Allosteric communication in the gating mechanism for controlled protein degradation by the bacterial ClpP peptidase.

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.

Exploring the Effect of Mechanical Anisotropy of Protein Structures in the Unfoldase Mechanism of AAA+ Molecular Machines.

Essential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of µm to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule-severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. Unfolding of wild-type DHFR requires disruption of mechanically strong ß-sheet interfaces near each terminal, which yields branched pathways associated with unzipping along soft directions and shearing along strong directions. By contrast, unfolding of circular permutant DHFR variants involves single pathways due to softer mechanical interfaces near terminals, but translocation hindrance can arise from mechanical resistance of partially unfolded intermediates stabilized by ß-sheets. For spastin, optimal severing action initiated by pulling on a tubulin subunit is achieved through specific orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine.

Unfolding and Translocation of Knotted Proteins by Clp Biological Nanomachines: Synergistic Contribution of Primary Sequence and Topology Revealed by Molecular Dynamics Simulations.

We use Langevin dynamics simulations to model, at an atomistic resolution, how various natively knotted proteins are unfolded in repeated allosteric translocating cycles of the ClpY ATPase. We consider proteins representative of different topologies, from the simplest knot (trefoil 31), to the three-twist 52 knot, to the most complex stevedore, 61, knot. We harness the atomistic detail of the simulations to address aspects that have so far remained largely unexplored, such as sequence-dependent effects on the ruggedness of the landscape traversed during knot sliding. Our simulations reveal the combined effect on translocation of the knotted protein structure, i.e., backbone topology and geometry, and primary sequence, i.e., side chain size and interactions, and show that the latter can dominate translocation hindrance. In addition, we observe that due to the interplay between the knotted topology and intramolecular contacts the transmission of tension along the polypeptide chain occurs very differently from that of homopolymers. Finally, by considering native and non-native interactions, we examine how the disruption or formation of such contacts can affect the translocation processivity and concomitantly create multiple unfolding pathways with very different activation barriers.