Molecular dynamics of the <scp>P</scp>450cam–<scp>P</scp>dx complex reveals complex stability and novel interface contacts

Hollingsworth, Scott A.; Poulos, T.L.

doi:10.1002/pro.2583

Cited by 19 publications

(24 citation statements)

References 41 publications

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“…To summarize, in our simulations, salt bridges between D251 and K178 or R186 are broken in the presence of Pdx, again in accordance with previous findings . On the other hand, in the Pdx‐free simulations (P‐FeOO and ‐Cpd(0)), we find that they are also broken in significant segments of the simulation, and are also less frequent than predicted, due to the fact that these interactions are weaker when D251 is protonated.…”

Section: Resultssupporting

confidence: 92%

Proton relay network in P450cam formed upon docking of putidaredoxin

Uğur

Chandrasekhar

2019

Proteins

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Cytochromes P450 are versatile heme‐based enzymes responsible for vital life processes. Of these, P450cam (substrate camphor) has been most studied. Despite this, precise mechanisms of the key O─O cleavage step remain partly elusive to date; effects observed in various enzyme mutants remain partly unexplained. We have carried out extended (to 1000 ns) MM‐MD and follow‐on quantum mechanics/molecular mechanics computations, both on the well‐studied FeOO state and on Cpd(0) (compound 0). Our simulations include (all camphor‐bound): (a) WT (wild type), FeOO state. (b) WT, Cpd(0). (c) Pdx (Putidaredoxin, redox partner of P450)‐docked‐WT, FeOO state. (d) Pdx‐docked WT, Cpd(0). (e) Pdx‐docked T252A mutant, Cpd(0). Among our key findings: (a) Effect of Pdx docking appears to go far beyond that indicated in prior studies: it leads to specific alterations in secondary structure that create the crucial proton relay network. (b) Specific proton relay networks we identify are: FeOO(H)⋯T252⋯nH 2O⋯D251 in WT; FeOO(H)⋯nH 2O⋯D251 in T252A mutant; both occur with Pdx docking. (c) Direct interaction of D251 with –FeOOH is, respectively, rare/frequent in WT/T252A mutant. (d) In WT, T252 is in the proton relay network. (e) Positioning of camphor appears significant: when camphor is part of H‐bonding network, second protonation appears to be facilitated.

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Section: Resultssupporting

confidence: 92%

Proton relay network in P450cam formed upon docking of putidaredoxin

Uğur

Chandrasekhar

2019

Proteins

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“…In addition to the PCAs presented here and in previous publications (19), and the several crystal structures of P450cam (18) in states between the open and closed forms, these results indicate that a potentially active intermediate state is sufficiently populated with a long enough lifetime to accommodate the much faster proton-coupled electron transfer processes required for regioselective and stereoselective substrate hydroxylation. (8) with the crosslinker removed and the system mutated to wild type (8), identical to the starting point used in our previous MD study (20). For P450cam(C)-Pdx simulations, we started with the published NMR structure of Pdx in complex with P450cam in the ferric, closed state (PDB ID code 2M56) (9).…”

Section: Discussionmentioning

confidence: 99%

“…The parameters used here were identical to those used in a previous study (20), but are discussed briefly here. The ff10 force fields were used to model protein, whereas the Cys-heme ligand parameters were taken from Shahokh et al (22).…”

Section: Discussionmentioning

confidence: 99%

“…Buried surface area over the course of each complex trajectory was calculated by subtracting the solvent-accessible surface area of the full complex as calculated by VMD using a probe radius of 1.4 Å from the combined surface areas of P450cam and Pdx separately. Analysis of the interface contacts was carried out by using locally developed analysis tools that have previously been used to document major atom-to-atom contacts (20), but is briefly described here. For every frame of a given trajectory, atom-to-atom contact pairs are recorded for every atom-to-atom pair that is within 3.5 Å.…”

Section: Discussionmentioning

confidence: 99%

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Conformational selectivity in cytochrome P450 redox partner interactions

Hollingsworth

Batabyal

Nguyen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein–protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis.

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“…Production runs were made with Amber14 using the same input parameters as described earlier. 10 The main difference is that 0.002 ps steps were taken rather than 0.001 ps, frames were saved every 20 ps, and Amber 14 rather than Amber 12 was used. Several 100 ns simulations were carried out that differed in the initial random starting velocity.…”

Section: Methodsmentioning

confidence: 99%

Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

2017

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Previous crystal structures of cytochrome P450cam complexed with its redox partner, putidaredoxin (Pdx), shows that P450cam adopts the open conformation. It has been hypothesized that the Pdx-induced shift toward the open state frees the essential Asp251 from salt bridges with Arg186 and Lys178 so that Asp251 can participate in a proton relay network required for O2 activation. This in part explains why P450cam has such a strict requirement for Pdx. One problem with this view is that looser substrate-protein interactions in the open state may not be compatible with the observed regio- and stereoselective hydroxylation. In the present study, molecular dynamics simulations show that Pdx binding favors a conformation that stabilizes the active site and decreases camphor mobility yet retains a partially open conformation compatible with the required proton relay network. The R186A mutant which frees Asp251 in the absence of Pdx retains good enzyme activity, and the crystal structure shows that product, 5-exo-hydroxycamphor, is bound. This indicates that rupture of the Asp251-Arg186 relaxes selectivity with respect to source of electrons and enables X-ray generated reducing equivalents to support substrate hydroxylation. These combined computational and experimental results are consistent with the proposed role of Pdx in assisting the release of Asp251 from ion pairs so that it can participate in proton-coupled electron transfer.

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Molecular dynamics of the P450cam–Pdx complex reveals complex stability and novel interface contacts

Cited by 19 publications

References 41 publications

Proton relay network in P450cam formed upon docking of putidaredoxin

Proton relay network in P450cam formed upon docking of putidaredoxin

Conformational selectivity in cytochrome P450 redox partner interactions

Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

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