“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.

Insights into the Dynamics and Dissociation Mechanism of a Protein Redox Complex Using Molecular Dynamics

“…Figure 6 shows a comparison of our previously determined wild type kinetics 14 with that of the Y134F LmP mutant. The Y134F LmP mutant exhibits a simple hyperbolic behavior, with a 3.6-fold increase in K m when compared to wild type (Fig.…”

“…The preparation of the MD simulation is similar to our previous study of the LmP-LmCytc system 14 and described briefly here. Hydrogen atoms were added to the crystal structure using the psfgen plugin of VMD 1.9.1.…”

“…3 Further emphasizing the importance of electrostatic interactions in the LmP system, we recently documented a secondary binding site for LmCytc on LmP. 14 This non-catalytic site is composed of four nearly consecutive negatively charged residues on helix A, adjacent to but separate from the active site. A combination of computational and experimental results has shown that helix A influences complex formation and dissociation for the LmP system, but not for CCP.…”

“…7 These computational results guided the mutagenesis studies where removing 3 negative charges on helix A was found to lower k cat by ≈3-fold and the rate of association of the two proteins by ≈6-fold. 14 …”

A cross-domain charge interaction governs the activity of NO synthase

“…The importance of this conserved Trp has not yet been tested in nNOS or in other NOS enzymes. In any case, it is important to note that rapid conformational sampling likely occurs during NOS FMN-NOSoxy domain docking (40,41) to generate many transiently-docked species, several of which still place the FMN and heme close enough for ET between them. That said, there also may be docking preferences among the NOS enzymes that are driven by their structural differences, such as iNOS having fewer surface charges on its oxygenase and reductase domains relative to nNOS or eNOS (30).…”