Road-blocker HSP disease mutation disrupts pre-organization for ATP hydrolysis in kinesin through a second sphere control

Kinesin motor proteins perform several essential cellular functions powered by the adenosine triphosphate (ATP) hydrolysis reaction. Several single-point mutations in the kinesin motor protein KIF5A have been implicated to hereditary spastic paraplegia disease (HSP), a lethal neurodegenerative disease in humans. In earlier studies, we have shown that a series of HSP-related mutations can impair the kinesin’s long-distance displacement or processivity by modulating the order–disorder transition of the linker connecting the heads to the coiled coil. On the other hand, the reduction of kinesin’s ATP hydrolysis reaction rate by a distal asparagine-to-serine mutation is also known to cause HSP disease. However, the molecular mechanism of the ATP hydrolysis reaction in kinesin by this distal mutation is still not fully understood. Using classical molecular dynamics simulations combined with quantum mechanics/molecular mechanics calculations, the pre-organization geometry required for optimal hydrolysis in kinesin motor bound to α/β-tubulin is determined. This optimal geometry has only a single salt-bridge (of the possible two) between Arg203-Glu236, putting a reactive water molecule at a perfect position for hydrolysis. Such geometry is also needed to create the appropriate configuration for proton translocation during ATP hydrolysis. The distal asparagine-to-serine mutation is found to disrupt this optimal geometry. Therefore, the current study along with our previous one demonstrates how two different effects on kinesin dynamics (processivity and ATP hydrolysis), caused by a different set of genotypes, can give rise to the same phenotype leading to HSP disease.

Kinesin-1 is a motor protein that walks along microtubule (MT) filaments toward the plus-ends using energy acquired from the adenosine triphosphate (ATP) hydrolysis reaction while performing various cellular activities. For instance, it is responsible for the intracellular transport of vesicles, organelles, and signaling complexes (1–3). Neuronal kinesin KIF5A, for example (4, 5), is particularly important for retrograde axonal transport inside neurons. Several single-point mutations of the KIF5A kinesin are found to be extremely pathological, leading to a lethal neurodegenerative disease in humans, hereditary spastic paraplegia (HSP) disease (6, 7).Generally, kinesin motor proteins function in a homodimeric state. In earlier studies (8, 9), we have shown that all kinesins have some structurally important regions: motor domains which perform the catalytic conversion of ATP as well as binding to the MT, the coiled-coil stalk region that is essential for dimerization, and the neck linker region that connects the motor domain to the coiled-coil stalk region. It was shown that the energetic balance between kinesin binding to the MT and coiled-coil interactions in the dimerization interface is crucially important for the required order–disorder transition of the neck linker (8). This transition mediates the coordination between two motor domains of the kinesin dimer faithfully, which is required for a long-distance run on the MT. This process is referred to as processivity (10, 11). On the other hand, the rate of hydrolysis of ATP determines the gliding velocity of the kinesin on the MT. It is important to note here that the processivity, directionality of stepping, and gliding velocity are crucial for their function (7–12).Mutations related to HSP disease are genotypic. While most of these mutations are found to be in the motor domain of the kinesin, some are also found to be in the dimerization region. As shown in our earlier studies, which investigated the effect of HSP disease-related mutations on the dynamics of kinesin, these mutations are either at the MT-binding interface or at the dimerization region. Therefore, they destroy the energetic balance between the relative strength of these interactions, impairing the kinesin processivity that leads to HSP disease (8, 9). However, some other mutations in the motor domain are found not to affect the MT-binding strength but also lead to the disease. Experiments have shown that those mutations actually reduce the rate of ATP hydrolysis and thereby affect the gliding velocity of the kinesin (6, 7). It has been proposed that such kinesins, due to their sluggish movement, act as road blockers for other normally moving kinesis. In this article, we will concentrate on one of such mutations which is distal to the ATP hydrolysis reaction center.Like other motor proteins (13–25), the hydrolysis of the ATP molecule is an essential step in the mechanochemical cycle of kinesin. Here we focus on the mutation of the asparagine residue at a distal position with respect to the hydrolysis reaction center that is changed to a serine residue (Asn255Ser for PDB ID: 4HNA) (26). This mutation causes a reduction in the kinesin gliding velocity on the MT and in the ATPase rate compared to the wild-type one (8). The large distance (~11.5 Å) between the asparagine residue and the terminal γ-phosphorus atom of the ATP in the kinesin-1 structure suggests a long-distance or second sphere control of the ATP hydrolysis reaction (Fig. 1). Similar large distance effects have been previously observed. Biochemical studies have shown that the rate of ATP hydrolysis of kinesin-1 increases by ~33-fold upon binding to the MT (6). The MT-binding site and ATP hydrolysis reaction center are also far apart from each other (Fig. 1). Herein, using all-atom explicit solvent molecular dynamics (MD) simulations, hybrid quantum mechanics/molecular mechanics (QM/MM) methods, and an enhanced sampling approach for calculating free energies, we provide an explanation for the molecular origin of this second sphere control of the ATP hydrolysis reaction in kinesin-1, which is needed to understand its connection to HSP disease. In this connection, it is important to note that an earlier study (23) on this topic focusing on the ATP hydrolysis mechanism of kinesin did not consider the effect of MT binding and was limited to aqueous kinesin only. Inclusion of the MT-bound state is required to understand the complete process of ATP hydrolysis which is attempted in this study.Open in a separate windowFig. 1.ATP-bound kinesin-α/β-tubulin complex. It is composed of the kinesin motor domain region, α-tubulin, and β-tubulin. The tubulins are the units of the microtubule filaments. The γ-phosphate group of the ATP is surrounded by the Glu236, Arg203, Ser202, Thr92, Mg²⁺ ion, and six water molecules. The HSP disease-related residue, Asn255, is also shown, and the proposed second sphere interactions are highlighted.In this study, we will first present the three different reaction schemes for the ATP hydrolysis reaction in the wild-type kinesin bound to MT. We will then discuss our findings on the reaction mechanism and energetics for those three reaction schemes using hybrid QM/MM calculations and most importantly determine the pre-organization geometry for the reaction. Next, the effect of mutation and MT unbinding on the pre-organization geometry will be described using all-atom explicit solvent MD simulations and an enhanced sampling approach for calculating free energies. A multiple sequence alignment will also be presented to show how important are the residues/interactions of pre-organization geometry. Finally, we will discuss our results in the context of the HSP disease.