Biophysical Society Thematic Meeting | Hamburg 2022

Biophysics at the Dawn of Exascale Computers

Poster Abstracts

44-POS Board 44 CHARACTERIZING THE ENERGY LANDSCAPE OF CRE RECOMBINASE CATALYTIC DOMAIN THROUGH MOLECULAR DYNAMICS SIMULATIONS Marco A Ramírez-Martínez 1 ; Nina Pastor 1 ; 1 Universidad Autónoma del Estado de Morelos, Laboratorio de Dinámica de Proteínas, Cuernavaca, Mexico Site-specific tyrosine recombinases are proteins that promote scission or insertion of DNA sequences, being the Cre-loxP system one of the most studied. It acts by forming a tetramer, in which two active and two inactive recombinase monomers bind and recombine two loxP sequences. The complex is stabilized by the interaction of the last catalytic (CAT) domain helix (helix N) with a pocket formed in the adjacent subunit. Despite its extensive use in genetic engineering, there is still missing information regarding target selection, intasome formation, and structural changes associated with the recombination reaction. Here we used molecular dynamics simulations to characterize the energy landscape of the two states of CAT domain from Cre recombinase with the helix N in three different positions (cat-delN: no helix N; cat-trans: helix N in the intasome pocket; and cat-cis: helix N in a proposed auto-inhibitory pocket). All simulations were carried out using GROMACS, the charmm36m force-field, TIP3P water model, 0.15 M NaCl, 300K, the hydrogen mass partitioning algorithm and a 4 femtoseconds time step. For each system we ran three replicas of 5 microseconds each. Our results show that cat-delN simulations in both states sample similar energetic landscapes, displaying a scissor-like movement in the helix N interaction pocket; this movement is reduced in the cat-trans and cat-cis simulations, suggesting that it might be involved in complex formation/disassembly. For the cat- cis simulations, helix N formed stable interactions with the active site residues, keeping this segment in the active site pocket. Also, all systems failed to form a complete active site, which suggests that quaternary interactions are necessary for activation. We acknowledge the computer resources provided by the LNS del Sureste de México through grant 202101023N, CONACyT (CVU: 858905, project INF-2014-02-231504), Laboratorio de Supercómputo y Visualización en Paralelo, and LANCAD through grants 99-2021 and 49-2022.

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