Biology subjects

Banwait, J. K.

Publications and source records attributed to Banwait, J. K..

4 recordsLinked to original sources

bioRxiv2026

Biased stochastic motor dynamics drive bidirectional, processive translocation by the AAA+ disaggregase Hsp104.

Protein aggregation disrupts proteostasis and drives neurodegeneration. Hsp104 is a hexameric, ring-shaped AAA+ ATPase that dissolves protein aggregates, yet how hexamers translocate and extract polypeptides trapped in mechanically resistant aggregates remains unclear. Using substrates that recapitulate the physical constraints of aggregates, we establish that Hsp104 is a processive, bidirectional translocase that can dynamically switch direction while threading a single polypeptide. On mechanically restrained substrates and prions, Hsp104 hexamers execute biased stochastic transitions among three conformational states at individual interprotomer interfaces: closed, extended, and a previously unobserved hyperextended form. These transitions follow kinetically favored paths rather than a rigid rotary sequence. The resulting biased stochastic stepping, enabled by the conformational plasticity of Hsp104 hexamers, underpins operational adaptability and redefines the functional logic of AAA+ motors.

biochemistry↗

bioRxiv2025

ClpA- and ClpAP-Catalyzed Unfolding and Translocation are Differentially Coupled to ATP Binding

ClpA is an ATP-dependent chaperone essential for protein quality control in E. coli. Upon ATP binding, ClpA forms hexameric rings capable of association with the tetradecameric ClpP protease. ClpA couples ATP binding and/or hydrolysis to the unfolding and translocation of protein substrates into the central cavity of ClpP for degradation. We previously developed a single-turnover stopped-flow method sensitive to ClpA-catalyzed translocation in the absence of ClpP-catalyzed proteolysis. This method was used on unstructured substrates so that the kinetics were reflective of translocation and not unfolding. We showed that at saturating [ATP], ClpA translocated at [~]20 aa s-1, with the kinetic step size, i.e., the average number of amino acids (aa) translocated between two rate-limiting steps being [~]14 aa step-1. Adding ClpP increased the rate to [~]36 aa s-1 and decreased the kinetic step-size to [~]5 aa step-1. Here we apply this method to substrates containing folded Titin I27 domains. We report that at saturating [ATP], ClpA unfolded and translocated at [~]12 aa s-1, nearly half the rate of translocation alone. However, in the presence of ClpP, ClpA exhibited a rate of [~]40 aa s-1, representing no reduction in rate over translocation alone. Interestingly, unlike translocation alone, the kinetic step-size for unfolding and translocation was [~]29 aa step-1 for both ClpA and ClpAP. Examining the [ATP]-dependence of the unfolding reactions revealed that the increased kinetic step-size results from the averaging of a large unfolding step-size of [~]97 aa, representing cooperative unfolding of a single Titin I27 domain, followed by multiple smaller translocation steps on the newly unfolded chain. Moreover, just like translocation alone, the introduction of folds into the substrate results in different kinetics between ClpA and ClpAP. These observations further support a model where ClpP allosterically impacts ClpA-catalyzed processes. SignificanceClpA is one of several AAA+ motors in E. coli. As part of the ATP-dependent protease ClpAP, it facilitates the removal of misfolded and properly folded proteins from the cell. Previously, we published the [ATP]-dependencies of kinetic parameters such as rate constants, kinetic step-sizes, and rates for ClpA- and ClpAP-catalyzed translocation. Here, for the first time, we make similar determinations for the unfolding and translocation cycle. We find both processes to be kinetically coupled to ATP binding, with unfolding being more sensitive to decreasing [ATP] compared to translocation. This coupling differs between ClpA and ClpAP. These findings reinforce the foundation for comparing how AAA+ motors respond to substrate folds, ATP levels, and allosteric regulation.

biophysics↗

bioRxiv2024

Quantitative Insights into Processivity of an Hsp100 Protein Disaggregase on Folded Protein Substrates

The Hsp100 family of proteins play important roles in maintaining protein homeostasis in cells. E. coli ClpB is an Hsp100 protein that remodels misfolded proteins or aggregates. ClpB is proposed to couple the energy from ATP binding and hydrolysis to processively unfold and translocate protein substrates through its axial channel in the hexameric ring structure. However, many of the details of this reaction remain obscure. We have recently developed a transient state kinetics approach to study ClpB catalyzed protein unfolding and translocation. In this work we have used this approach to begin to examine how ATP is coupled to the protein unfolding reaction. Here we show that at saturating [ATP], ClpB induces the cooperative unfolding of a complete TitinI27 domain of 98 amino acids, which is represented by the kinetic step-size m [~]100 amino acids. This unfolding event is followed by rapid and undetected translocation up to the next folded domain. At sub-saturating [ATP], ClpB still induces cooperative unfolding of a complete TitinI27 domain but translocation becomes partially rate-limiting, which leads to an apparent reduced kinetic step-size as small as [~] 50 amino acids. Further, we show that ClpB exhibits an unfolding processivity of P = (0.74 {+/-} 0.06) independent of [ATP]. These findings advance our understanding of the elementary reactions catalyzed by E. coli ClpB but are broadly applicable to a variety of Hsp100 family members. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/617403v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@cb70ceorg.highwire.dtl.DTLVardef@5b1607org.highwire.dtl.DTLVardef@34cbaorg.highwire.dtl.DTLVardef@a280c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

E. coli ClpB is a Robust and Processive Protein Unfoldase

E. coli ClpB, and S. cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of [~]0.9 aa s-1 with a step-size of [~]60 amino acids. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered.

biophysics↗