Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL pro ) to cleave the virus-encoded polyproteins. We report here that the 3CL pro containing additional N-and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln The data indicate that immature 3CL pro can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL pro from the polyprotein and design of new structure-based inhibitors.

Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interface is more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composed of four structural domains, which show a different folding topology from those of the other oxidative decarboxylases. The active center is located at the interface between domains B and C. For human mitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and II, which is mediated by the movements of domains A and D. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analogue at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-112 and lysine-183 as the general acid and base, respectively. In addition, a divalent metal ion (Mn(2+) or Mg(2+)) is essential in helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternary structural integrity of the enzyme.

Cellular entry of human immunodeficiency virus type 1 (HIV-1) requires binding to both CD4 (ref, 1, 2) and to one of the chemokine receptors recently discovered to act as coreceptors. Viruses that infect T-cell lines to form syncytia (syncytium-inducing, SI) are frequently found in late-stage HIV disease and utilize the chemokine receptor CXCR-4; macrophage-tropic viruses are non-syncytium-inducing (NSI), found throughout disease and utilize CCR-5 (ref. 3-11). We postulated that CCR-5 gene defects might reduce infection risk in seronegative subjects and prolong AIDS-free survival in seropositive subjects with NSI but not SI virus. Homozygous (delta ccr5/delta ccr5) and heterozygous (CCR5/delta ccr5) CCR-5 deletions (delta ccr5) were found in 7 (2.7%) and 51 (19.5%), respectively, of 261 seronegative subjects from the San Francisco Men's Health Study. CCR-5/delta ccr5 genotype was identified in 33 of 172 (19.2%) nonprogressors and 25 of 234 (10.7%) progressors from the seropositive arm of this cohort. The delta ccr5 allele conferred a significant protective effect against HIV-1 infection (P = 0.001) and a survival advantage against disease progression (P = 0.02). Although both progressing and nonprogressing CCR5/delta ccr5 subjects were identified, a distinct survival advantage was shown for those with NSI virus (P < 0.0001). Thus, the protective effect of delta ccr5 against disease progression is lost when the infecting virus uses CXCR-4 as a coreceptor.

SARS (severe acute respiratory syndrome) has been one of the most severe viral infectious diseases last year and still remains as a highly risky public health problem around the world. Exploring the types of interactions responsible for structural stabilities of its component protein molecules constitutes one of the approaches to find a destabilization method for the virion particle. In this study, we performed a series of experiments to characterize the quaternary structure of the dimeric coronavirus main protease (M(pro), 3CL(pro)). By using the analytical ultracentrifuge, we demonstrated that the dimeric SARS coronavirus main protease exists as the major form in solution at protein concentration as low as 0.10 mg/mL at neutral pH. The enzyme started to dissociate at acidic and alkali pH values. Ionic strength has profound effect on the dimer stability indicating that the major force involved in the subunit association is ionic interactions. The effect of ionic strength on the protease molecule was reflected by the drastic change of electrostatic potential contour of the enzyme in the presence of NaCl. Analysis of the crystal structures indicated that the interfacial ionic interaction was attributed to the Arg-4...Glu-290 ion pair between the subunits. Detailed examination of the dimer-monomer equilibrium at different pH values reveals apparent pK(a) values of 8.0 +/- 0.2 and 5.0 +/- 0.1 for the Arg-4 and Glu-290, respectively. Mutation at these two positions reduces the association affinity between subunits, and the Glu-290 mutants had diminished enzyme activity. This information is useful in searching for substances that can intervene in the subunit association, which is attractive as a target to neutralize the virulence of SARS coronavirus.

The severe acute respiratory syndrome (SARS) coronavirus (CoV) main protease represents an attractive target for the development of novel anti-SARS agents. The tertiary structure of the protease consists of two distinct folds. One is the N-terminal chymotrypsin-like fold that consists of two structural domains and constitutes the catalytic machinery; the other is the C-terminal helical domain, which has an unclear function and is not found in other RNA virus main proteases. To understand the functional roles of the two structural parts of the SARS-CoV main protease, we generated the fulllength of this enzyme as well as several terminally truncated forms, different from each other only by the number of amino acid residues at the C-or N-terminal regions. The quaternary structure and K d value of the protease were analyzed by analytical ultracentrifugation. The results showed that the N-terminal 1-3 amino acid-truncated protease maintains 76% of enzyme activity and that the major form is a dimer, as in the wild type. However, the amino acids 1-4-truncated protease showed the major form to be a monomer and had little enzyme activity. As a result, the fourth amino acid seemed to have a powerful effect on the quaternary structure and activity of this protease. The last C-terminal helically truncated protease also exhibited a greater tendency to form monomer and showed little activity. We concluded that both the C-and the N-terminal regions influence the dimerization and enzyme activity of the SARS-CoV main protease.

