Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.
Substitute for another bond. Docking simulations of two potent inhibitors that bear the 1,2,3‐triazole moiety produced two conformations of approximately equal energy. Further analysis of the protease by X‐ray crystallography solved the ambiguity of the binding mode and revealed that the triazole ring is an effective amide surrogate and retains all the hydrogen bonds in the active site (see figure).
Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN. This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site. Only a single divalent cation was observed even at extremely high concentrations of the metals. We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism.
Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2؉ , whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn 2؉ or Mg 2؉, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. . Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences. The retroviral integrase (IN)1 is a virus-encoded enzyme that catalyzes integration of viral DNA into host DNA (1-3). As DNA integration is an essential step in the virus life cycle, IN is an important target for the design of drugs that block the replication of pathogenic retroviruses such as the human immunodeficiency virus (HIV). The integration reaction occurs in two distinct steps. First, IN nicks the viral DNA near the 3Ј-ends of both strands (the "processing" reaction); it then inserts these ends into host target DNA (the joining reaction). Both reactions comprise a nucleophilic attack by a hydroxyl oxygen on a phosphorous atom in the DNA backbone; the hydroxyl is derived from a water molecule in the processing reaction and from the newly formed 3Ј-OH at the end of the viral DNA in the joining reaction. Divalent cations Mn 2ϩ or Mg 2ϩ are known to be required as cofactors. In vitro the reactions are most efficient in the presence of Mn 2ϩ , but as Mg 2ϩ is more abundant in living cells, it is generally presumed to be the physiologically relevant cation.Retroviral integrases contain approximately 300 amino acids and are composed of three domains (4). The first two domains are highly conserved, and both include metal-binding sites. The N-terminal domain (amino acids ϳ1-50) contains a zinc fingerlike motif, HHCC. Binding of Zn 2ϩ at this site stabilizes the structure of HIV-1 IN and enhances multimerization and activity (5-7). The central, catalytic domain (amino acids ϳ50 -200) is characterized by a triad of invariant acidic amino acids (Asp-64, Asp-121, and Glu-157 in ASV IN), the last two separated by 35 amino acids comprising the D,D(35)E motif. These three acidic residues are essential for both processing and joining activity and have been proposed to bind the divalent metal cofactors during catalysis (8).Solution of the crystal structures of the isolated catalytic core domains of HIV-1 (9, 10) and ASV IN (11,12) have revealed that these retroviral enzymes belong to a superfamily of nucleases and polynucleotidyltransferases, all of which contain a cluster of conserved acidic amino acids at their presumed active sites. HIV-1 reverse transcriptase ribonuclease H (RNase H) domain, another member of this superfamily, was shown to bind two divalent cations in this site (13), prompting the suggestion (14) that all members of this ...
The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab fragment of a monoclonal antibody 7C11 was solved at 2.8-Å resolution. Bla g 2 binds to the antibody through four loops that include residues 60 -70, 83-86, 98 -100, and 129 -132. Cation-interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and several tyrosines in 7C11. In the complex with Fab, Bla g 2 forms a dimer, which is stabilized by a quasi-four-helix bundle comprised of an ␣-helix and a helical turn from each allergen monomer, exhibiting a novel dimerization mode for an aspartic protease. A disulfide bridge between C51a and C113, unique to the aspartic protease family, connects the two helical elements within each Bla g 2 monomer, thus facilitating formation of the bundle. Mutation of these cysteines, as well as the residues Asn-52, Gln-110, and Ile-114, involved in hydrophobic interactions within the bundle, resulted in a protein that did not dimerize. The mutant proteins induced less -hexosaminidase release from mast cells than the wild-type Bla g 2, suggesting a functional role of dimerization in allergenicity. Because 7C11 shares a binding epitope with IgE, the information gained by analysis of the crystal structure of its complex provided guidance for site-directed mutagenesis of the allergen epitope. We have now identified key residues involved in IgE antibody binding; this information will be useful for the design of vaccines for immunotherapy.Cockroach allergy is associated with the development of asthma and is a risk factor for emergency room admission of asthmatic patients, especially among inner city children living in low-income houses infested with cockroaches (1, 2). Cockroaches release allergens to the environment, which are carried by particles (5-40 m of diameter) that reach the lung by inhalation. Bla g 2 is one of the most important cockroach allergens, eliciting production of specific IgE in ϳ70% of cockroach-allergic patients at exposure levels that are 10 -100-fold lower than those from other common indoor allergens from dust mite and cat (3-5). Exposure to Bla g 2 results in cross-linking of IgE bound to the surface of mast cells or basophils from sensitized patients (i.e. in immunological terms, cross-linking refers to the non-covalent linkages between the allergen and two IgE molecules at the surface of mast cells or basophils), and induces release of potent mediators (histamine, leukotrienes, prostaglandins, etc.) of allergic reactions.We recently solved the crystal structure of Bla g 2, confirming that the overall fold of this allergen corresponds to that of pepsin-like aspartic proteases, and revealing structural elements that explain why it is enzymatically inactive (6, 7). Bla g 2 contains important amino acid substitutions in the area corresponding to the catalytic site. These modifications impair enzymatic function, and the allergen did not show proteolytic activity in standard in vitro assays using casein and hemoglobin as substrates (7). Bla g 2 belongs to a gro...
Although the existence of retroviruses and their ability to cause diseases have been known for almost a century [1], it was the emergence of AIDS in the early 1980s that provided a huge impetus to structural studies of their protein and nucleic acid components. Retroviruses, most notably HIV-1, are enveloped in a glycoprotein coat and lack the high degree of internal and external symmetry that makes it possible to crystallize many relatively simple viruses, such as picornaviruses, exemplified by the viruses that cause common cold and polio. It is thus unlikely that high-resolution information about the structural organization of intact retroviruses could be obtained with the currently available methods such as crystallography, although
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