A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR

Nikolaos G Sgourakis,Nathan A May,Lisa F Boyd,Jinfa Ying,Ad Bax,David H Margulies

doi:10.1074/jbc.m115.689661

Abstract

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L(d) by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L(d), "mini-H2-L(d)," consisting of only the α1α2 platform domain. Mini-H2-L(d) refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β2-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β2-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L(d) MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.

Highlights

The m06/gp48 protein of murine cytomegalovirus (MCMV) binds to major histocompatibility complex class I (MHC-I) proteins, diverting them to lysosomes
Guided by previously reported co-immunoprecipitation results [5, 36], we probed for binding of the MHC-I allotype H2-Ld to the recombinant m06 (Fig. 2a)
MHC-I interference by viral proteins is a multifaceted process involving the targeting of a number of distinct intermediates along the peptide loading and presentation pathway [1]

Summary

Background

The m06/gp protein of MCMV binds to MHC-I proteins, diverting them to lysosomes. Results: Recombinant m06 binds weakly to H2-Ld MHC-I and tightly to mini-H2-Ld, which provides excellent NMR spectra for mapping the binding site. MiniH2-Ld refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the ␤2-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and ␤2-microglobulin-associated MHC-I. The ability to make complete backbone assignments of mini-H2-Ld permitted us to characterize its interaction with m06 through a discrete site located under the peptide-binding platform that partially overlaps with the ␤2m interface on the MHC-I heavy chain The identification of this region is consistent with in vitro binding experiments showing much weaker complex formation between m06 and ␤2m-associated MHC-I. This study defines an MHC-I binding site for a member of the m02-m16 family and suggests a novel strategy exploited by a viral immunoevasin to bind MHC-I molecules

Experimental Procedures

Results

Discussion