Yuichi Kokabu

Fission Yeast Swi5-Sfr1 Protein Complex, an Activator of Rad51 Recombinase, Forms an Extremely Elongated Dogleg-shaped Structure

Background: The Swi5-Sfr1 protein complex is an activator of Rad51 recombinase, which mediates DNA strand exchange in homologous recombination. Results: Swi5 and Sfr1 form a 1:1 complex, which exhibits an extremely elongated dogleg-shaped structure in solution. Conclusion: The Swi5-Sfr1 structure is suitable for binding within the helical groove of the Rad51 filament. Significance: A structural model will advance our understanding of the molecular mechanism of homologous recombination. In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f0) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.

Homology‐modelled structure of the βB2B3‐crystallin heterodimer studied by ion mobility and radical probe MS

Ion mobility MS was employed to study the structure of the βB2B3‐crystallin heterodimer following its detection by ESI‐TOF MS. The results demonstrate that the heterodimer has a similar cross‐section (3 165 Å2) and structure to the βB2B2‐crystallin homodimer. Several homology‐modelled structures for the βB2B3 heterodimer were constructed and assessed in terms of their calculated collision cross‐sections and whether the solvent accessibilities of reactive amino acid side chains throughout the βB3 subunit are in accord with measured oxidation levels in radical probe MS protein footprinting experiments. The βB2B3 heterodimer AD model provides the best representation of the heterodimer’s structure overall following a consideration of both the ion mobility and radical probe MS data.

Rotation Mechanism of Molecular Motor V1-ATPase Studied by Multiscale Molecular Dynamics Simulation

Enterococcus hirae V1-ATPase is a molecular motor composed of the A3B3 hexamer ring and the central stalk. In association with ATP hydrolysis, three catalytic AB pairs in the A3B3 ring undergo conformational changes, which lead to a 120° rotation of the central stalk. To understand how the conformational changes of three catalytic pairs induce the 120° rotation of the central stalk, we performed multiscale molecular dynamics (MD) simulations in which coarse-grained and all-atom MD simulations were combined using a fluctuation matching methodology. During the rotation, a catalytic AB pair spontaneously adopted an intermediate conformation, which was not included in the initial inputs of the simulations and was essentially close to the “bindable-like” structure observed in a recently solved crystal structure. Furthermore, the creation of a space between the bindable-like and tight pairs was required for the central stalk to rotate without steric hindrance. These cooperative rearrangements of the three catalytic pairs are crucial for the rotation of the central stalk.

Molecular Modeling and Molecular Dynamics Simulations of Recombinase Rad51

The Rad51 ATPase plays central roles in DNA homologous recombination. Yeast Rad51 dimer structure in the active form of the filament was constructed using homology modeling techniques, and all-atom molecular dynamics (MD) simulations were performed using the modeled structure. We found two crucial interaction networks involving ATP: one is among the γ-phosphate of ATP, K(+) ions, H352, and D374; the other is among the adenine ring of ATP, R228, and P379. Multiple MD simulations were performed in which the number of bound K(+) ions was changed. The simulated structures suggested that K(+) ions are indispensable for the stabilization of the active dimer and resemble the arginine and lysine fingers of other P-loop containing ATPases and GTPases. MD simulations also showed that the adenine ring of ATP mediates interactions between adjacent protomers. Furthermore, in MD simulations starting from a structure just after ATP hydrolysis, the opening motion corresponding to dissociation from DNA was observed. These results support the hypothesis that ATP and K(+) ions function as glue between protomers.