Kinesin's moonwalk

Kinesin-1 is a single-molecule walking machine, driven by ATP turnover. Recent optical trapping experiments show that pulling backwards on a walking kinesin-1 molecule causes the mechanical walking action to reverse, while the coupled chemical cycle of ATP turnover continues, apparently, to run forwards -- kinesin can moonwalk. Individual forward- and back-steps are fast, and each appears to be a single event, complete in a few tens of microseconds, with no substeps. Between steps, kinesin pauses, waiting for the next ATP to arrive. Several lines of evidence indicate that during these between-step dwells, only one of the two heads is strongly attached to the microtubule. The position of the other head during the dwells is less certain, and more controversial.     

Molecular motors: Kinesin's string variable

A recent model suggests that the walking action of kinesin is due to a 13 residue 'fundamental engine' called the neck linker domain, which cyclically zips and unzips to the main part of the heads. New experiments confirm one prediction of the model: that crosslinking the neck linker to the head should block motility.     

驱动蛋白的颈链结构与功能关系

驱动蛋白的颈链是驱动蛋白力产生的关键元件。颈链与驱动蛋白马达结构域的对接过程为驱动蛋白沿着微管的向前运动提供了驱动力。驱动蛋白的颈链由14~18个氨基酸组成,它连接着马达结构域和由缠绕螺旋组成的茎部。颈链与驱动蛋白马达结构域的对接过程是通过多种弱键相互作用实现的,这些弱键相互作用多数都直接或间接与水分子有关。颈链及其相关区域的极为巧妙的氨基酸结构使得这些弱键能够在细胞环境下有效地发挥作用。对颈链的结构与功能关系的认识大大提高了我们对驱动蛋白运动机制的理解。     

Molecular motors: Kinesin's dynamically dockable neck

Kinesin is a molecular walking machine with two identical motor heads connected to a coiled-coil tail. Details of the coordination mechanism, which causes kinesin to walk directionally, and the tracking mechanism, which guides each detaching head to its next site on the microtubule, are beginning to emerge.     

Processivity of single-headed kinesin motors

The processive movement of single-headed kinesins is studied by using a ratchet model of non-Markov process, which is built on the experimental evidence that the strong binding of kinesin to microtubule in rigor state induces a large apparent change in the local microtubule conformation. In the model, the microtubule plays a crucial active role in the kinesin movement, in contrast to the previous belief that the microtubule only acts as a passive track for the kinesin motility. The unidirectional movement of single-headed kinesin is resulted from the asymmetric periodic potential between kinesin and microtubule while its processivity is determined by its binding affinity for microtubule in the weak ADP state. Using the model, various experimental results for monomeric kinesin KIF1A, such as the mean step size, the step-size distribution, the long run length and the mean velocity versus load, can be well explained quantitatively. This local conformational change of the microtubule may also play important roles in the processive movement of conventional two-headed kinesins. An experiment to verify the model is suggested.     

Processivity of ribozyme-catalyzed RNA polymerization

The "RNA world" hypothesis proposes that early in the evolution of life, before the appearance of DNA or protein, RNA was responsible both for encoding genetic information and for catalyzing biochemical reactions. Ribo-organisms living in the RNA world would have replicated their RNA genomes by using an RNA polymerase ribozyme. Efforts to provide experimental support for the RNA world hypothesis have focused on producing such a polymerase, and in vitro evolution methods have led to the isolation of a polymerase ribozyme that catalyzes primer extension which is accurate and general, but slow. To understand the reaction of this ribozyme, we developed a method of measuring polymerase processivity that is particularly useful in the case of an inefficient polymerase. This method allowed us to demonstrate that the polymerase ribozyme, despite its inefficiency, is partially processive. It is currently limited by a low affinity for the primer-template duplex, but once it successfully binds the primer-template duplex in the productive alignment, it catalyzes an extension reaction that is so rapid that it can occur multiple times during the short span of a single binding event. This finding contributes to the understanding of one of the more sophisticated activities yet to be generated de novo in the laboratory and sheds light on the parameters to be targeted for further optimization.     

