Coordination and control inside simple biomolecular machines

Motor proteins, sometimes referred to as mechanoenzymes, are a group of proteins that maintain a large part of intracellular motion. Being enzymes, they undergo chemical reactions leading to energy conversion and changes of their conformation. Being mechanodevices, they use the chemical energy to perform mechanical work, leading to the phenomena of motion. Over the past 20 years a series of novel experiments (e.g. single molecule observations) has been performed to gain the deeper knowledge about chemical states of molecular motors as well as their dynamics in the presence or absence of an external force. At the same time, many theoretical models have been proposed, offering various insights into the nano-world dynamics. They can be divided into three main categories: mechanochemical models, ratchet models and molecular dynamics simulations. We demonstrate that by combining those complementary approaches a deeper understanding of the dynamics and chemistry of the motor proteins can be achieved. As a working example, we choose kinesin -a motor protein responsible for directed transport of organelles and vesicles along microtubule tracts.

Current Opinion in Structural Biology, 2013

Journal of Biological Chemistry, 2007

The P4 protein of bacteriophage 12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small ϳ1 Å changes in the ATP-binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at the atomic level. Here we study in atomic detail the mechanochemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, consisting of superfamily 4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNAbinding site that moves along the mechanical reaction coordinate. The lever is flanked by ␥-phosphate sensors (Asn-234 and Ser-252) that report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg-279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This ensures cooperative sequential hydrolysis that is tightly coupled to mechanical motion. Given the structural conservation, the mutated residues may play similar roles in other hexameric helicases and related molecular motors.

Atomistic Approaches in Modern Biology

Molecular motors are adenosine tri-phosphate (ATP) hydrolysis-driven, cellular proteins responsible for a wide variety of different tasks, such as transport, energy metabolism, and DNA processing. Their operation cycle spans a wide range of length and time scales, from the localized and fast chemical reaction in the catalytic site(s) to the large scale and much slower conformational motions involved in the motors' physiological function. From a computational point of view, this means that currently there exists no single approach capable of capturing the whole spectrum of events during molecular motor function. In the present review, we show for PcrA helicase, a molecular motor involved in the unwinding of double-stranded DNA, how a combination of computational approaches can be used to examine PcrA's function in its entirety as well as in detail. Combined quantum mechanical/molecular mechanical simulations are used to study the catalytic ATP hydrolysis event and its coupling to protein conformational changes. Molecular dynamics simulations then provide a means of studying overall PcrA function on a nanosecond time scale. Finally, to reach physiologically relevant time scales, i.e., milliseconds, stochastic simulations are employed. We show that by combining the three stated approaches one can obtain insight into PcrA helicase function.

Journal of Statistical Physics, 2007

Molecular motors are considered that convert the chemical energy released from the hydrolysis of adenosine triphosphate (ATP) into mechanical work. Such a motor represents a small system that is coupled to a heat reservoir, a work reservoir, and particle reservoirs for ATP, adenosine diphosphate (ADP), and inorganic phosphate (P). The discrete state space of the motor is defined in terms of the chemical composition of its catalytic domains. Each motor state represents an ensemble of molecular conformations that are thermally equilibrated. The motor states together with the possible transitions between neighboring states define a network representation of the motor. The motor dynamics is described by a continuous-time Markov process (or master equation) on this network. The consistency between thermodynamics and network dynamics implies (i) local and nonlocal balance conditions for the transition rates of the motor and (ii) an underlying landscape of internal energies for the motor states. The local balance conditions can be interpreted in terms of constrained equilibria between neighboring motor states; the nonlocal balance conditions pinpoint chemical and/or mechanical nonequilibrium.

2022

We used single-molecule nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of Superfamily 1 helicase PcrA as it translocates on, or unwinds, several kb-long DNA molecules. We recorded over 2 million enzyme steps under various assisting and opposing forces in diverse ATP and ADP conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences in vivo, such as when the helicase encounters bound proteins or duplex DNA; we show how PcrA’s kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base-pairs being unwound. Instead, the sequence of the single stranded DNA on which the PcrA walks de...

Biophysical Journal, 2007

PcrA helicase from Bacillus stearothermophilus is one of the smallest motor proteins structurally known in full atomic detail. It translocates progressively from the 39 end to the 59 end of single-stranded DNA utilizing the free energy from ATP hydrolysis. The similarities in structure and reaction pathway between PcrA helicase and F1-ATPase suggest a similar mechanochemical mechanism at work in both systems. Previous studies of PcrA translocation demonstrated a domain stepping mechanism in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of two translocation domains is synchronized with alternating mobilities of the individual domains such that PcrA moves unidirectionally along single-stranded DNA. To substantiate this translocation mechanism, this study applies molecular dynamics simulations, elastic network theory, and multiple sequence alignment to analyze the system. The analysis provides further evidence that directional translocation of PcrA is regulated allosterically through synchronization of ATP hydrolysis and domain mobilities. We identify a set of essential residues coevolutionarily coupled in related helicases that should be involved in the allosteric regulation of these motor proteins.

Biophysical Journal, 1996

The typical biochemical paradigm for coupling between hydrolysis of ATP and the performance of chemical or mechanical work involves a well-defined sequence of events (a kinetic mechanism) with a fixed stoichiometry between the number of ATP molecules hydrolyzed and the turnover of the output reaction. Recent experiments show, however, that such a deterministic picture of coupling may not be adequate to explain observed behavior of molecular motor proteins in the presence of applied forces. Here we present a general model in which the binding of ATP and release of ADP serve to modulate the binding energy of a motor protein as it travels along a biopolymer backbone. The mechanism is loosely coupled-the average number of ATPs hydrolyzed to cause a single step from one binding site to the next depends strongly on the magnitude of an applied force and on the effective viscous drag force. The statistical mechanical perspective described here offers insight into how local anisotropy along the "track" for a molecular motor, combined with an energyreleasing chemical reaction to provide a source of nonequilibrium fluctuations, can lead to macroscopic motion.

1998

We investigate theoretically a novel mechanism of transducing chemical energy into a directed force. A hypothetical motor protein is considered such that conformational changes induced by nucleotide binding and/or hydrolysis lead to asymmetric internal velocity fluctuations. We demonstrate that these fluctuations result in unidirectional motion if rectified by protein friction. The motor protein force-velocity relationship and other characteristics are computed based on analogy with known molecular motors. @ 1998 Elsevier Science B.V.

2009

Hexameric ring-shaped AAA؉ molecular motors have a key function of active translocation of a macromolecular chain through the central pore. By performing multiscale molecular dynamics (MD) simulations, we revealed that HslU, a AAA؉ motor in a bacterial homologue of eukaryotic proteasome, translocates its substrate polypeptide via paddling mechanism during ATP-driven cyclic conformational changes. First, fully atomistic MD simulations showed that the HslU pore grips the threaded signal peptide by the highly conserved Tyr-91 and Val-92 firmly in the closed form and loosely in the open form of the HslU. The grip depended on the substrate sequence. These features were fed into a coarse-grained MD, and conformational transitions of HslU upon ATP cycles were simulated. The simulations exhibited stochastic unidirectional translocation of a polypeptide. This unidirectional translocation is attributed to paddling motions of Tyr-91s between the open and the closed forms: downward motions of Tyr-91s with gripping the substrate and upward motions with slipping on it. The paddling motions were caused by the difference between the characteristic time scales of the pore-radius change and the up-down displacements of Tyr-91s. Computational experiments on mutations at the pore and the substrate were in accord with several experiments. coarse-grained model ͉ fully atomistic simulation ͉ HslU ͉ molecular dynamics ͉ multiple basin model