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 ...

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.

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.

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Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A–ssDNA complex defines the 5′–3′ directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.