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.

Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.drug design | hepatitis C | substrate envelope D rug resistance is a major obstacle in the treatment of quickly evolving diseases. Hepatitis C virus (HCV) is a genetically diverse Hepacivirus of the Flaviviridae family infecting an estimated 180 million people worldwide (1). The viral RNA genome is translated as a single polyprotein and subsequently processed by host-cell and viral proteases into structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (2). The viral RNA-dependent RNA polymerase, NS5B, is inherently inaccurate and misincorporation of bases accounts for a very high mutation rate (3). While some mutations are neutral, others will alter the viability of the virus and propagate with varying efficiencies in each patient. Thus HCV infected individuals will develop a heterogeneous population of virus variants known as quasispecies (4). As patients begin treatment, the selective pressures of antiviral drugs will favor drug resistant variants (5). Therefore, an inhibitor must not only recognize one protein variant, but an ensemble of related enzymes. A detailed understanding of the atomic mechanisms of resistance is essential to effectively combat drug resistance against HCV antivirals.The essential HCV NS3/4A protease is an attractive therapeutic target responsible for cleaving at least four sites along the viral polyprotein. These sites share little sequence homology except for an acid at position P6, Cys or Thr at P1, and Ser or Ala at P1′ (Table S1). The first cleavage event at the 3-4A junction occurs in cis as a unimolecular process, while the remaining substrates are processed bimolecularly in trans. The NS3/4A protease also cleaves the human cellular targets TRIF and MAVS, which confounds the innate immune response to viral infection (6-8).Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone f...

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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One of the most remarkable structural aspects of Scapharca dimeric hemoglobin is the disruption of a very well-ordered water cluster at the subunit interface upon ligand binding. We have explored the role of these crystallographically observed water molecules by site-directed mutagenesis and osmotic stress techniques. The isosteric mutation of Thr-72 3 Val in the interface increases oxygen affinity more than 40-fold with a surprising enhancement of cooperativity. The only significant structural effect of this mutation is to destabilize two ordered water molecules in the deoxy interface. Wild-type Scapharca hemoglobin is strongly sensitive to osmotic conditions. Upon addition of glycerol, striking changes in Raman spectrum of the deoxy form are observed that indicate a transition toward the liganded form. Increased osmotic pressure, which lowers the oxygen affinity in human hemoglobin, raises the oxygen affinity of Scapharca hemoglobin regardless of whether the solute is glycerol, glucose, or sucrose. Analysis of these results provides an estimate of six water molecules lost upon oxygen binding to the dimer, in good agreement with eight predicted from crystal structures. These experiments suggest that the observed cluster of interfacial water molecules plays a crucial role in communication between subunits.Water plays a unique and ubiquitous role in biology. Folding, stability, and function of protein molecules are all strongly influenced by their interactions with water molecules. Despite the importance of these interactions, the precise role of water in structure and function is often difficult to elucidate. Ordered water molecules are nearly universally observed in macromolecular crystallographic analysis (1, 2), and biologically important roles for these observed water molecules have been implicated in a few cases including substrate specificity (3), catalysis (4), antigen-antibody association (5), and DNA recognition (6).High resolution x-ray crystallography on the dimeric hemoglobin (HbI) from Scapharca inaequivalvis has revealed a striking ligand-linked rearrangement of water molecules at the subunit interface (7,8). HbI binds oxygen cooperatively with a Hill coefficient of 1.5 and shows no change in oxygen affinity or cooperativity as pH varies from 5.5 to 9.0 (9). Quite unlike mammalian hemoglobins, where cooperativity is mediated by large quaternary structural changes (10), HbI displays minimal ligand-linked quaternary changes. The oxygen affinity of each subunit appears to depend largely on the position of the side chain of Phe-97 which packs in the heme pocket in the deoxy state, but upon heme ligation is extruded into the subunit interface where it expels several water molecules (see Fig. 1). The core of the deoxy interface includes a cluster of 17 very well-ordered water molecules, 6 of which are lost upon oxygenation. (An additional two water molecules within the heme pocket are directly displaced by the bound oxygen.)We have undertaken the present studies to determine the functional import...

Interferon regulatory factors (IRFs) are essential in the innate immune response and other physiological processes. Activation of these proteins in the cytoplasm is triggered by phosphorylation of Ser/Thr residues in a C-terminal autoinhibitory region, which stimulates dimerization, transport into the nucleus, assembly with the coactivator CBP/p300 and initiation of transcription. The novel crystal structure of the transactivation domain of pseudophosphorylated human IRF5 reveals a striking dimer in which the bulk of intersubunit interactions involve a highly extended C-terminal region. The corresponding region has previously been shown to block CBP/p300 binding to unphosphorylated IRF3. Mutation of key interface residues supports the observed dimer as the physiologically activated state of IRF5 and IRF3. Thus phosphorylation likely activates IRF5 and other family members by triggering remarkable conformational rearrangements that switch the C-terminal segment from an autoinihibitory to a dimerization role.

Annelid erythrocruorins are highly cooperative extracellular respiratory proteins with molecular masses on the order of 3.6 million Daltons. We report here the 3.5 A crystal structure of erythrocruorin from the earthworm Lumbricus terrestris. This structure reveals details of symmetrical and quasi-symmetrical interactions that dictate the self-limited assembly of 144 hemoglobin and 36 linker subunits. The linker subunits assemble into a core complex with D(6) symmetry onto which 12 hemoglobin dodecamers bind to form the entire complex. Although the three unique linker subunits share structural similarity, their interactions with each other and the hemoglobin subunits display striking diversity. The observed diversity includes design features that have been incorporated into the linker subunits and may be critical for efficient assembly of large quantities of this complex respiratory protein.