Dataset Information

Molecular Mechanism for Conformational Dynamics of Ras·GTP Elucidated from In-Situ Structural Transition in Crystal.

ABSTRACT: Ras•GTP adopts two interconverting conformational states, state 1 and state 2, corresponding to inactive and active forms, respectively. However, analysis of the mechanism for state transition was hampered by the lack of the structural information on wild-type Ras state 1 despite its fundamental nature conserved in the Ras superfamily. Here we solve two new crystal structures of wild-type H-Ras, corresponding to state 1 and state 2. The state 2 structure seems to represent an intermediate of state transition and, intriguingly, the state 1 crystal is successfully derived from this state 2 crystal by regulating the surrounding humidity. Structural comparison enables us to infer the molecular mechanism for state transition, during which a wide range of hydrogen-bonding networks across Switch I, Switch II and the ?3-helix interdependently undergo gross rearrangements, where fluctuation of Tyr32, translocation of Gln61, loss of the functional water molecules and positional shift of GTP play major roles. The NMR-based hydrogen/deuterium exchange experiments also support this transition mechanism. Moreover, the unveiled structural features together with the results of the biochemical study provide a new insight into the physiological role of state 1 as a stable pool of Ras•GTP in the GDP/GTP cycle of Ras.

SUBMITTER: Matsumoto S

PROVIDER: S-EPMC4867591 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Molecular Mechanism for Conformational Dynamics of Ras·GTP Elucidated from In-Situ Structural Transition in Crystal.

Matsumoto Shigeyuki S Miyano Nao N Baba Seiki S Liao Jingling J Kawamura Takashi T Tsuda Chiemi C Takeda Azusa A Yamamoto Masaki M Kumasaka Takashi T Kataoka Tohru T Shima Fumi F

Scientific reports 20160516

Ras•GTP adopts two interconverting conformational states, state 1 and state 2, corresponding to inactive and active forms, respectively. However, analysis of the mechanism for state transition was hampered by the lack of the structural information on wild-type Ras state 1 despite its fundamental nature conserved in the Ras superfamily. Here we solve two new crystal structures of wild-type H-Ras, corresponding to state 1 and state 2. The state 2 structure seems to represent an intermediate of sta ...[more]

PMID: 27180801

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Project description:BACKGROUND: Cathepsin B (catB) is a promising target for anti-cancer drug design due to its implication in several steps of tumorigenesis. catB activity and inhibition are pH-dependent, making it difficult to identify efficient inhibitor candidates for clinical trials. In addition it is known that heparin binding stabilizes the enzyme in alkaline conditions. However, the molecular mechanism of stabilization is not well understood, indicating the need for more detailed structural and dynamic studies in order to clarify the influence of pH and heparin binding on catB stability. RESULTS: Our pKa calculations of catB titratable residues revealed distinct protonation states under different pH conditions for six key residues, of which four lie in the crucial interdomain interface. This implies changes in the overall charge distribution at the catB surface, as revealed by calculation of the electrostatic potential. We identified two basic surface regions as possible heparin binding sites, which were confirmed by docking calculations. Molecular dynamics (MD) of both apo catB and catB-heparin complexes were performed using protonation states for catB residues corresponding to the relevant acidic or alkaline conditions. The MD of apo catB at pH 5.5 was very stable, and presented the highest number and occupancy of hydrogen bonds within the inter-domain interface. In contrast, under alkaline conditions the enzyme's overall flexibility was increased: interactions between active site residues were lost, helical content decreased, and domain separation was observed as well as high-amplitude motions of the occluding loop - a main target of drug design studies. Essential dynamics analysis revealed that heparin binding modulates large amplitude motions promoting rearrangement of contacts between catB domains, thus favoring the maintenance of helical content as well as active site stability. CONCLUSIONS: The results of our study contribute to unraveling the molecular events involved in catB inactivation in alkaline pH, highlighting the fact that protonation changes of few residues can alter the overall dynamics of an enzyme. Moreover, we propose an allosteric role for heparin in the regulation of catB stability in such a manner that the restriction of enzyme flexibility would allow the establishment of stronger contacts and thus the maintenance of overall structure.

Dataset Information

Molecular Mechanism for Conformational Dynamics of Ras·GTP Elucidated from In-Situ Structural Transition in Crystal.

Publications

Molecular Mechanism for Conformational Dynamics of Ras·GTP Elucidated from In-Situ Structural Transition in Crystal.

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