CD4 glycoprotein on the surface of T cells helps in the immune response and is the receptor for HIV infection. The structure of a soluble fragment of CD4 determined at 2.3 Å resolution reveals that the molecule has two intimately associated immunoglobulin-like domains. Residues implicated in HIV recognition by analysis of mutants and antibody binding are salient features in domain D1. Domain D2 is distinguished by a variation on the β-strand topologies of antibody domains and by an intra-sheet disulphide bridge.CD4, a cell-surface glycoprotein found primarily on T lymphocytes, is required to shape the T-cell repertoire during thymic development and to permit appropriate activation of mature T cells 1 . T cells that recognize antigens associated with class II major histocompatibility complex (MHC) molecules, mainly T helper cells, express CD4. Evidence is accumulating that CD4 and the T-cell receptor coordinately engage class II molecules on antigenpresenting cells to mediate an efficient cellular immune response, and that engaged CD4 may transmit a signal to an associated cytoplasmic tyrosine kinase, p56 lck .CD4 belongs to the immunoglobulin superfamily of molecules which generally serve in recognition processes 2,3 . The sequence of CD4 4,5 indicates that it consists of a large (~370 residues) extracellular segment composed of four tandem immunoglobulin-like domains, a single transmembrane span, and a short (38 residues) C-terminal cytoplasmic tail. The first domain (D1) shares several features with immunoglobulin variable domains, but the sequence similarities between immunoglobulins and the other extracellular domains (D2, D3 and D4) are more remote.In humans, CD4 can be subverted from its normal immuno-supportive role to become the receptor for infection by the human immunodeficiency virus (HIV) 1,6,7 . Recombinant soluble CD4 proteins bind to the HIV envelope glycoprotein gp120, and can thus inhibit viral infection and virus-mediated cell fusion in vitro (refs 8, 9 and references therein). (refs 21-23 and unpublished results), the main flexibility seems to be at the D2 to D3 junction. We have now crystallized a truncated derivative of CD4 that diffracts well, and here we report its atomic structure. This recombinant fragment 8 as secreted from Chinese hamster ovary (CHO) cells consists of residues 1-183 of human CD4 plus two missense residues, Asp-Thr; and it is unglycosylated. This molecule, which we refer to as D1D2, is as active as sCD4 in binding to gp120 (dissociation constant K d ≃ 3 nM) and retains all antibody epitopes mapped to these domains of CD4 (ref. 8 and unpublished results). Others have crystallized similar fragments from the N-terminal half of sCD4 24,25 and the structure of one is reported in the accompanying paper 25 . HHS Public AccessHere we describe the D1D2 structure in comparison with that of immunoglobulin domains, provide a geometrical definition for HIV recognition sites, and discuss implications of the structure for normal CD4 function and evolution of the immunoglobu...

Hydrogen peroxide (H2O2) has been implicated recently as an intracellular messenger that affects cellular processes including protein phosphorylation, transcription and apoptosis. A set of novel peroxidases, named peroxiredoxins (Prx), regulate the intracellular concentration of H2O2 by reducing it in the presence of an appropriate electron donor. The crystal structure of a human Prx enzyme, hORF6, reveals that the protein contains two discrete domains and forms a dimer. The N-terminal domain has a thioredoxin fold and the C-terminal domain is used for dimerization. The active site cysteine (Cys 47), which exists as cysteine-sulfenic acid in the crystal, is located at the bottom of a relatively narrow pocket. The positively charged environment surrounding Cys 47 accounts for the peroxidase activity of the enzyme, which contains no redox cofactors.

Cancer cells express tumour-specific antigens derived via genetic and epigenetic alterations, which may be targeted by T-cell-mediated immune responses. However, cancer cells can avoid immune surveillance by suppressing immunity through activation of specific inhibitory signalling pathways, referred to as immune checkpoints. In recent years, the blockade of checkpoint molecules such as PD-1, PD-L1 and CTLA-4, with monoclonal antibodies has enabled the development of breakthrough therapies in oncology, and four therapeutic antibodies targeting these checkpoint molecules have been approved by the FDA for the treatment of several types of cancer. Here, we report the crystal structures of checkpoint molecules in complex with the Fab fragments of therapeutic antibodies, including PD-1/pembrolizumab, PD-1/nivolumab, PD-L1/BMS-936559 and CTLA-4/tremelimumab. These complex structures elucidate the precise epitopes of the antibodies and the molecular mechanisms underlying checkpoint blockade, providing useful information for the improvement of monoclonal antibodies capable of attenuating checkpoint signalling for the treatment of cancer.

The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines. Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial. By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein. In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s(-1). The disulfide bond-mediated conformation switch results in a metastable form that is locally strained by approximately 3 kcal mol(-1). On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of ␥H2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.Unlike kinases, which are derived from a common ancestor, the opposing phosphatases have evolved in separate, structurally distinct families. In fact, a variety of catalytic mechanisms have been harnessed to facilitate protein dephosphorylation. The protein-Ser/Thr phosphatases, such as the PPP and PPM families, mediate dephosphorylation by using two metal ions at the active site, activating a water molecule for nucleophilic attack on the substrate phosphate in a single-step reaction (1). The family of Cys-dependent protein-tyrosine phosphatases (PTPs) 4 utilizes a two-step catalytic mechanism involving an essential nucleophilic cysteinyl residue, which forms a Cysphosphate intermediate (2). More recently, a third family of Asp-dependent phosphatases belonging to the haloacid dehalogenase (HAD) superfamily (3) has been gaining prominence.Various HAD phosphatases have been linked to fundamental aspects of control of cell function. One of the best characterized is FCP1, which is an important regulator of transcription through dephosphorylation of the C-terminal domain of RNA polymerase II (4). Chronophin has been shown to dephosphorylate Ser-3 in cofilin and thereby function as a regulator of the actin cytoskeleton (5). More recently, a HAD phosphatase known as Dullard has been shown to dephosphorylate the phosphatidic acid phosphatase lipin, functioning in a phosphatase cascade that regulates nuclear membrane biogenesis (6). In these cases, the HADs are functioning as protein-Ser/Thr phosphatases. However, there is now also an example of a member of the HAD superfamily with the capacity to dephosphorylate phosphotyrosyl residues in substrates.Eyes Absent (EYA) was identified initially as a component of a network of transcription regulators, the retinal determination gene network that is responsible for eye development in Drosophila. It is now known to be involved in tissue and organ developme...