N. Watanabe

Structural study of the pressure adaptation of proteins from deep-sea bacteria

In recent years, significant development in the high-pressure macromolecular crystallography (HPMX) using a diamond anvil cell (DAC) has been performed especially by Prof. R. Fourmes group in combination with shorter wavelength X-ray of synchrotron radiation [1]. We are also trying to establish HPMX experimental environment at the Photon Factory, Japan [2]. HPMX is a unique method that provides high-resolution structural informations under pressure including hydration waters at a molecular surface and an internal cavity. One of the important applications is studying functional sub-states of biological macromolecules, and we are attempting to elucidate a mechanism of pressure tolerance of proteins from several organisms living in deep seas such as the Mariana Trench. For example, 3-isopropylmalate dehydrogenase (IPMDH) from the deep-sea bacterium Shewanella benthica DB21MT-2 is much more tolerant to the pressure stress than its counterpart from the land bacterium S. oneidensis MR-1 (So-IPMDH), even though these two enzymes share about 85% amino-acid identity. Crystal structures of So-IPMDH have been determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa. Waters penetrating into the internal cavity at the dimer interface and squeezing into a molecular surface cleft opposite the active site are observed at above 410 MPa and 580 MPa, respectively [3]. The bottom of the cleft of So-IPMDH is characterized by the presence of Ser266 at the bottom, which is able to form a hydrogen bond to the squeezed water molecule. On the other hand, IPMDHs from deep-sea bacterium favors an alanine at the same position (Ala266). As expected, no water penetration is observed there at the same pressure range for the S266A mutated So-IPMDH, and the mutation develops tolerance to the pressure. In addition, some results of the high-pressure structure analysis of other proteins, and pressure-induced phase transitions in some protein crystals will also be mentioned.

Crystal structure of the Vif-interaction domain of the anti-viral APOBE3F

Human cells express a family of cytidine deaminases, called APOBEC3 (A3) (A3A, B, C, D, F, G, and H). The family enzymes, especially A3G and A3F potentially inhibit replication of retroviruses including HIV-1. However, HIV-1 overcomes the A3-mediated antiviral system by expressing a virus-encoded antagonist, viral infectivity factor (Vif) protein. In HIV-1-infected cells, Vif specifically binds with A3 followed by proteasomal degradation of A3. Hence, inhibition of the interaction between A3 and Vif is an attractive strategy for developing novel anti-HIV-1 drugs. To date, we have determined the first crystal structure of A3 with Vif-binding interface, A3C (PDB ID: 3VOW). In addition, our extensive mutational analysis, based on the A3C structure, revealed that structural features of the Vifbinding interface are highly conserved among A3C, DE, and F [1]. However, more recently, Bohn et al. and Karen et al. have shown the crystal structures of mutant A3F C-terminal domain (CTD) which is responsible for the Vif interaction, and have predicted more extended area, including our identified residues, for the interface on the A3F CTD [2][3]. To clarify the Vif-binding interface of A3F, we sought to determine the crystal structure of the wild-type A3F CTD and evaluated contributions of the additional residues for the Vifinteraction interface by virological method. First, we have successfully determined the crystal structure of A3F CTD at 2.75 Å resolution. Furthermore, we have identified four additional residues unique on the A3F CTD but not A3C for Vif interaction, which are located in the vicinity of our previously reported interface. These results demonstrated that the structural features of Vif-binding interface are indeed conserved between A3C and A3F. Taken together, these results will provide the fine-tuned structure information to understand the binding between A3 and Vif and to facilitate a development of novel anti-HIV-1 compounds targeting A3 proteins.

Structural studies of the multidrug-responsible transcriptional repressor protein CgmR

4 successful to produce crystals in our screening. Initial diffraction tests were performed using a home-source X-ray generator (Rigaku FR-E) equipped with a Rigaku R-AXIS detector at 100 K. The best crystal was found to diffract up to 2.75 Å resolution. The structure was determined by molecular replacement using our previous structure of human FEN1 in the FEN1-PCNA complex crystal (PDB code 1UL1) as a search model. The structure shows that the enzyme holds both the upstream and downstream duplexes and induces a sharp kink of the DNA by embedding the kinked template strand in a basic cleft.

Structural studies on the promoter recognition of transcription factor HNF-6

4 of the N and C termini of the domain were found to participate in the domain architecture by forming an extended portion of the first helix alpha-1, and a novel looping motif that traverses straight along the domain surface, respectively. The motifs combine to increase the domain surface of WRN HRDC, which is larger than that of Sgs1 and E. coli. In WRN HRDC, neither of the proposed DNA-binding surfaces in Sgs1 or E. coli is conserved, and the domain was shown to lack DNA-binding ability in vitro. Moreover, the domain was shown to be thermostable and resistant to protease digestion, implying independent domain evolution in WRN. Coupled with the unique long linker region in WRN, the WRN HRDC may be adapted to play a distinct function in WRN that involves protein-protein interactions.

Structure determination of a novel protein by sulphur SAD using novel crystal mounting method

ScRh3Bx(x=0.0-1.0) has been investigated recently as an ultrahard material. Crystal structure refinements and electron density analyses of this material were carried out by synchrotron X-ray powder diffraction. The powder diffraction data were collected using Multi-Detector System powder diffractometer at the BL-4B2 experimental station of the Photon Factory. The crystal structure refinements were performed using the Rietveld method and the electron density maps were calculated with the Maximum Entropy Method (MEM). The results of the refinements show that the crystal structure of ScRh3Bx is cubic with Pm3m space group, which has same atomic arrangement with perovskite structure. The lattice constant increases linearly according to the increase of B amount. In the electron density maps obtained by MEM analysis, electron density raises are obviously observed between B and Rh atoms. The rises of electron density show the existence of covalent bond between B and Rh atom. In spite of the liner increase of lattice constant according to the increase of B amount, the hardness of this series of compounds have a minimum between 0.4 and 0.7 of B contents. This change of hardness is supposed to be related to the amounts of the covalent bond in the crystal structure. The bond character of this series of compounds is also discussed based on the results of electron density analyses.