Formation of infectious HIV-1 involves assembly of Gag polyproteins into immature particles and subsequent assembly of mature capsids after proteolytic disassembly of the Gag shell. We report a 12-mer peptide, capsid assembly inhibitor (CAI), that binds the capsid (CA) domain of Gag and inhibits assembly of immature- and mature-like capsid particles in vitro. CAI was identified by phage display screening among a group of peptides with similar sequences that bind to a single reactive site in CA. Its binding site was mapped to CA residues 169-191, with an additional contribution from the last helix of CA. This result was confirmed by a separate X-ray structure analysis showing that CAI inserts into a conserved hydrophobic groove and alters the CA dimer interface. The CAI binding site is a new target for antiviral development, and CAI is the first known inhibitor directed against assembly of immature HIV-1.

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Type II polyketide synthases (PKSs) utilize a dedicated and essential acyl carrier protein (ACP) in the biosynthesis of a specific polyketide product. As part of our ongoing studies into the mechanisms and control of polyketide biosynthesis, we report the second structure of a polyketide synthase ACP. In this work, multidimensional, heteronuclear NMR was employed to investigate the structure and dynamics of the ACP involved in the biosynthesis of the commonly prescribed polyketide antibiotic, oxytetracycline (otc). An ensemble of 28 structures of the 95 amino acid otc ACP (9916Da) was computed by simulated annealing with the inclusion of 1132 experimental restraints. Atomic RMSDs about the mean structure for all 28 models is 0.66 A for backbone atoms, 1.15 A for all heavy atoms (both values calculated for the folded part of the protein (residues 3-80)), and 0.41 A for backbone atoms within secondary structure. Otc ACP adopts the typical right-handed, four-helix fold of currently known ACPs but with the addition of a 13-residue flexible C-terminus. A comparison of the global folds of all structurally characterized ACPs is described, illustrating that PKS ACPs show clear differences as well as similarities to FAS ACPs. (15)N relaxation experiments for the protein backbone also reveal that the long loop between helices I and II is flexible and helix II, a proposed site of protein-protein interactions, shows conformational exchange. The helices of the ACP form a rigid scaffold for the protein, but these are interspersed with an unusual proportion of flexible linker regions.

Malonylation of an acyl carrier protein (ACP) by malonyl Coenzyme A-ACP transacylase (MCAT) is fundamental to bacterial fatty acid biosynthesis. Here, we report the structure of the Steptomyces coelicolor (Sc) fatty acid synthase (FAS) ACP and studies of its binding to MCAT. The carrier protein adopts an alpha-helical bundle structure common to other known carrier proteins. The Sc FAS ACP shows close structural homology with other fatty acid ACPs and less similarity with Sc actinorhodin (act) polyketide synthase (PKS) ACP where the orientation of helix I differs. NMR experiments were used to map the binding of ACP to MCAT. This data suggests that Sc FAS ACP interacts with MCAT through the negatively charged helix II of ACP, consistent with proposed models for ACP recognition by other FAS enzymes. Differential roles for residues at the interface are demonstrated using site-directed mutagenesis and in vitro assays. MCAT has been suggested, moreover, to participate in bacterial polyketide synthesis in vivo. We demonstrate that the affinity of the polyketide synthase ACP for MCAT is lower than that of the FAS ACP. Mutagenesis of homologous helix II residues on the polyketide synthase ACP suggests that the PKS ACP may bind to MCAT in a different manner than the FAS counterpart.