terized [19-25], understanding the subtle balance of molecular recognition events that confer drug resistance in HIV-1 is crucial to the development of second generation drugs in the treatment of HIV-1 infection. Summary HIV protease is the aspartyl protease that processes the Gag and Pol polyproteins and allows for the matura-The homodimeric HIV-1 protease is the target of some tion of the immature HIV virion, thus allowing the spread of the most effective antiviral AIDS therapy, as it facili-of the virus. Remarkably, the precise physical parame-tates viral maturation by cleaving ten asymmetric and ters that govern how HIV-1 protease binds to its ten nonhomologous sequences in the Gag and Pol poly-natural, nonhomologous substrates [26-31] (Table 1) re-proteins. Since the specificity of this enzyme is not main poorly understood. The active site of the homodi-easily determined from the sequences of these cleav-meric protease is at the dimer interface [18, 32]. Despite age sites alone, we solved the crystal structures of the symmetry conferred on its active site because it is complexes of an inactive variant (D25N) of HIV-1 prote-a homodimer, the enzyme recognizes asymmetric sub-ase with six peptides that correspond to the natural strate sites within the Gag and Pol polyproteins. The substrate cleavage sites. When the protease binds to amino acid sequences of these substrates are asymmet-its substrate and buries nearly 1000 A ˚ 2 of surface area, ric around the cleavage sites in both size and charge the symmetry of the protease is broken, yet most inter-distribution. In addition, these sites share little sequence nal hydrogen bonds and waters are conserved. How-homology. How then does the protease recognize a ever, no substrate side chain hydrogen bond is con-particular peptide sequence as being a substrate? There served. Specificity of HIV-1 protease appears to be must be a breakdown in the symmetry within the individ-determined by an asymmetric shape rather than a par-ual protease dimer when it binds to its substrates. This ticular amino acid sequence. breakdown has often been difficult to characterize, however , since many of the complexes of HIV protease Introduction bound to asymmetric ligands do not uniquely orient the protease dimer in the crystal cell. This lack of unique As the worldwide AIDS epidemic continues into its third orientation resulted in protease-substrate structures decade, a cure for HIV-1 still eludes the medical commu-with 50% of the ligand oriented in one direction and nity [1]. In the absence of a cure for HIV-1 pathogenesis, 50% in the other, thus averaging out the asymmetry suppressing viral replication and maintaining it at low within the protease. To elucidate how HIV-1 protease to undetectable levels have become critical goals in the recognizes its substrates, we determined the crystal field of HIV-1 research [2-5]. To this end, highly active structures of six complexes of HIV-1 protease with de-antiretroviral therapy (HAART) has become a successful cameric peptides that correspond ...

TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (K d ‫؍‬ 4.5 ؋ 10 ؊12 M) than APV (K d ‫؍‬ 3.9 ؋ 10 ؊10 M). Our X-ray data (resolution ranging from 2.2 to 1.2 Å) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (؊12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope.

Summary APOBEC3G is a DNA cytidine deaminase that has anti-viral activity against HIV-1 and other pathogenic viruses. In this study the crystal structure of the catalytically active C-terminal domain was determined to 2.25 Å. This structure corroborates features previously observed in NMR studies, a bulge in the second β-strand and a lengthening of the second α-helix. Oligomerization is postulated to be critical for the function of APOBEC3G. In this structure, four extensive intermolecular interfaces are observed suggesting potential models for APOBEC3G oligomerization. The structural and functional significance of these interfaces was probed by solution NMR and disruptive variants were designed and tested for DNA deaminase and anti-HIV activities. The variant designed to disrupt the most extensive interface lost both activities. NMR solution data provides evidence that another interface, which coordinates a novel zinc site, also exists. Thus, the observed crystallographic interfaces of APOBEC3G may be important for both oligomerization and function.

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Deaminase activity mediated by the human APOBEC3 family of proteins contributes to genomic instability and cancer. APOBEC3A is by far the most active in this family and can cause rapid cell death when overexpressed, but in general how the activity of APOBEC3s is regulated on a molecular level is unclear. In this study the biochemical and structural basis of APOBEC3A substrate binding and specificity is elucidated. We find that specific binding of single-stranded DNA is regulated by the cooperative dimerization of APOBEC3A. The crystal structure elucidates this homo-dimer as a symmetric domain swap of the N-terminal residues. This dimer interface provides insights into how cooperative protein-protein interactions may impact function in the APOBEC3 enzymes, and provides a potential scaffold for strategies aimed at reducing their mutation load.