Deciphering Intrinsic Inter-subunit Couplings that Lead to Sequential Hydrolysis of F 1 -ATPase Ring

Abstract

Rotary sequential hydrolysis of the metabolic machine F1-ATPase is a prominent manifestation of high coordination among multiple chemical sites in ring-shaped molecular machines, and it is also functionally essential for F1 to tightly couple chemical reactions and central γ-shaft rotation. High-speed AFM experiments have identified that sequential hydrolysis is maintained in the F1 stator ring even in the absence of the γ-rotor. To explore the origins of intrinsic sequential performance, we computationally investigated essential inter-subunit couplings on the hexameric ring of mitochondrial and bacterial F1. We first reproduced in stochastic Monte Carlo simulations the experimentally determined sequential hydrolysis schemes by kinetically imposing inter-subunit couplings and following subsequent tri-site ATP hydrolysis cycles on the F1 ring. We found that the key couplings to support the sequential hydrolysis are those that accelerate neighbor-site ADP and Pi release upon a certain ATP binding or hydrolysis reaction. The kinetically identified couplings were then examined in atomistic molecular dynamics simulations at a coarse-grained level to reveal the underlying structural mechanisms. To do that, we enforced targeted conformational changes of ATP binding or hydrolysis to one chemical site on the F1 ring and monitored the ensuing conformational responses of the neighboring sites using structure-based simulations. Notably, we found asymmetrical neighbor-site opening that facilitates ADP release upon enforced ATP binding. We also captured a complete charge-hopping process of the Pi release subsequent to enforced ATP hydrolysis in the neighbor site, confirming recent single-molecule analyses with regard to the role of ATP hydrolysis in F1. Our studies therefore elucidate both the coordinated chemical kinetics and structural dynamics mechanisms underpinning the sequential operation of the F1 ring.

Designed Elastic Networks: Models of Complex Protein Machinery

“…Despite obvious simplifications, such models can reproduce conformational fluctuations in proteins, and, remarkably, normal modes of elastic networks have been shown to be able to describe even the large-amplitude motions related to ATP binding and hydrolysis in various molecular machines and motors (e.g., [ 6 , 7 , 8 ]). Moreover, in several previous studies, protein elastic networks have been considered as dynamical systems, and conformational dynamics in response to external forcing has been probed in order to investigate functional mechanochemical motions in protein machines [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ].…”

Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA in association with Cas2

“…In order to obtain the individual residue correlations with the active site1, we calculated first the pairwise correlations for all amino acids and nucleic acids residues. The correlation between each pair of residues is given by , where R i and R j are the position vectors of residue i and j , taking at C α atom of an amino acid or the center of mass (COM) of the nitrogen atoms (N1 to N4, N6, N7, N9) in the base of a nucleotide and the backbone P atom; 〈…〉 represents averaging over the simulation trajectory (51,52). Accordingly, we obtained a pairwise N x N correlation matrix, with N =1624 counting all residues from protein and DNA, by averaging over 200 snapshots from each of the 200-ns equilibrium simulation.…”

“…, where " and # are the position vectors of residue i and j, taking at . atom of an amino acid or the center of mass (COM) of the nitrogen atoms (N1 to N4, N6, N7, N9) in the base of a nucleotide and the backbone P atom; á…ñ represents averaging over the simulation trajectory (51,52). Accordingly, we obtained a pairwise N x N correlation matrix, with N =1624 counting all residues from protein and DNA, by averaging over 200 snapshots from each of the 200-ns equilibrium simulation.…”

Coarse-Grained Protein Dynamics Studies Using Elastic Network Models

“…As ENM-MD is effective at simulating long-time behavior, it could be used together with short all-atom MD simulations for studying structural details. A combination of targeted all-atom MD simulations and targeted ENM-MD simulations including the effect of ATP binding was adopted to investigate the inter-subunit coupling of F1-ATPase [73].…”