Processivity of chimeric class V myosins

Unconventional myosin V takes many 36-nm steps along an actin filament before it dissociates, thus ensuring its ability to move cargo intracellularly over long distances. In the present study we assessed the structural features that affect processive run length by analyzing the properties of chimeras of mouse myosin V and a non-processive class V myosin from yeast (Myo4p) (Reck-Peterson, S. L., Tyska, M. J., Novick, P. J., and Mooseker, M. S. (2001) J. Cell Biol. 153, 1121-1126). Surprisingly a chimera containing the yeast motor domain on the neck and rod of mouse myosin V (Y-MD) showed longer run lengths than mouse wild type at low salt. Run lengths of mouse myosin V showed little salt dependence, whereas those of Y-MD decreased steeply with ionic strength, similar to a chimera containing yeast loop 2 in the mouse myosin V backbone. Loop 2 binds to acidic patches on actin in the weak binding states of the cycle (Volkmann, N., Liu, H., Hazelwood, L., Krementsova, E. B., Lowey, S., Trybus, K. M., and Hanein, D. (2005) Mol. Cell 19, 595-605). Constructs containing yeast loop 2, which has no net charge compared with +6 for wild type, showed a higher K(m) for actin in steady-state ATPase assays. The results imply that a positively charged loop 2 and a high affinity for actin are important to maintain processivity near physiologic ionic strength.     

Processivity of single-headed kinesin motors

The processive movement of single-headed kinesins is studied by using a ratchet model of non-Markov process, which is built on the experimental evidence that the strong binding of kinesin to microtubule in rigor state induces a large apparent change in the local microtubule conformation. In the model, the microtubule plays a crucial active role in the kinesin movement, in contrast to the previous belief that the microtubule only acts as a passive track for the kinesin motility. The unidirectional movement of single-headed kinesin is resulted from the asymmetric periodic potential between kinesin and microtubule while its processivity is determined by its binding affinity for microtubule in the weak ADP state. Using the model, various experimental results for monomeric kinesin KIF1A, such as the mean step size, the step-size distribution, the long run length and the mean velocity versus load, can be well explained quantitatively. This local conformational change of the microtubule may also play important roles in the processive movement of conventional two-headed kinesins. An experiment to verify the model is suggested.     

Processivity of DNA exonucleases.

A homopolymer system has been developed to examine the digestion strategies of DNA exonucleases. Escherichia coli exonuclease I and lambda-exonuclease, are processive enzymes. However, T7 exonuclease, spleen exonuclease, E. coli exonuclease III, the 3' leads to 5'-exonuclease of T4 DNA polymerase, and both the 3' leads to 5' and the 5' leads to 3' activity of E. coli DNA polymerase I dissociate frequently from the substrate during the course of digestion. Regions of duplex DNA are a dissociation signal for exonuclease I.     

On the Processivity of DNA Replication

Abstract

In this paper we describe the nature and importance of processive enzymatic reactions in biological processes. A model is set up to describe the processive synthetic process in DNA replication, and experiments are presented to define and test the model, using the components of the T4 phage-coded five-protein (in vitro) DNA replication system of Alberts, Nossal and coworkers. These experiments are performed either with a homogeneous oligo dT-poly dA primer-template system, or with a natural primer-template system using phage M13 DNA. The results are used to define some molecular aspects of the microscopic “processivity cycle”.     

Kinesin's IAK tail domain inhibits initial microtubule-stimulated ADP release

Kinesin undergoes a global folding conformational change from an extended active conformation at high ionic concentrations to a compact inhibited conformation at physiological ionic concentrations. Here we show that much of the observed ATPase activity of folded kinesin is due to contamination with proteolysis fragments that can still fold, but retain an activated ATPase function. In contrast, kinesin that contains an intact IAK-homology region exhibits pronounced inhibition of its ATPase activity (140-fold in 50 mM KCl) and weak net affinity for microtubules in the presence of ATP, resulting from selective inhibition of the release of ADP upon initial interaction with a microtubule. Subsequent processive cycling is only partially inhibited. Fusion proteins containing residues 883-937 of the kinesin alpha-chain bind tightly to microtubules; exposure of this microtubule-binding site in proteolysed species is probably responsible for their activated ATPase activities at low microtubule concentrations.     

