Ikuo Kurisaki

Bound Na(+) is a Negative Effecter for Thrombin-Substrate Stereospecific Complex Formation.

Thrombin has been studied as a paradigmatic protein of Na(+)-activated allosteric enzymes. Earlier structural studies suggest that Na(+)-binding promotes the thrombin-substrate association reaction. However, it is still elusive because (1) the structural change, driven by Na(+)-binding, is as small as the thermal fluctuation, and (2) the bound Na(+) is close to Asp189 in the primary substrate binding pocket (S1-pocket), possibly preventing substrate access via repulsive interaction. It still remains a matter of debate whether Na(+)-binding actually promotes the reaction. To solve this problem, we examined the effect of Na(+) on the reaction by employing molecular dynamics (MD) simulations. By executing independent 210 MD simulations of apo and holo systems, we obtained 80 and 26 trajectories undergoing substrate access to S1-pocket, respectively. Interestingly, Na(+)-binding results in a 3-fold reduction of the substrate access. Furthermore, we examined works for the substrate access and release, and found that Na(+)-binding is disadvantageous for the presence of the substrate in the S1-pocket. These observations provide the insight that the bound Na(+) is essentially a negative effecter in thrombin-substrate stereospecific complex formation. The insight rationalizes an enigmatic feature of thrombin, relatively low Na(+)-binding affinity. This is essential to reduce the disadvantage of Na(+)-binding in the substrate-binding.

Spontaneous adjustment mechanism in an RNA-binding protein: cooperation between energetic stabilization and target search enhancement.

We propose a novel concept associated with the relationship between structure and function in biomolecular systems. We performed a 75 nanoseconds molecular dynamics (MD) simulation for an RNA-binding protein, neuro-oncological ventral antigen (NOVA), and examined its physico-chemical properties. NOVA dissociated from the NOVA-RNA complex showed a large conformational change: formation of intra-molecular hydrogen bonds between the C-terminal region and the loop structure located at the middle of amino acid sequence. The free energy analysis suggests that the deformed structure is more stabilized in macromolecular crowding environment where the dielectric constant is smaller than 5. The solvent accessible surface area (SASA) analysis indicates that NOVA enhances the efficiency of association with RNA by changing the relative SASA for the target sequence in RNA molecules. Based on the obtained results, we propose a novel concept of spontaneous adjustment mechanism to explain the structural and energetic changes observed for NOVA in the free state.

Multiple oxygen entry pathways in globin proteins revealed by intrinsic pathway identification method

Each subunit of human hemoglobin (HbA) stores an oxygen molecule (O2) in the binding site (BS) cavity near the heme group. The BS is buried in the interior of the subunit so that there is a debate over the O2 entry pathways from solvent to the BS; histidine gate or multiple pathways. To elucidate the O2 entry pathways, we executed ensemble molecular dynamics (MD) simulations of T-state tetramer HbA in high concentration O2 solvent to simulate spontaneous O2 entry from solvent into the BS. By analyzing 128 independent 8 ns MD trajectories by intrinsic pathway identification by clustering (IPIC) method, we found 141 and 425 O2 entry events into the BS of the α and β subunits, respectively. In both subunits, we found that multiple O2 entry pathways through inside cavities play a significant role for O2 entry process of HbA. The rate constants of O2 entry estimated from the MD trajectories correspond to the experimentally observed values. In addition, by analyzing monomer myoglobin, we verified that the high ...