Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.

YciF is a protein that is up-regulated when bacteria experience stress conditions, and is highly conserved in a range of bacterial species. YciF has no known structure or biochemical function. To learn more about its potential molecular function and its role in the bacterial stress response, we solved the crystal structure of YciF at 2.0 Å resolution by the multiple wavelength anomalous diffraction (MAD) technique. YciF is a dimer in solution, and forms a homodimer in the crystal asymmetric unit. The two monomers form a dimer with a molecular twofold axis, with a significant burial of solvent-accessible surface area. The protein is an all-alpha protein composed of five helices: a four-helix bundle, and a short additional helix at the dimer interface. The protein is structurally similar to portions of the diiron-containing proteins, rubrerythrin and the Bacillus anthracis Dlp-2.

Using a multiple alignment of 175 cytochrome P450 (CYP) family 2 sequences, 20 conserved sequence motifs (CSMs) were identified with the program PCPMer. Functional importance of the CSM in CYP2B enzymes was assessed from available data on site-directed mutants and genetic variants. These analyses suggested an important role of the CSM 8, which corresponds to 187 RFDYKD 192 in CYP2B4. Further analysis showed that residues 187, 188, 190, and 192 have a very high rank order of conservation compared with 189 and 191. Therefore, eight mutants (R187A, R187K, F188A, D189A, Y190A, K191A, D192A, and a negative control K186A) were made in an N-terminal truncated and modified form of CYP2B4 with an internal mutation, which is termed 2B4dH/H226Y. Function was examined with the substrates 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone and with the inhibitors 4-(4-chlorophenyl)imidazole (4-CPI) and bifonazole (BIF). Compared with the template and K186A, the mutants R187A, R187K, F188A, Y190A, and D192A showed >2-fold altered substrate specificity, k cat , K m , and/or k cat /K m for 7-MFC and 7-EFC and 3-to 6-fold decreases in differential inhibition (IC 50,BIF /IC 50 , 4-CPI ). Subsequently, these mutants displayed 5-12°C decreases in thermal stability (T m ) and 2-8°C decreases in catalytic tolerance to temperature (T 50 ) compared with the template and K186A. Furthermore, when R187A and D192A were introduced in CYP2B1dH, the P450 expression and thermal stability were decreased. In addition, R187A showed increased activity with 7-EFC and decreased IC 50 One of the most intriguing recent discoveries about mammalian cytochromes P450 (CYP)3 is the remarkable conformational plasticity exhibited by a number of the enzymes (1-4). This plasticity allows a single P450 to adapt its ligand binding site to a wide variety of compounds of different size, shape, and chemistry. The rabbit CYP2B4 provides some of the most striking examples of an adaptable active site as inferred from x-ray crystal structures of ligand-free, 4-(4-chlorophenyl)imidazole (4-CPI)-bound, 1-(4-chlorophenyl)imidazole-bound, and bifonazole (BIF)-bound x-ray crystal structures as well as solution thermodynamics derived from isothermal titration calorimetry (5-10). In particular, comparisons of three structures identified five plastic regions in CYP2B4 (8), which account for almost one-third of the protein and contribute to broad substrate specificity by allowing different conformations in response to ligands. These results suggest an important role of non-active site regions/residues in ligand-induced conformational transitions and differential substrate binding and catalysis. However, predicting the role of non-active site regions/residues is currently very difficult.Recently, directed evolution of CYP2B1 has been carried out to locate important non-active site amino acid residues. , and Ser 334 were found to contribute to enzyme catalysis and/or stabil...

No abstract