title: Effect of Glu12-His89 Interaction on Dynamic Structures in HIV-1 p17 Matrix Protein Elucidated by NMR.
abstract: To test the existence of the salt bridge and stability of the HIV-1 p17 matrix protein, an E12A (mutated at helix 1) was established to abolish possible electrostatic interactions. The chemical shift perturbation from the comparison between wild type and E12A suggested the existence of an electrostatic interaction in wild type between E12 and H89 (located in helix 4). Unexpectedly, the studies using urea denaturation indicated that the E12A substitution slightly stabilized the protein. The dynamic structure of E12A was examined under physiological conditions by both amide proton exchange and relaxation studies. The quick exchange method of amide protons revealed that the residues with faster exchange were located at the mutated region, around A12, compared to those of the wild-type protein. In addition, some residues at the region of helix 4, including H89, exhibited faster exchange in the mutant. In contrast, the average values of the kinetic rate constants for amide proton exchange for residues located in all loop regions were slightly lower in E12A than in wild type. Furthermore, the analyses of the order parameter revealed that less flexible structures existed at each loop region in E12A. Interestingly, the structures of the regions including the alpha1-2 loop and helix 5 of E12A exhibited more significant conformational exchanges with the NMR time-scale than those of wild type. Under lower pH conditions, for further destabilization, the helix 1 and alpha2-3 loop in E12A became more fluctuating than at physiological pH. Because the E12A mutant lacks the activities for trimer formation on the basis of the analytical ultra-centrifuge studies on the sedimentation distribution of p17 (Fledderman et al. Biochemistry 49, 9551-9562, 2010), it is possible that the changes in the dynamic structures induced by the absence of the E12-H89 interaction in the p17 matrix protein contributes to a loss of virus assembly.
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Konagaya
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo, Japan.
authors.forename: Rina
authors.initials: R
authors.lastname: Miyakawa
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo, Japan.
authors.forename: Masumi
authors.initials: M
authors.lastname: Sato
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Akimasa
authors.initials: A
authors.lastname: Matsugami
authors.affiliation: Laboratory for Biomolecular Structure and Dynamics, RIKEN Quantitative Biology Center, Yokohama, Kanagawa, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Hayashi
authors.affiliation: Laboratory for Biomolecular Structure and Dynamics, RIKEN Quantitative Biology Center, Yokohama, Kanagawa, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano, Tokyo, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nishimura
mesh_terms: D000577:Amides; D003062:Codon; D005973:Glutamine / Q000737:chemistry* / Q000235:genetics; D015488:HIV Antigens / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011522:Protons; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics
chemical_list: D000577:Amides; D003062:Codon; D015488:HIV Antigens; D011522:Protons; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1; D005973:Glutamine; D006639:Histidine