Biology subjects

Dayananda, A.

Publications and source records attributed to Dayananda, A..

3 recordsLinked to original sources

bioRxiv2023

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 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. 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 intraand inter-protomer coupling in the open - close 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 removal of ADEP molecules or point mutations, alter the allosteric network to weaken the coupling.

biophysics↗

bioRxiv2023

Single-molecule FRET probes the allosteric effect of ATP on the protein-translocating pore loops of a AAA+ machine

AAA+ proteins (ATPases associated with various cellular activities) comprise a family of powerful ring-shaped ATP-dependent translocases that carry out numerous vital substrate-remodeling functions. ClpB is a AAA+ protein disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops protruding into its center and binding to substrate-proteins. It remains unknown whether these pore loops contribute only passively to substrate-protein threading or have a more active role. Recently, we have applied single-molecule FRET (smFRET) spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the microsecond timescale that are likely to be mechanistically important for disaggregation. Here, using smFRET, we study the allosteric coupling between the pore loops and the two nucleotide binding domains of ClpB (NBD1-2). By mutating the conserved Walker B motifs within the NBDs to abolish ATP hydrolysis, we demonstrate how the nucleotide state of each NBD tunes pore loop dynamics. This effect is surprisingly long-ranged; in particular, PL2 and PL3 respond differentially to a Walker B mutation in either NBD1 or NBD2, as well as to mutations in both. We characterize the conformational dynamics of pore loops and the allosteric paths connecting NBDs to pore loops by molecular dynamics simulations and find that both principal motions and allosteric paths can be altered by changing the ATPase state of ClpB. Remarkably, PL3, which is highly conserved in AAA+ machines, is found to favor an upward conformation when only NBD1 undergoes ATP hydrolysis, but a downward conformation when NBD2 is active. These results explicitly demonstrate a significant long-range allosteric effect of ATP hydrolysis sites on pore-loop dynamics. Pore loops are therefore established as active participants that undergo ATP-dependent conformational changes to translocate substrate proteins through the central pores of AAA+ machines. Statement of SignificanceMolecular machines function by coupling the energy of ATP hydrolysis to mechanical motion. How this coupling occurs and what timescales are involved remains an open question. In this study, we use a powerful single-molecule FRET technique to measure the real-time dynamics of pore loops, which are essential protein-translocating elements of the ATP-dependent disaggregation machine ClpB. Using a series of mutations of the ATP-hydrolysis motifs of ClpB, we find that, although the motions of these pore loops take place on the microsecond time scale, they are markedly affected by the much slower changes in the nucleotide state of the machine. Generally, this study shows that protein machines, such as ClpB, are wired to harness ATP binding and hydrolysis to allosterically affect distal events, such as the function-related mechanics of pore-loops.

biophysics↗

bioRxiv2020

Factors Underlying Asymmetric Dynamics of Disaggregase and Microtubule Severing AAA+ Machines

Disaggregation and microtubule-severing nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily assemble into ring-shaped hexamers that enable protein remodeling by coupling large-scale conformational changes with application of mechanical forces within a central pore by loops protruding within the pore. We probed these motions and intra-ring interactions that support them by performing extensive explicit solvent molecular dynamics simulations of single-ring severing proteins and the double-ring disaggregase ClpB. Simulations reveal that dynamic stability of hexamers of severing proteins and of the nucleotide binding domain 1 (NBD1) ring of ClpB, which belong to the same clade, involves a network of salt bridges that connect conserved motifs of central PL1 loops of the hexamer. Clustering analysis of ClpB highlights correlated motions of domains of neighboring protomers supporting strong inter-protomer collaboration. Severing proteins have weaker inter-protomer coupling and stronger intra-protomer stabilization through salt bridges formed between PL2 and PL3 loops. Distinct mechanisms are identified in the NBD2 ring of ClpB involving weaker inter-protomer coupling through salt bridges formed by non-canonical loops and stronger intra-protomer coupling. Pore width fluctuations associated with the PL1 constriction in the spiral states, in the presence of a substrate peptide, highlight stark differences between narrowing of channels of severing proteins and widening of the NBD1 ring of ClpB. This indicates divergent substrate processing mechanisms of remodeling and translocation by ClpB and substrate tail-end gripping and possible wedging on microtubule lattice by severing enzymes. Relaxation dynamics of the distance between the PL1 loops and the centers of mass of protomers reveals observation-time-dependent dynamics, leading to predicted relaxation times of tens of microseconds on millisecond experimental timescales. For ClpB the predicted relaxation time is in excellent agreement with the extracted time from smFRET experiments.

biophysics↗