鈥淏ind and Crawl鈥?Association Mechanism of Leishmania major Peroxidase and Cytochrome c Revealed by Brownian and Molecular Dynamics Simulations
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- 作者：James B. Fields ; Scott A. Hollingsworth ; Georges Chreifi ; Matthias Heyden ; Anton P. Arce ; Hugo I. Maga帽a-Garcia ; Thomas L. Poulos ; Douglas J. Tobias
- 刊名：Biochemistry
- 出版年：2015
- 出版时间：December 15, 2015
- 年：2015
- 卷：54
- 期：49
- 页码：7272-7282
- 全文大小：500K
- ISSN：1520-4995
文摘
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 (鈭?08 M鈥? s鈥?), 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鈥揕mCytc 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 鈥渂ind and crawl鈥?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.