ABSTRACT
Cytomegalovirus (CMV) is a widespread opportunistic pathogen that causes birth defects when transmitted transplacentally and severe systemic illness in immunocompromised individuals. MSL-109, a human monoclonal IgG isolated from a CMV seropositive individual, binds to the essential CMV entry glycoprotein H (gH) and prevents infection of cells. Here, we suggest a mechanism for neutralization activity by MSL-109. We define a genetic basis for resistance to MSL-109 and have generated a structural model of gH that reveals the epitope of this neutralizing antibody. Using surface-based, time-resolved FRET, we demonstrate that gH/gL interacts with glycoprotein B (gB). Additionally, we detect homodimers of soluble gH/gL heterodimers and confirm this novel oligomeric assembly on full-length gH/gL expressed on the cell surface. We show that MSL-109 perturbs the dimerization of gH/gL:gH/gL, suggesting that dimerization of gH/gL may be required for infectivity. gH/gL homodimerization may be conserved between alpha- and betaherpesviruses, because both CMV and HSV gH/gL demonstrate self-association in the FRET system. This study provides evidence for a novel mechanism of action for MSL-109 and reveals a previously undescribed aspect of viral entry that may be susceptible to therapeutic intervention.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Cytomegalovirus Infections/drug therapy , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , Antibodies, Viral/pharmacology , Base Sequence , CHO Cells , COS Cells , Chlorocebus aethiops , Cricetulus , Dimerization , Drug Resistance, Viral/immunology , Epitope Mapping , Human Umbilical Vein Endothelial Cells , Humans , Molecular Sequence Data , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/geneticsABSTRACT
Nectins (nectin1-4) and Necls [nectin-like (Necl1-5)] are Ig superfamily cell adhesion molecules that regulate cell differentiation and tissue morphogenesis. Adherens junction formation and subsequent cell-cell signaling is initiated by the assembly of higher-order receptor clusters of cognate molecules on juxtaposed cells. However, the structural and mechanistic details of signaling cluster formation remain unclear. Here, we report the crystal structure of poliovirus receptor (PVR)/Nectin-like-5/CD155) in complex with its cognate immunoreceptor ligand T-cell-Ig-and-ITIM-domain (TIGIT). The TIGIT/PVR interface reveals a conserved specific "lock-and-key" interaction. Notably, two TIGIT/PVR dimers assemble into a heterotetramer with a core TIGIT/TIGIT cis-homodimer, each TIGIT molecule binding one PVR molecule. Structure-guided mutations that disrupt the TIGIT/TIGIT interface limit both TIGIT/PVR-mediated cell adhesion and TIGIT-induced PVR phosphorylation in primary dendritic cells. Our data suggest a cis-trans receptor clustering mechanism for cell adhesion and signaling by the TIGIT/PVR complex and provide structural insights into how the PVR family of immunoregulators function.
Subject(s)
Cell Adhesion , Receptors, Immunologic/metabolism , Receptors, Virus/metabolism , Signal Transduction , Crystallography, X-Ray , Humans , Models, Molecular , Protein Conformation , Receptors, Immunologic/chemistryABSTRACT
Secretory and membrane proteins carry amino-terminal signal sequences that, in cotranslational targeting, are recognized by the signal recognition particle protein SRP54 without sequence specificity. The most abundant membrane proteins on Earth are the light-harvesting chlorophyll a/b binding proteins (LHCPs). They are synthesized in the cytoplasm, imported into the chloroplast, and posttranslationally targeted to the thylakoid membrane by cpSRP, a heterodimer formed by cpSRP54 and cpSRP43. We present the 1.5 angstrom crystal structure of cpSRP43 characterized by a unique arrangement of chromodomains and ankyrin repeats. The overall shape and charge distribution of cpSRP43 resembles the SRP RNA, which is absent in chloroplasts. The complex with the internal signal sequence of LHCPs reveals that cpSRP43 specifically recognizes a DPLG peptide motif. We describe how cpSPR43 adapts the universally conserved SRP system to posttranslational targeting and insertion of the LHCP family of membrane proteins.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/metabolism , Light-Harvesting Protein Complexes/metabolism , Signal Recognition Particle/chemistry , Signal Recognition Particle/metabolism , Amino Acid Motifs , Amino Acid Sequence , Ankyrin Repeat , Arabidopsis/chemistry , Arabidopsis Proteins/metabolism , Calorimetry , Chloroplast Proteins , Crystallography, X-Ray , Dimerization , Hydrophobic and Hydrophilic Interactions , Light-Harvesting Protein Complexes/chemistry , Models, Biological , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Protein Subunits , RNA, Plant/chemistry , RNA, Plant/metabolism , Thylakoids/metabolismABSTRACT
Two GTPases in the signal recognition particle and its receptor (FtsY) regulate protein targeting to the membrane by formation of a heterodimeric complex. The activation of both GTPases in the complex is essential for protein translocation. We present the crystal structure of chloroplast FtsY (cpFtsY) at 1.75 A resolution. The comparison with FtsY structures in different nucleotide bound states shows structural changes relevant for GTPase activation and provides insights in how cpFtsY is pre-organized for complex formation with cpSRP54. The structure contains an amino-terminal amphipathic helix similar to the membrane targeting sequence of Escherichia coli FtsY. In cpFtsY this motif is extended, which might be responsible for the enhanced attachment of the protein to the thylakoid membrane.