The maturation of SARS coronavirus involves the autocleavage of polyproteins 1a and 1ab by the main protease (Mpro) and a papain-like protease; these represent attractive targets for the development of anti-SARS drugs. The functional unit of Mpro is a homodimer, and each subunit has a His-41cdots, three dots, centeredCys-145 catalytic dyad. Current thinking in this area is that Mpro dimerization is essential for catalysis, although the influence of the substrate binding on the dimer formation has never been explored. Here, we delineate the contributions of the peptide substrate to Mpro dimerization. Enzyme kinetic assays indicate that the monomeric mutant R298A/L exhibits lower activity but in a cooperative manner. Analytical ultracentrifugation analyses indicate that in the presence of substrates, the major species of R298A/L shows a significant size shift toward the dimeric form and the monomer-dimer dissociation constant of R298A/L decreases by 12- to 17-fold, approaching that for wild-type. Furthermore, this substrate-induced dimerization was found to be reversible after substrates were removed. Based on the crystal structures, a key residue, Glu-166, which is responsible for recognizing the Gln-P1 of the substrate and binding to Ser-1 of another protomer, will interact with Asn-142 and block the S1 subsite entrance in the monomer. Our studies indicate that mutation of Glu-166 in the R298A mutant indeed blocks the substrate-induced dimerization. This demonstrates that Glu-166 plays a pivotal role in connecting the substrate binding site with the dimer interface. We conclude that protein-ligand and protein-protein interactions are closely correlated in Mpro.

Viral proteases are essential for pathogenesis and virulence of severe acute respiratory syndrome coronavirus (SARS-CoV). Little information is available on SARS-CoV papain-like protease 2 (PLP2), and development of inhibitors against PLP2 is attractive for antiviral therapy. Here, we report the characterization of SARS-CoV PLP2 (from residues 1414 to 1858) purified from baculovirus-infected insect cells. We demonstrate that SARS-CoV PLP2 by itself differentially cleaves between the amino acids Gly180 and Ala181, Gly818 and Ala819, and Gly2740 and Lys2741 of the viral polypeptide pp1a, as determined by reversed-phase high-performance liquid chromatography analysis coupled with mass spectrometry. This protease is especially selective for the P1, P4, and P6 sites of the substrate. The study demonstrates, for the first time among coronaviral PLPs, that the reaction mechanism of SARS-CoV PLP2 is characteristic of papain and compatible with the involvement of the catalytic dyad (Cys)-S(-)/(His)-Im(+)H ion pair. With a fluorogenic inhibitor-screening platform, we show that zinc ion and its conjugates potently inhibit the enzymatic activity of SARS-CoV PLP2. In addition, we provided evidence for evolutionary reclassification of SARS-CoV. The results provide important insights into the biochemical properties of the coronaviral PLP family and a promising therapeutic way to fight SARS-CoV.

Cleavage stimulation factor (CstF) is a heterotrimeric protein complex essential for polyadenylation of mRNA precursors. The 77 kDa subunit, CstF-77, is known to mediate interactions with the other two subunits of CstF as well as with other components of the polyadenylation machinery. We report here the crystal structure of the HAT (half a TPR) domain of murine CstF-77, as well as its C-terminal subdomain. Structural and biochemical studies show that the HAT domain consists of two subdomains, HAT-N and HAT-C domains, with drastically different orientations of their helical motifs. The structures reveal a highly elongated dimer, spanning 165 A, with the dimerization mediated by the HAT-C domain. Light-scattering studies, yeast two-hybrid assays, and analytical ultracentrifugation measurements confirm this self-association. The mode of dimerization and the relative arrangement of the HAT-N and HAT-C domains are unique to CstF-77. Our data support a role for CstF dimerization in pre-mRNA 3' end processing.