Search

Grp94, an ER-localized molecular chaperone, is required for the folding and activation of many membrane and secretory proteins. Client activation by Grp94 is mediated by nucleotide and conformational changes. In this work, we aim to understand how microscopic changes from nucleotide hydrolysis can potentiate large-scale conformational changes of Grp94. We performed all-atom molecular dynamics simulations on the ATP-hydrolysis competent state of the Grp94 dimer in four different nucleotide bound states. We found that Grp94 was the most rigid when ATP was bound. ATP hydrolysis or nucleotide removal enhanced mobility of the N-terminal domain and ATP lid, resulting in suppression of interdomain communication. In an asymmetric conformation with one hydrolyzed nucleotide, we identified a more compact state, similar to experimental observations. We also identified a potential regulatory role of the flexible linker, as it formed electrostatic interactions with the Grp94 M-domain helix near the region where BiP is known to bind. These studies were complemented with normal-mode analysis of an elastic network model to investigate Grp94's large-scale conformational changes. SPM analysis identified residues that are important in signaling conformational change, many of which have known functional relevance in ATP coordination and catalysis, client binding, and BiP binding. Our findings suggest that ATP hydrolysis in Grp94 alters allosteric wiring and facilitates conformational changes.

Proteolysis is essential for the control of metabolic pathways and the cell cycle. Bacterial caseinolytic proteases (Clp) use peptidase components, such as ClpP, to degrade defective substrate proteins and to regulate cellular levels of stress-response proteins. To ensure selective degradation, access to the proteolytic chamber of the double-ring ClpP tetradecamer is controlled by a critical gating mechanism of the two axial pores. The binding of conserved loops of the Clp ATPase component of the protease or small molecules, such as acyldepsipeptide (ADEP), at peripheral ClpP ring sites, triggers axial pore opening through dramatic conformational transitions of flexible N-terminal loops between disordered conformations in the "closed" pore state and ordered hairpins in the "open" pore state. In this study, we probe the allosteric communication underlying these conformational changes by comparing residue-residue couplings in molecular dynamics simulations of each configuration. Both principal component and normal mode analyses highlight large-scale conformational changes in the N-terminal loop regions and smaller amplitude motions of the peptidase core. Community network analysis reveals a switch between intra- and inter-protomer coupling in the open-closed pore transition. Allosteric pathways that connect the ADEP binding sites to N-terminal loops are rewired in this transition, with shorter network paths in the open pore configuration supporting stronger intra- and inter-ring coupling. Structural perturbations, either through the removal of ADEP molecules or point mutations, alter the allosteric network to weaken the coupling.

Although it is known that majority of kinesin motors walk predominantly toward the plus end of microtubules (MTs) in a hand-over-hand manner, the structural origin of the stepping directionality is not understood. To resolve this issue, we modeled the structures of kinesin-1 (Kin1), MT, and the Kin1-MT complex using the elastic network model and calculated the residue-dependent responses to a local perturbation in the constructs. Kin1 binding elicits an asymmetric response that is pronounced in α/β-tubulin dimers in the plus end of the MT. Kin1 opens the clefts of multiple plus end α/β-tubulin dimers, creating binding-competent conformations, which is required for processivity. Reciprocally, MT induces correlations between switches I and II in the motor and enhances fluctuations in adenosine 5'-diphosphate and the residues in the binding pocket. Our findings explain both the directionality of stepping and MT effects on a key step in the catalytic cycle of kinesin.