No abstract

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 intra- and 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.

The current pandemic has shown that we need sensitive and deployable diagnostic technologies. Surface-enhanced Raman scattering (SERS) sensors can be an ideal solution for developing such advanced point-of-need (PON) diagnostic tests. Homogeneous (reagentless) SERS sensors work by directly responding to the target without any processing step, making them capable for simple one-pot assays, but their limitation is the achievable sensitivity, insufficient compared to what is needed for sensing of viral biomarkers. Noncovalent DNA catalysis mechanisms have been recently exploited for catalytic amplification in SERS assays. These advances used catalytic hairpin assembly (CHA) and other DNA self-assembly processes to develop sensing mechanisms with improved sensitivities. However, these mechanisms have not been used in OFF-to-ON homogeneous sensors, and they often target the same biomarker, likely due to the complexity of the mechanism design. There is still a strong need for a catalytic SERS sensor with a homogeneous mechanism and a rationalization of the catalytic sensing mechanism to translate this sensing strategy to different targets and applications. We developed and investigated a homogeneous SERS sensing mechanism that uses catalytic amplification based on DNA self-assembly. We systematically investigated the role of three domains in the fuel strand (internal loop, stem, and toehold), which drives the catalytic mechanism. The thermodynamic parameters determined in our studies were used to build an algorithm for automated design of catalytic sensors that we validated on target sequences associated with malaria and SARS-CoV-2 strains. With our mechanism, we were able to achieve an amplification level of 20-fold for conventional DNA and of 36-fold using locked nucleic acids (LNAs), with corresponding improvements observed in the sensor limit of detection (LOD). We also show a single-base sequence specificity for a sensor targeting a sequence associated with the omicron variant, tested against a delta variant target. This work on catalytic amplification of homogeneous SERS sensors has the potential to enable the use of this sensing modality in new applications, such as infectious disease surveillance, by improving the LOD while conserving the sensor's homogeneous character.

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 ranging from protein unfolding and disaggregation to DNA melting and unwinding. ClpB is a AAA+ disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops which protrude to its center and bind to substrate-proteins, and two nucleotide-binding domains (NBD1-2), responsible for ATP binding and hydrolysis. It remains unknown whether and how the two nucleotide-binding sites interact with each other and affect the pore loops. Recently, we applied single-molecule FRET (smFRET) spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB in aqueous solution. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the timescale of microseconds and change their conformations in response to substrate-protein binding. Here, using smFRET, we study the allosteric coupling between the two ATP-binding sites and the pore loops in ClpB. Using Walker mutations that perturb either ATP hydrolysis or binding, we demonstrate that nucleotide states of the NBDs tune the pore loop dynamic equilibrium. This communication is remarkably long-range, and in particular, PL2 and PL3 are each affected by mutations in both NBD1 and NBD2. We characterize the allosteric paths connecting the NBDs to the pore loops by molecular dynamics simulations, and find that these paths can be altered by changing the ATPase state of ClpB. Further, a rigorous thermodynamic double-mutant cycle analysis reveals the coupling of the two ATP-binding sites in their effects on pore loop dynamics. Surprisingly, abolishing ATP hydrolysis in both NBDs results in an order of magnitude stronger substrate-protein binding to PL2, but not to the other pore loops. Importantly, PL3 which is highly conserved in AAA+ machines, is found to favor either an upward or a downward conformation depending on which of the NBDs remains active. These results explicitly demonstrate a significant long-range allosteric communication between the ATP binding sites and the pore loops, and shed new light on the Brownian ratchet substrate translocation mechanism of 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.

No abstract

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.

No abstract