A structural pathway for activation of the kinesin motor ATPase


Molecular motor proteins use the energy from ATP hydrolysis to move along cytoskeletal filaments, either actin or microtubules, producing work that can be used to contract muscles (Cooke, 1986), transport organelles and vesicles (Hirokawa, 1998; Brown, 1999), assemble spindles and move chromosomes (Endow, 1999) or drive cytokinesis (Goldberg et al., 1998). The mechanism by which motor proteins generate force and move on actin or microtubules is not yet understood. The ATPase activity of the motor is believed to increase greatly upon binding of the motor to its filament, enabling the motor to move along the filament, but the conformational changes associated with activation of the motor ATPase, and therefore required for motor movement, are not known.

Molecular motors undergo transitions between several distinct conformations that correspond to different nucleotide states as they hydrolyze ATP (reviewed in Cooke, 1986), e.g. a Mg⋅ATP prehydrolysis state, a Mg⋅ADP-Pi transition state and a Mg⋅ADP + Pi post-hydrolysis state. Workers anticipate that these nucleotide states and transitions between them will be detectable by spectroscopic or other analytical methods, or observed in crystal structures. Binding to actin or microtubules is expected to further change motor structure by causing structural changes in the filament-binding interface that are transmitted to the nucleotide-binding cleft, resulting in the greatly elevated ATPase activity that is observed when actin or microtubules are added to motors in ATPase reactions. For myosin, the structural changes associated with ATPase activation by actin are predicted to accelerate dissociation of ADP or Pi, the rate-limiting step in ATP hydrolysis (Lymn and Taylor, 1971), whereas for the kinesin motors, the structural changes induced by binding to microtubules should enable the motor to overcome the rate-limiting step, under non-saturating microtubule concentrations, of ADP release (Hackney, 1988).

Three different conformations of myosin have been observed in proteins crystallized with no nucleotide, a transition state analog or Mg⋅ADP (Rayment et al., 1993; Fisher et al., 1995; Dominguez et al., 1998; Houdusse et al., 1999). The models, interpreted to represent the motor in different states of the ATP hydrolysis cycle, have allowed workers to identify structural elements that undergo conformational changes during nucleotide hydrolysis (Fisher et al., 1995; Houdusse et al., 1999). In addition to a dramatic rotation of the lever arm, the ‘switch I’ and ‘switch II’ regions, which are structurally analogous to G protein elements that change in conformation upon nucleotide hydrolysis and exchange (Sablin et al., 1996), undergo movements that change the structure of the active site. The crystal structures of the kinesin motors have been less informative than those of myosin because, to date, they all contain Mg⋅ADP, despite attempts to crystallize the motors with different nucleotides or nucleotide analogs to obtain motors in different states. Comparisons of the available models have revealed only small differences (Sack et al., 1999), which are too small to classify the structures as distinct conformations. The kinesin crystal structures have therefore yielded little information thus far about the elements of the motor that move or change in conformation during nucleotide hydrolysis.

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Identifying the conformational changes that convert the motor into a filament-activated state will be critical to understanding the mechanism of motility of the myosins and kinesins. Although the elevated ATPase produced by adding actin or microtubules to ATPase assays has been well documented, the mechanism by which the enhancement of nucleotide hydrolysis occurs is not known. Recently, the relationship between the basal and microtubule-activated ATPase of the kinesin motors has been elucidated with the report of a mutant that separates the two activities by decoupling nucleotide and microtubule binding by the motor, preventing activation of the motor ATPase by microtubules (Song and Endow, 1998). The decoupling mutant binds tightly to both ADP and microtubules, unlike wild-type kinesin proteins, which bind weakly to microtubules in the presence of ADP, indicating that communication between the nucleotide- and microtubule-binding regions of the motor is disrupted. The change of a single amino acid residue in the mutant blocks activation of its ATPase by microtubules but does not block its basal ATPase activity, demonstrating that the basal and microtubule-activated ATPase activities of the motor can be separated from one another. Separation of the basal and microtubule-stimulated ATPase of the motor could occur by preventing structural changes that are required for ATPase activation and occur when the motor binds to microtubules.

