Biology subjects

Ghanbarpour, A.

Publications and source records attributed to Ghanbarpour, A..

5 recordsLinked to original sources

bioRxiv2026

Electrostatic Complementarity at the ClpX Substrate-EntryChannel Governs ATP-Driven Protein Unfolding

AAA+ proteases maintain proteostasis by mechanically unfolding protein substrates before degradation, yet how ATP-driven pulling is converted into productive unfolding remains poorly understood. Here, using ssrA-tagged GFP substrates spanning a range of surface charges, we show that electrostatic complementarity between the positively charged ClpX substrate-entry channel and folded substrate domains governs unfolding efficiency. Positively charged substrates unfolded inefficiently despite substrate recognition, thermal stability, ATPase activation, and pore-loop engagement comparable to negatively charged substrates. Cryo-EM structures of a positively charged substrate revealed multiple substrate-engagement states arising from electrostatic incompatibility at the ClpX substrate-entry channel, whereas negatively charged substrates formed favorable electrostatic interactions that likely stabilize partially unfolded intermediates during successive ATP-driven unfolding attempts. Together, these findings identify electrostatic complementarity as a key determinant of productive ATP-driven protein unfolding and suggest that tuning substrate or substrate-entry electrostatics provides a mechanism for regulating AAA+ protease activity.

biochemistry↗

bioRxiv2025

Structural Plasticity of the Membrane-Bound Protein Degradation Assembly Supports Bacterial Adaptation to Stress

Protein degradation by AAA+ proteases is essential for bacterial adaptation to environmental stress. The membrane-bound AAA+ protease FtsH forms a large inner-membrane complex with the SPFH (Stomatin, Prohibitin, Flotillin, HflK/C) family transmembrane proteins HflK and HflC, playing a key role in bacterial recovery from aminoglycoside antibiotic stress. Recent structural studies have revealed both open, asymmetric and closed, symmetric conformations of the HflK/C assembly under different sample-preparation conditions, suggesting two distinct models for how this complex modulates FtsH proteolysis. To determine which conformation reflects the biologically active state, we engineered a disulfide-crosslinked HflK/C variant to stabilize the closed conformation and resolved its structure using high-resolution cryo-EM. Phenotypic assays showed that cells expressing either this stabilized, closed HflK/C variant or an HflK/C mutant that disrupts interactions with FtsH exhibit significantly impaired growth under aminoglycoside stress. Surprisingly, the cryo-EM structure of the FtsH*HflK/C complex from cells challenged with the aminoglycoside antibiotic tobramycin revealed a novel HflK/C arrangement, characterized by two openings on opposite sides that may facilitate substrate access to FtsH during proteotoxic stress. Together, our results suggest that both the dynamic open conformation of HflK/C and its specific interactions with FtsH are critical for adaptation to aminoglycoside-induced stress. Given the conserved structural and functional features of SPFH family members, our findings may offer a broader framework for understanding how this protein family operates under both basal and stress conditions.

biophysics↗

bioRxiv2024

An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins

FtsH, a AAA protease, associates with HflK/C subunits to form a megadalton complex that spans the inner membrane and extends into the periplasm of E. coli. How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. The membrane curvature in our FtsH{middle dot}HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsHs function in the degradation of membrane-embedded proteins.

biochemistry↗

bioRxiv2023

A proteolytic AAA+ machine poised to unfold a protein substrate

AAA+ proteolytic machines unfold proteins prior to degradation. Cryo-EM of a ClpXP-substrate complex reveals a postulated but heretofore unseen intermediate in substrate unfolding/degradation. The natively folded substrate is drawn tightly against the ClpX channel by interactions between axial pore loops and the substrate degron tail, and by contacts with the native substrate that are, in part, enabled by movement of one ClpX subunit out of the typically observed hexameric spiral.

molecular biology↗

bioRxiv2022

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

Intracellular proteases must be specific to avoid degrading the wrong proteins. Here, we present cryo-EM structures of E. coli ClpXP, a AAA+ protease, which reveal that the axial channel of ClpX is closed prior to the binding and subsequent translocation of a protein substrate. An open-channel ClpX mutation stimulates degradation of casein, a non-specific substrate, indicating that channel closure contributes to increased degradation specificity. We demonstrate that ClpX activates ClpP cleavage of a degron-free decapeptide by a channel-independent mechanism, in which the peptide substrate appears to pass through a symmetry mismatched gap in the interface between ClpX and ClpP before entering the degradation chamber via the axial portal of ClpP. The peptide products of ClpXP protein degradation are likely to exit the chamber by the reverse route.

biochemistry↗