Basic and translational applications of engineered MHC class I proteins

Abstract

Major histocompatibility complex (MHC) class I molecules can be engineered as single chain trimers (SCTs) that sequentially incorporate all three subunits of the fully assembled proteins, namely peptide, β2 microglobulin, and heavy chain. SCTs have been made with many different MHC-peptide complexes and are used as novel diagnostic and therapeutic reagents, as well as probes for diverse biological questions. Here, we review the recent and diverse applications of SCTs. These applications include new approaches to enumerate disease-related T cells, DNA vaccines, eliciting responses to pre-assembled MHC-peptide complexes, and unique probes of lymphocyte development and activation. Future applications of SCTs will be driven by their further engineering and the ever-expanding identification of disease-related peptides using chemical, genetic and computational approaches.

Figure 1. SCT design and structural features

(a) The original SCT design incorporated all three pMHC components into a single polypeptide chain by sequentially attaching OVA peptide (OVAp) (yellow) to β2m (magenta) to HC (cyan) with flexible linkers (orange). Two new generations of SCTs improve peptide binding within the SCT. The SCT^Y84A variant improves accommodation of the first linker, whereas the dtSCT variant contains a disulfide trap to staple peptide into the MHC ligand binding cleft. Disulfide bonds bridging the indicated cysteine residues are represented as green brackets. (b) The crystal structure of SCT^WT shows that SCTs adopt the same ectodomain structure as native pMHCI. This is represented in the ribbon diagram of SCT^WT, where OVAp and PBL are rendered as ball and stick models and colored as follows: OVAp and PBL carbon atoms, yellow and orange, respectively; nitrogen atoms, blue; oxygen atoms, red. The protein is oriented with the OVAp N terminus on the left and the membrane-proximal α3 domain at the bottom. A possible conformation for the BHL is represented and rendered as small orange balls. PBL: peptide-β₂m linker, BHL: β₂m-heavy-chain linker. Figure adapted from Mitaksov et al (14).

Figure 2. Linker accommodation in three generations of SCT

The panels in this figure illustrate native MHCI-OVAp binding and three generations of SCTs: SCT^WT, SCT^Y84A and dtSCT. In the native pMHCI, the HC residue Tyr84 is a steric impediment to C terminal peptide extensions. Thus in the SCT^WT, the linker extending from the C terminus of the peptide must bulge above Tyr84, which impairs peptide anchoring to F pocket residues in the HC ligand binding cleft (15). In the SCT^Y84A, the Ala84 substitution allows for better linker accommodation. In dtSCT, the dual substitution of Cys residues in position 84 of the HC and the second linker position (L2) introduces a disulfide trap. This trap essentially staples the peptide into the MHC groove to increase thermal stability and render the complex refractory to peptide exchange. The solvent-accessible surfaces of the peptide-binding grooves are displayed as blue dotted surfaces and the peptide (P1-P8) and first nine residues (SCT^wt; L1-L9) or six residues (SCT^Y84A and dtSCT; L1-L6) of PBL are represented as SPK models. The heavy chain pocket locations (A – F) are indicated. Figure adapted from Mitaksov et al (14).