Processivity errors of gene expression in Escherichia coli

Not all ribosomes that initiate translation of an mRNA sequence will successfully complete it and produce a full-length protein product. By comparing the amounts of lacZ monomer and lacZ dimer protein expressed from a plasmid in a strictly controlled assay, we calculate a dimer to monomer ratio of 0.76. We interpret this to mean that ribosomes have a 76% chance of completing the synthesis of a beta-galactosidase polypeptide. The remaining 24% of the initiated chains end in processivity accidents. For the wild-type, premature RNA polymerase termination is found to account for roughly one-third of the processivity accidents. For the hyperaccurate SmP mutant, we observe a processivity of 0.28, but the presence of streptomycin improves this to 0.50. Thus, the hyperaccuracy with respect to missense substitutions for this mutant is accompanied by a reduced processivity. Addition of streptomycin increase the first error class and reduces the second one. This finding is relevant to the optimization of ribosome function and the growth performance of ribosome mutants.     

Kinesin's tail domain is an inhibitory regulator of the motor domain

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.     

Kinesin's biased stepping mechanism: amplification of neck linker zippering

A physically motivated model of kinesin's motor function is developed within the framework of rectified Brownian motion. The model explains how the amplification of neck linker zippering arises naturally through well-known formulae for overdamped dynamics, thereby providing a means to understand how weakly-favorable zippering leads to strongly favorable plus-directed binding of a free kinesin head to microtubule. Additional aspects of kinesin's motion, such as head coordination and rate-limiting steps, are directly related to the force-dependent inhibition of ATP binding to a microtubule bound head. The model of rectified Brownian motion is presented as an alternative to power stroke models and provides an alternative interpretation for the significance of ATP hydrolysis in the kinesin stepping cycle.     

The UvrD303 Hyper-helicase Exhibits Increased Processivity

DNA helicases use energy derived from nucleoside 5′-triphosphate hydrolysis to catalyze the separation of double-stranded DNA into single-stranded intermediates for replication, recombination, and repair. Escherichia coli helicase II (UvrD) functions in methyl-directed mismatch repair, nucleotide excision repair, and homologous recombination. A previously discovered 2-amino acid substitution of residues 403 and 404 (both Asp → Ala) in the 2B subdomain of UvrD (uvrD303) confers an antimutator and UV-sensitive phenotype on cells expressing this allele. The purified protein exhibits a “hyper-helicase” unwinding activity in vitro. Using rapid quench, pre-steady state kinetic experiments we show the increased helicase activity of UvrD303 is due to an increase in the processivity of the unwinding reaction. We suggest that this mutation in the 2B subdomain results in a weakened interaction with the 1B subdomain, allowing the helicase to adopt a more open conformation. This is consistent with the idea that the 2B subdomain may have an autoregulatory role. The UvrD303 mutation may enable the helicase to unwind DNA via a “strand displacement” mechanism, which is similar to the mechanism used to processively translocate along single-stranded DNA, and the increased unwinding processivity may contribute directly to the antimutator phenotype.     

Processivity of cellobiohydrolases is limited by the substrate

Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exo- and endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (k(cat)) and dissociation rate constant (k(off)). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their k(off) values, whereas the k(cat) values were similar. Furthermore, the k(off) values for endoglucanases (EGs) were much higher than the k(off) values for CBHs, whereas the k(cat) values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of k(off) may be the primary target for the selection of cellulases.     

Processivity and substrate-binding in family 18 chitinases

Enzymatic depolymerisation of polysaccharides is a key technology in the biorefining of biomass. The enzymatic conversion of the abundant insoluble polysaccharides cellulose and chitin is of particular interest and complexity, because of the bi-phasic nature of the process, the seemingly complicated tasks faced by the enzymes, and the importance of these conversions for the future biorefinery. Here we review recent work on family 18 chitinases that sheds light on important aspects of the catalytic action of these depolymerising enzymes, including the structural basis of processivity and its direction, the energies involved in substrate-binding and displacement.