Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV).The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 Å resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 Å resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor-and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.Severe acute respiratory syndrome (SARS), 3 a newly emerged infectious disease, claimed 813 lives from ϳ8000 patients during a 2003 global epidemic. In severe illness, influenza-like symptoms quickly progress to pneumonia, hypoxia, and acute respiratory distress and failure, resulting in 10% overall death rate with exceptionally high mortality among the elderly (1). A novel coronavirus (SARS-CoV) has been identified as the etiological agent of SARS. The SARS-CoV surface spike protein S mediates viral entry into the host cell (2) and includes two functional domains as follows: S1 (Gly 13 -Arg 667 ) and S2 (Ser 668 -Thr 1255 ). S1 contains the host-specific receptor binding domain (RBD), whereas S2 mediates fusion between viral and host cell membranes (3). Angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for the SARSCoV (4). The recently determined structure of the S1-RBD in complex with the extracellular domain of ACE2 (5) illustrates the structural basis for the initial step of virus-host recognition.As the mediator of host-specific SARS infection and a major viral surface antigen, the S protein is an attractive candidate for both vaccine development and immunotherapy. Marasco and co-workers (6) previously identified a potent neutralizing human monoclonal antibody against the S1 RBD, designated "80R," from two nonimmune (i.e. not restricted by B cell recombination) human antibody libraries. 80R binds S1 with nanomolar affinity, blocks the binding of S1 to ACE2, prevents the formation of syncytia in vitro (6), and inhibits viral replication in vivo (7). Deletion studies have shown that the 80R epitope on S1 is located in the minimal ACE2 binding domain, between residues 324 and 503 (6, 7).Here, we rep...
Cell cycle progression is controlled at several different junctures by the targeted destruction of cell cycle regulatory proteins. These carefully orchestrated events include the destruction of the securin protein to permit entry into anaphase, and the destruction of cyclin B to permit exit from mitosis. These destruction events are mediated by the ubiquitin/proteasome system. The human ubiquitin-conjugating enzyme, UbcH10, is an essential mediator of the mitotic destruction events. We report here the 1.95-Å crystal structure of a mutant UbcH10, in which the active site cysteine has been replaced with a serine. Functional analysis indicates that the mutant is active in accepting ubiquitin, although not as efficiently as wild-type. Examination of the crystal structure reveals that the NH 2 -terminal extension in UbcH10 is disordered and that a conserved 3 10 -helix places a lysine residue near the active site. Analysis of relevant mutants demonstrates that for ubiquitin-adduct formation the presence or absence of the NH 2 -terminal extension has little effect, whereas the lysine residue near the active site has significant effect. The structure provides additional insight into UbcH10 function including possible sites of interaction with the anaphase promoting complex/cyclosome and the disposition of a putative destruction box motif in the structure.Ubiquitin-mediated proteolysis regulates cell cycle progression at several key control points. At least two such control points occur in mitosis. One is at the transition from metaphase to anaphase and the other is at the exit from mitosis (for reviews, see Refs. 1-5). At the transition from metaphase to anaphase, the securin protein in the securin-separase protein complex is destroyed to release separase. The freed separase cleaves the protein complexes binding the sister chromatids together. Cleavage of these protein complexes is thought to facilitate sister chromatid segregation, and hence entry into anaphase. To exit from mitosis, cyclin B in the cyclin B-cdc2 complex must be destroyed. Destruction of cyclin B results in the inactivation of the cdc2 kinase. The inactivation of cdc2 is an essential event for resetting the cell cycle machinery (6).To accomplish the ubiquitination of securin and cyclin B (and other proteins targeted for ubiquitin-mediated destruction), three enzyme activities, designated E1 1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme, Ubc), and E3 (ubiquitin ligase), must work in concert (for review, see Ref. 7). The E1 protein activates ubiquitin and then transfers it to the E2 protein. The ubiquitin forms an adduct to the E2 protein via a thiol ester linkage between the active site cysteine of E2 and the carboxyl terminus of ubiquitin. The E2 then donates the ubiquitin to the target protein, either directly or in conjunction with the E3 activity. In some instances, the same protein possesses both the E2 and E3 activity. Ultimately, a polyubiquitin-target protein conjugate is formed that then is recognized by the proteasome. The...
The proteins securin and cyclin B are destroyed in mitosis by the ubiquitin/proteasome system. This destruction is important to mitotic progression. The N-terminal regions of these proteins contain the sequence features recognized by the ubiquitination system. We have demonstrated using circular dichroism and 1-D and 2-D nuclear magnetic resonance that these rather substantial regions are natively unfolded. Based on these ¢ndings, we propose a model that helps to explain previously enigmatic observations. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
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