title: A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR.
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.
authors.affiliation: From the Laboratory of Chemical Physics, NIDDK, and.
authors.forename: Nikolaos G
authors.initials: NG
authors.lastname: Sgourakis
authors.affiliation: the Molecular Biology Section, Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: May
authors.affiliation: the Molecular Biology Section, Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Lisa F
authors.initials: LF
authors.lastname: Boyd
authors.affiliation: From the Laboratory of Chemical Physics, NIDDK, and.
authors.forename: Jinfa
authors.initials: J
authors.lastname: Ying
authors.affiliation: From the Laboratory of Chemical Physics, NIDDK, and bax@nih.gov.
authors.forename: Ad
authors.initials: A
authors.lastname: Bax
authors.affiliation: the Molecular Biology Section, Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892 dhm@nih.gov.
authors.forename: David H
authors.initials: DH
authors.lastname: Margulies
mesh_terms: D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018146:Muromegalovirus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D015395:Histocompatibility Antigens Class I; D014759:Viral Envelope Proteins
keywords: MCMV; antigen presentation; herpesvirus; immunoevasin; m06/gp48; major histocompatibility complex (MHC); nuclear magnetic resonance (NMR); protein-protein interaction; surface plasmon resonance (SPR)