain, the SCT design is truncated at the C-terminus of the ectodomain and tagged with a 6× histidine purification tag. (B) Two views of HLA SCT^H74L/Y84C crystal structure (PDB accession code 7SR0) shown as a gray transparent surface with an underlying carton ribbon representation, with secondary structure elements indicated. Ribbons are colored as a blue-to-red spectrum (blue: N-terminus, red: C-terminus) for the heavy chain and dark grey for β₂m. The bound peptide is shown as purple spheres. (C) The SCT peptide binding cleft is shown in a molecular surface representation highlighting the A-F pockets. The YML 9-mer peptide from the SCT^H74L/Y84C crystal structure is shown in a purple stick representation with p2 (Met) and p9 (Thr) bound in the B- and F-pockets of the HLA binding cleft respectively. Ordered linker residues are colored orange in the stick representation and the location of the Y84 cysteine mutation is shown in orange on the surface.

Figure 2

Evaluation of SCT design thermostabilities and comparison of experimental stability with predicted peptide binding ranks. (A) Thermostabilities are plotted for a subset of peptide-specific SCTs and peptide-matched pHLA-I^RFs including averaged thermostabilities for each class of SCT design (Y84: n=14; H74L Y84: n= 14; Y84A: n= 31; H74L Y84A: n= 14; Y84C: n= 18; H74L Y84C: n= 23; Y84C A139C: n= 14) along with the averaged thermostabilities for pHLA-I^RFs (n=20). Data points shown for peptide-specific SCT designs and matched pHLA-I^RFs are calculated as an average of three replicate measurements (shown in colored circles). Data points for the “Average” represent an average of all SCTs in the specified SCT class or pHLA-I^RF with Tm measurements run in triplicate for each SCT or pHLA-I^RF included in the average. (B) NetMHCpan predicted binding ranks are plotted versus thermostability measurements for SCTs and pHLA-I^RFs: Considering pHLA-I^RF Tms as the most representative of “native” pHLA-I stability, the measured Tms over 20 examples across six alleles, ranged from 42.1 to 67.4° C, with an average of 53 ± 7° C (vertical pink line); the average Tms for Y84A SCT (n=29) and H74L/Y84C (n=25) constructs are 47 ± 3°C (vertical blue line) and 54 ± 3°C (vertical green line), respectively, noting that this was a very non-random selection of SCTs and pHLA-I^RFs with few matched peptide/allele pairs.

Figure 3

Structural evaluation of SCT designs including the VHH co-crystallization reagent VHH-AD01 (A) Recognition of β₂m by anti-HLA-I VHH-AD01 is shown in cartoon ribbon representation, with the VHH colored pink overall with CDR1 highlighted in lime, CDR2 in yellow, and CDR3 in blue. The SCT heavy chain is shown in black-and-white cartoon representation with β₂m in solid gray (PDB accession code 7SQP). Secondary structure elements are indicated. (B) The native structure of HLA-A*02:01 (PDB accession code 6RSY) is shown on the left with the superposition of all 11 SCTs determined herein shown on the right. Structures are shown in putty representations where the backbone is displayed as a tube with a diameter and color correlating with the experimentally determined B-factors (blue/narrow = low B-factor, red/fat = high B-factor) on a relative scale. (C) Superposition of all HLA-A*02:01 SCT structures reported in this work plus a reference native HLA-I structure are shown in cartoon ribbon representations: SCT^Y84A in grey; SCT^H74L/Y84C in teal; SCT^Y84C/A139C in blue; χSCT in lavender; native HLA-A*02:01 (PDB accession code 6RSY) in yellow. The YML 14mer peptide from SCT^H74L/Y84C (PDB accession code 7SR4) is shown as a teal noodle. The side-chains of the Y84C and A139C mutations are shown in orange stick representations. Illustrative RMSD values from global alignments performed with PyMOL (25) on all main chain heavy atoms in the α1α2 domains are: 0.19 Å between SCT^Y84A (PDB accession code 7SQP) and SCT^H74L/Y84C (PDB accession code 7SR3), and 0.18 Å between SCT^H74L/Y84C (PDB accession code 7SR4) and SCT^Y84C/A139C (PDB accession code 7ST3). For comparison, the RMSD for two identical molecules in the AU of PDB accession code 7ST3 is 0.11 Å, and the RMSD between the reference HLA-A*02:01 structure and SCT^Y84C/A139C (PDB accession code 7SR5) with the same WT-1 peptide is 0.45 (Å) Pairing of murine H-2K^d α3 with human HLA-A*02:01 α1/α2 domains in the murine/human χSCT also had little effect on the overall structure of the binding cleft, with an alignment yielding an RMSD of 0.24 Å between SCT^{Y84A χ} (PDB accession code 6E1I) and SCT^Y84A (PDB accession code 7SQP). (D) Top: bird’s eye view of the α1α2 domain, shown as a cartoon ribbon, showing the bound peptide in a stick representation colored by atom type and the side-chain of residue 74, H or L, circled with a dashed line. Bottom: stereo view of the side-chains of residues neighboring the H74L mutation from the superposition of the YML12-mer SCT^H74L/Y84C (teal; PDB accession code 7SR3) and YML12-mer SCT^Y84A (blue; PDB accession code 7SQP) structures. The main chain of the YML 12-mer peptide is shown with carbon atoms in orange in both views for reference.

Figure 4

Peptide binding in different SCT designs. (A) Left: peptide binding modes observed across the YML series, with peptides shown as main-chain backbones in cartoon representations, colored according to peptide length and SCT design as indicated. Right: three orthogonal views of the superposition of all peptides are shown with the α1α2 domain shown as a black-and-white cartoon ribbon. (B) The YML 14-mer peptide is shown in two different SCT design-dependent conformations in a superposition of the SCT^Y84A and SCT^H74L/Y84C structures. The peptides are shown in molecular surface representations colored as indicated. The α1α2 domain, colored dark gray with the α2 helix removed for clarity, is shown as a cartoon ribbon oriented with the peptide C-terminus on the left. (C) The electron density map (blue mesh) is shown for the YML 14-mer SCT^Y84C/A139C structure, illustrating the two alternative binding modes present in the structure. The YML 14-mer peptides are shown in stick representations, oriented with N-termini on the left, colored with carbon atoms either in black or dark gray.

Figure 5

The observed noncanonical 8-mer binding mode. (A) Sequence logo generated from 1,883 8-mer peptides isolated from HLA-A*24:02 via the Artemis mass-spec procedure² is shown. The p1-p8 peptide positions are graphed on the abscissa and information content at each position in bits is graphed on the ordinate. Note that the tyrosine constituting the p2 anchor residue for HLA-A*24:02 9-mer and longer peptides partially shifts register to p1 for 8-mers (arrow), indicating utilization of an alternate binding mode where 8-mer p1 occupies the B-pocket. (B) The 8-mer YPPVPETF peptide/SCT crystal structure (tyrosine at p1; PDB accession code 7SRK) is shown as a gray molecular surface, with the peptide shown in blue in a main-chain cartoon/side-chain stick representation, binding in the non-canonical mode. The logo peptide (YLAAAAAAV, tyrosine at p2) from the A*02:01 SCT^Y84C/A139C crystal structure (PDB accession code 7STG) is superimposed in black to contrast canonical binding.