The Structure of HLA-DM, the Peptide Exchange Catalyst that Loads Antigen onto Class II MHC Molecules during Antigen Presentation

AbstractThe three-dimensional structure of the soluble ectodomain of HLA-DM has been determined to 2.5 Å resolution by X-ray crystallography. HLA-DM has both peptide exchange activity and acts as a chaperone to peptide-free class II MHC molecules. As predicted, the structure is similar to that of classical class II MHC molecules except that the peptide-binding site is altered to an almost fully closed groove. An unusual cavity is found at the center of the region that binds peptides in class II MHC molecules, and a tryptophan-rich lateral surface is identified that is a candidate both for binding to HLA-DR, to effect catalysis, and to HLA-DO, an inhibitor

Identification of the Lateral Interaction Surfaces of Human Histocompatibility Leukocyte Antigen (HLA)-DM with HLA-DR1 by Formation of Tethered Complexes That Present Enhanced HLA-DM Catalysis

Human histocompatibility leukocyte antigen (HLA)-DM is a major histocompatibility complex (MHC)-like protein that catalyzes exchange of antigenic peptides from MHC class II molecules. To investigate the molecular details of this catalysis we created four covalent complexes between HLA-DM and the MHC class II allele DR1. We introduced a disulfide bond between the naturally occurring cysteine β46 on HLA-DM and an engineered cysteine on the end of a linker attached to either the NH2- or the COOH terminus of an antigenic peptide that is tightly bound on DR1. We find that when DM is attached to the NH2 terminus of the peptide, it can, for all linker lengths tested, catalyze exchange of the peptide with a half-life a few minutes (compared with uncatalyzed t1/2 > 100 h). This rate, which is several orders of magnitude greater than the one we obtain in solution assays using micromolar concentrations of HLA-DM, is dominated by a concentration independent factor, indicating an intramolecular catalytic interaction within the complex. A similar complex formed at the COOH terminus of the peptide shows no sign of DM-specific intramolecular catalysis. Restrictions on the possible interaction sites imposed by the length of the linkers indicate that the face of DR1 that accommodates the NH2 terminus of the antigenic peptide interacts with the lateral face of HLA-DM that contains cysteine β46

Structure of phenylalanyl-tRNA synthetase from Thermus thermophilus

International audienceThe crystal structure of phenylalanyl-tRNA synthetase from Thermus thermophilus, solved at 2.9 A resolution, displays (alpha beta)2 subunit organization. Unexpectedly, both the catalytic alpha- and the non-catalytic beta-subunits comprise the characteristic fold of the class II active-site domains. The alpha beta heterodimer contains most of the building blocks so far identified in the class II synthetases. The presence of an RNA-binding domain, similar to that of the U1A spliceosomal protein, in the beta-subunit is indicative of structural relationships among different families of RNA-binding proteins. The structure suggests a plausible catalytic mechanism which explains why the primary site of tRNA aminoacylation is different from that of the other class II enzymes

Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39

The ecto-enzymatic domain of rat E-NTPDase1 CD39 was expressed and purified and diffraction-quality crystals of the enzyme were obtained

Molecular insights into recognition of GUCY2C by T-cell engaging bispecific antibody anti-GUCY2CxCD3

Abstract The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody’s activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb

Structure and functional interaction of the extracellular domain of human GABAB receptor GBR2

International audienceInhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABA(B) receptor is a G protein-coupled receptor central to mammalian brain function. Malfunction of GABA(B) receptor has been implicated in several neurological disorders. GABA(B) receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimeric interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABA(B) receptor that is unique to the GABAergic system

High Resolution Mapping of Bactericidal Monoclonal Antibody Binding Epitopes on <i>Staphylococcus aureus</i> Antigen MntC

<div><p>The <i>Staphylococcus aureus</i> manganese transporter protein MntC is under investigation as a component of a prophylactic <i>S</i>.<i>aureus</i> vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce <i>S</i>. <i>aureus</i> burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups (mAB 305-72-5 – group 1, mAB 305-78-7 – group 2, and mAB 305-101-8 – group 3) were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS). All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7) with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst <i>S</i>. <i>aureus</i> infection by preventing the capture and transport of Mn²⁺, a key element that the pathogen uses to evade host immunity.</p></div