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 Vif-binding 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 Vif-interaction 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.

SAD experiment using the wavelength of 2.7 Å or longer

Single-wavelength anomalous dispersion (SAD) experiment with light atoms as anomalous scatterers has been carried out using longer wavelengths up to 2.3 Å. We have been developing a synchrotron beamline dedicated to the SAD experiments where wavelengths longer than 2.7 Å are available to enhance weak anomalous signals. Larger background noise due to the longer wavelength, which is one of the major problems in the experiment, is reduced by introducing a standing helium chamber surrounding both the whole diffractometer and the X-ray detector. The system allows to perform experiments with normal and long waveldngths under the same environment. Helium cold stream is fed into the chamber at the sample position and reused after removing contaminants to keep the temperature of the stream at 30 K or below economically. Capillary-top-mount method [1] was improved to further reduce the background noise and to accommodate with smaller or needle-shape crystals. Several results on de-novo structural solutions with sulfur-SAD phasing will be reported in addition to the current performance of the beamline and its future plan.

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. Fourme's 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.