Here we report the crystal structures of three kinesin mutants that block the microtubule-stimulated ATPase of the motor by decoupling nucleotide and microtubule binding by the motor, but do not block the basal ATPase activity of the motor. We interpret the decoupling mutants as destabilizing the ADP conformation and/or stabilizing conformations required to convert the motor into the microtubule-activated state. The structures show the first major changes in conformation of the kinesin motors to be observed in crystal structures. The structural changes that we observe identify elements of the motor that undergo movements during activation by microtubules and transmit the changes between the nucleotide- and microtubule-binding regions of the motor. The structural elements affected by the mutants define a signaling pathway for activation of the kinesin motor ATPase by microtubules.

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— Update: 06-01-2023 — cohaitungchi.com found an additional article How Kinesin-1 Utilize the Energy of Nucleotide: The Conformational Changes and Mechanochemical Coupling in the Unidirectional Motion of Kinesin-1 from the website www.mdpi.com for the keyword kinsesin-1 power stroke stage of atpase cycle.

1. Introduction

Kinesin is a molecular walking motor protein inside cells. The size of kinesin is smaller than the other two members of motor proteins, myosin and dynein. The kinesin proteins can be divided into 14 subfamilies according to their structural and functional similarities (from kinesin-1 to kinesin-14) [1,2,3,4]. The kinesin-1 subfamily (also called conventional kinesin) is the founding member of the kinesin family [5,6] and mainly exists in the nerve axons to transport membranous organelles along microtubule lattice. Different from the kinesin-3 (monomer, but can also form a dimer [7]) and the kinesin-5 (tetramer) subfamily, the members of kinesin-1 form a dimer structure in vivo to “walk” toward the microtubule’s plus end. The entire structure of kinesin-1 can be mainly divided into three domains, i.e., the motor domain, the tail domain and the stalk domain (the motor domain and a part of stalk domain of kinesin-1 are shown in Figure 1). The motor domain (also called motor head), which contains the nucleotide-binding and microtubule-binding sites, is highly conserved among the kinesin family. The tail domain of kinesin-1 is used to bind with the “cargo”. Kinesin-1 proteins have different tail domains, which can bind the light chain to interact with different cargos [8]. The motor domain and the tail domain are connected by a single long α-helix, which is called the stalk domain. The two stalk domains of two kinesin-1 monomers coil together to form a coiled-coil structure and constitute a functional dimer. It is worthwhile to note that ~14 residues constitute the neck linker of kinesin-1, which connects the motor domain and the stalk domain. The conformational changes of the neck linker in different nucleotide-binding states are the key processes in the walking movement of kinesin-1 [9,10,11,12,13]. Because the motor domain locates in the N-terminal part of the protein, kinesin-1 belongs to the N-type kinesin. Kinesins with the motor domain located in the middle and the C-terminal part of the protein are the M-type and C-type, respectively. The walking directionality of kinesin varies with different locations of the motor domain. N-type kinesins (most members of the kinesin family) walk toward the plus end of the microtubule (some of the kinesin-5 proteins with N-terminal motor domain show bidirectional motility, as reviewed in Ref. [14]). In contrast, the C-type kinesin (mainly kinesin-14 subfamily [15]) walks toward the minus end of the microtubule. The M-type kinesin (mainly kinesin-13 subfamily) is relatively special because it takes one-dimensional diffusion toward the two ends of the microtubule [16,17,18,19].The kinesin-1 dimer walks along a single protofilament of the microtubule in a hand-over-hand manner. There are some noteworthy features of kinesin-1 walking movement: (1) Kinesin-1 can transform chemical energy of the adenosine triphosphate (ATP) binding and hydrolysis to mechanical energy of the walking along the microtubule with a cargo. (2) The chemical cycle and the mechanical cycle of kinesin-1 are highly coupled to ensure only one ATP molecule is consumed in one step [20,21]. The futile ATP hydrolysis rarely happens in kinesin-1 normal walking process. (3) The microtubule not only provides the track for the motility of kinesin-1 but also directly participates in the regulation of the kinesin-1 chemical cycle. The microtubule can catalyze the release of adenosine diphosphate (ADP), which is the product of ATP hydrolysis. In this way, the mechanochemical process of kinesin-1 is dramatically accelerated. The key process of energy transformation is from ATP entering the nucleotide-binding pocket to the docking movement of the neck linker, which pulls the other motor domain to the next binding site on the microtubule. How the conformational changes induced by the ATP binding can transmit to the neck linker region and finally drive the neck linker docking is an essential question in the walking mechanism of kinesin-1. In this paper, research on the conformational changes from the ATP binding to the neck liner docking and the coupling of mechanochemical cycle of kinesin-1 is reviewed.

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