"bind and Crawl" Association Mechanism of Leishmania major Peroxidase and Cytochrome c Revealed by Brownian and Molecular Dynamics Simulations

James B. Fields, Scott A. Hollingsworth, Georges Chreifi, Matthias Heyden, Anton P. Arce, Hugo I. Magaña-Garcia, Thomas L. Poulos, Douglas J. Tobias

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Leishmania major, the parasitic causative agent of leishmaniasis, produces a heme peroxidase (LmP), which catalyzes the peroxidation of mitochondrial cytochrome c (LmCytc) for protection from reactive oxygen species produced by the host. The association of LmP and LmCytc, which is known from kinetics measurements to be very fast (∼108 M-1 s-1), does not involve major conformational changes and has been suggested to be dominated by electrostatic interactions. We used Brownian dynamics simulations to investigate the mechanism of formation of the LmP-LmCytc complex. Our simulations confirm the importance of electrostatic interactions involving the negatively charged D211 residue at the LmP active site, and reveal a previously unrecognized role in complex formation for negatively charged residues in helix A of LmP. The crystal structure of the D211N mutant of LmP reported herein is essentially identical to that of wild-type LmP, reinforcing the notion that it is the loss of charge at the active site, and not a change in structure, that reduces the association rate of the D211N variant of LmP. The Brownian dynamics simulations further show that complex formation occurs via a "bind and crawl" mechanism, in which LmCytc first docks to a location on helix A that is far from the active site, forming an initial encounter complex, and then moves along helix A to the active site. An atomistic molecular dynamics simulation confirms the helix A binding site, and steady state activity assays and stopped-flow kinetics measurements confirm the role of helix A charges in the association mechanism.

Original languageEnglish (US)
Pages (from-to)7272-7282
Number of pages11
JournalBiochemistry
Volume54
Issue number49
DOIs
StatePublished - Nov 24 2015
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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