title: "Bind and Crawl" Association Mechanism of Leishmania major Peroxidase and Cytochrome c Revealed by Brownian and Molecular Dynamics Simulations.
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 (∼10(8) 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.
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: James B
authors.initials: JB
authors.lastname: Fields
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Hollingsworth
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: Georges
authors.initials: G
authors.lastname: Chreifi
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: Matthias
authors.initials: M
authors.lastname: Heyden
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: Anton P
authors.initials: AP
authors.lastname: Arce
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: Hugo I
authors.initials: HI
authors.lastname: Magaña-Garcia
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Poulos
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biology and Biochemistry, and §Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Tobias
mesh_terms: D020134:Catalytic Domain; D003198:Computer Simulation; D045304:Cytochromes c / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D056004:Molecular Dynamics Simulation; D009195:Peroxidase / Q000737:chemistry*; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry
chemical_list: D015800:Protozoan Proteins; D017382:Reactive Oxygen Species; D045304:Cytochromes c; D009195:Peroxidase