Single-molecule FRET probes allosteric effects on protein-translocating pore loops of a AAA+ machine
Year: 2024
Type: article
Abstract: 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 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 single-molecule FRET, 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.
Source: Biophysical Journal
Institutions: Weizmann Institute of Science, University of Cincinnati
Language: English
Topic: Heat shock proteins research
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $2,500
Funders: National Science Foundation, National Sleep Foundation, European Molecular Biology Organization, European Commission, Council for Higher Education, Yükseköğretim Kurulu, Horizon 2020 Framework Programme, Institute for Advanced Studies in the Humanities, University of Edinburgh, European Research Council, Horizon 2020, Planning and Budgeting Committee of the Council for Higher Education of Israel, Office of Advanced Cyberinfrastructure, H2020 European Research Council
Awards: G1258419064, G1453181110, Track 4: Advanced CI Coordination Ecosystem: Monitoring and Measurement Services, G257040694, G3562386289, Track 1: ACCESS Resource Allocations Marketplace and Platform Services (RAMPS), G4010157413, G434337789, ACO: An Open CI Ecosystem to Advance Scientific Discovery (OpenCI), NSF-BSF: Synergistic Multiscale Modeling and Single-Molecule Fluorescence Studies of the Dynamics and Function of AAA+ Protein Disaggregation Machines, Single-molecule spectroscopy of coordinated motions in allosteric proteins, Track 3: COre National Ecosystem for CyberinfrasTructure (CONECT), G5367127336, G5464919783, Track 2: Customized Multi-tier Assistance, Training, and Computational Help (MATCH) for End User ACCESS to CI, G8953989493
OpenAlex ID: W4390685702