Influenza viruses are global threat to humans, and the development of new antiviral agents are still demanded to prepare for pandemics and to overcome the emerging resistance to the current drugs. Influenza polymerase acidic protein N-terminal domain (PAN) has endonuclease activity and is one of the appropriate targets for novel antiviral agents. First, we performed X-ray cocrystal analysis on the complex structures of PAN with two endonuclease inhibitors. The protein crystallization and the inhibitor soaking were done at pH 5.8. The binding modes of the two inhibitors were different from a common binding mode previously reported for the other influenza virus endonuclease inhibitors. We additionally clarified the complex structures of PAN with the same two endonuclease inhibitors at pH 7.0. In one of the crystal structures, an additional inhibitor molecule, which chelated to the two metal ions in the active site, was observed. On the basis of the crystal structures at pH 7.0, we carried out 100 ns molecular dynamics (MD) simulations for both of the complexes. The analysis of simulation results suggested that the binding mode of each inhibitor to PAN was stable in spite of the partial deviation of the simulation structure from the crystal one. Furthermore, crystal structure analysis and MD simulation were performed for PAN in complex with an inhibitor, which was already reported to have a high compound potency for comparison. The findings on the presence of multiple binding sites at around the PAN substrate-binding pocket will provide a hint for enhancing the binding affinity of inhibitors.

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Some proteins are easily crystallized by utilizing ammonium sulfate (AS) as a precipitant, while others are not. To investigate the difference of AS behavior in protein crystallization between both types of proteins, crystals were grown for two proteins in the former type; carbonic anhydrase II (CAII) and myoglobin (Mb), and also for two proteins in the latter one; hen egg white lysozyme (HEWL) and human serum albumin (HSA). In particular, CAII and Mb were crystallized at high AS concentrations around 3.0 M. In contrast, single crystals were grown at a lower AS concentration of 1.2 M both for HEWL and HSA. Molecular dynamics simulations were carried out for all the proteins with calculation models, including AS at the concentrations of the respective crystallization conditions. The motion of the protein during the simulation was reduced in the presence of AS for all the proteins. Ammonium and sulfate ions (AS ions) were anisotropically distributed around the protein molecules, especially for the proteins in the former type, CAII and Mb, under the condition of high AS concentrations. The electrostatic potential around CAII and Mb was almost equally divided into the positive and negative areas, and the AS anisotropic distributions observed in the simulations were compatible with the shape of the iso-surface of the electrostatic potential. In contrast, AS ions were sparsely distributed under the low AS concentration for HEWL and HSA. Either positive or negative area of the electrostatic potential was dominant for HEWL and HSA. Hence, the surrounding space of the latter-type protein was not so distinctively polarized as that of the former-type one. AS ions were anisotropically distributed even for HEWL and HSA, when simulations were performed at high AS concentrations corresponding to 2.0 and 3.0 M in precipitant solution. The AS distributions were, however, different between the former-type proteins and the latter-type ones. Two AS dense areas appeared around CAII and Mb, while AS ions were crowded at one area for HEWL and HSA.

With the increasing number of solved protein crystal structures, much information on protein shape and atom geometry has become available. It is of great interest to know the structural diversity for a single kind of protein. Our preliminary study suggested that multiple crystal structures of a single kind of protein can be classified into several groups from the viewpoint of structural similarity. In order to broadly examine this finding, cluster analysis was applied to the crystal structures of hemoglobin (Hb), myoglobin (Mb), human serum albumin (HSA), hen egg-white lysozyme (HEWL), and human immunodeficiency virus type 1 protease (HIV-1 PR), downloaded from the Protein Data Bank (PDB). As a result of classification by cluster analysis, 146 crystal structures of Hb were separated into five groups. The crystal structures of Mb (n = 284), HEWL (n = 336), HSA (n = 63), and HIV-1 PR (n = 488) were separated into six, five, three, and six groups, respectively. It was found that a major factor causing these structural separations is the space group of crystals and that crystallizing agents have an influence on the crystal structures. Amino acid mutation is a minor factor for the separation because no obvious point mutation making a specific cluster group was observed for the five kinds of proteins. In the classification of Hb and Mb, the species of protein source such as humans, rabbits, and mice is another significant factor. When the difference in amino sequence is large among species, the species of protein source is the primary factor causing cluster separation in the classification of crystal structures.

Information on many protein crystal structures has recently become available due to developments in crystallographic techniques. Even for a single kind of protein, several and sometimes many crystal structures are available. Human immunodeficiency virus type 1 (HIV-1) protease is one of the most extensively studied viral proteins, and about six hundred crystal structures have been determined. In this work, we examined the structural diversity of HIV-1 protease, classifying crystal structures into several groups from the viewpoint of similarity in atom geometry. Using 499 crystal structures downloaded from the Protein Data Bank (PDB), cluster analysis was applied to the whole body of HIV-1 protease and also to a limited number of residues at the binding pocket. As a consequence of clustering with regard to the whole body, 499 crystal structures were separated into 6 groups. It was found that a major factor for this separation is the space group of the crystals and that the space group strongly depends on the agents used in the protein crystallization. Amino acid mutation is a minor factor for separation in clustering. In cluster analysis for a limited number of residues at the binding pocket, crystal structures were not distinctly separated, and no clear factor linked to the separation was clarified. The results suggest that amino acid mutations have little effect on the coordinates of the main-chain atoms of HIV-1 protease. Hence, the changes in drug efficacy or substrate fitness caused by mutations are mainly due to the physicochemical features of amino acid side chains.Key words clustering analysis; crystal structure; amino acid mutation; drug resistance; space group Due to the progress in techniques for protein crystallization and in software for model building, crystal structures of many proteins have been elucidated. The reliability of solved protein structures has also increased.1) The development of a high-energy X-ray source has also been important for advancing crystallographic studies.2) Due to the progress in crystallographic technology, the availability of crystal structures in good quality has enabled us to examine structural differences in proteins in detail. Even for a single kind of protein, an amino acid mutation will cause the difference in its activity. Mutagenesis is one of the best approaches for clarifying the relationship between the protein activity and the mutated amino acid residue. Since protein activity has a close relation with its structure, 3,4) it is of great interest in molecular biology that the influence of amino acid mutation induces the change in protein structure.Apart from artificial mutagenesis, amino acid mutation ceaselessly occurs in the process of evolution. There is a common consensus that the accumulation of amino acid mutations generates a genetic diversity and that the diversity is observed as a difference in protein function in phenotype. 5,6) In general, the mutation rate of viral proteins is much higher than that of protein in eukaryotes. Variants of a virus som...