Biology subjects

Kanamaru, S.

Publications and source records attributed to Kanamaru, S..

4 recordsLinked to original sources

bioRxiv2026

In-Cell Protein Crystallization via a Locally Flexible 24-mer Assembly Precursor

In-cell protein crystallization (ICPC) produces ordered protein crystals within living cells, but the mechanisms used by proteins to acquire long-range crystalline order in the cellular environment remains poorly understood. Here, we define the assembly pathway of CipB, a crystalline inclusion protein from Photorhabdus luminescens. CipB crystals formed in cells dissolve under mild acidic conditions into a predominant 24-mer species, supporting a model in which an in-cell crystal is built from a discrete 24-mer assembly precursor rather than through direct packing of smaller oligomeric states. Structural analysis of recrystallized CipB shows that the same 24-mer architecture packs into a body-centered cubic lattice, consistent with the lattice observed for the in-cell crystals. Cryo-EM and molecular dynamics analyses indicate that the 24-mer assembly precursor preserves its overall architecture while retaining local conformational flexibility at the N-terminal and surface-loop regions. Mutation analyses further link the N-terminal region to the formation of the 24-mer precursor and surface residues to lattice assembly. These observations support a stepwise crystallization model in which N-terminal flexibility facilitates the formation of an assembly-competent 24-mer precursor, whereas defined hydrophobic surface contacts subsequently organize these precursors into a long-range-ordered lattice.

biophysics↗

bioRxiv2022

Cell-free Protein Crystallization for Nanocrystal Structure Determination

In-cell protein crystallization (ICPC) has attracted attention as a next-generation structural biology tool because it does not require multistep purification processes and large-scale crystallization screenings. However, significant issues remain to be solved in context of obtaining various protein crystals in sufficient amounts and quality for structure determination by ICPC. Here, we report the development of cell-free protein crystallization (CFPC), a direct protein crystallization technique which uses cell-free protein synthesis. The most crucial advantages of CFPC are that the reaction scale and time can be minimized and that various reagents can be added during the reaction. We obtained high-quality nano-sized polyhedra crystals, which are produced in insect cells by infection with cytoplasmic polyhedrosis virus, at a 200 L reaction scale within 6 h. We applied this technology to structure determination of crystalline inclusion protein A (CipA) by suppressing twin crystal formation with addition of an inhibitor to the reaction solution. We succeeded in determining a 2.11 [A] resolution structure from the nanocrystals of CipA. This technology, which integrates in-cell and in vitro crystallizations significantly expands the tools available for high throughput protein structure determination, particularly in context of unstable, low-yield, or substrate-binding proteins, which are difficult to analyze by conventional methods.

biophysics↗

bioRxiv2019

Real-time tracking reveals the catalytic process of Rad51-driven DNA strand exchange

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

molecular biology↗

bioRxiv2019

Rad51 Interaction Analysis Reveals a Functional Interplay Among Recombination Auxiliary Factors

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. Here, we present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a widely conserved auxiliary factor. NMR and site-specific crosslinking experiments revealed two distinct sites within the intrinsically disordered N-terminus of Sfr1 that cooperatively bind to Rad51. Although disruption of this binding severely impaired Rad51 stimulation in vitro, interaction mutants did not show any defects in DNA repair. Unexpectedly, in the absence of the Rad51 paralogs Rad55-Rad57, which constitute another auxiliary factor complex, these interaction mutants were unable to promote DNA repair. Our findings provide molecular insights into Rad51 stimulation by Swi5-Sfr1 and suggest that, rather than functioning in an independent subpathway of HR as was previously proposed, Rad55-Rad57 facilitates the recruitment of Swi5-Sfr1 to Rad51.

molecular biology↗