Abstract
Most AAA+ remodeling motors denature proteins by pulling on the peptide termini of folded substrates, but it is not well-understood how motors produce grip when resisting a folded domain. Here, at single amino-acid resolution, we identify the determinants of grip by measuring how substrate tail sequences alter the unfolding activity of the unfoldase-protease ClpXP. The seven amino acids abutting a stable substrate domain are key, with residues 2-6 forming a core that contributes most significantly to grip. ClpX grips large hydrophobic and aromatic side chains strongly and small, polar, or charged side chains weakly. Multiple side chains interact with pore loops synergistically to strengthen grip. In combination with recent structures, our results support a mechanism in which unfolding grip is primarily mediated by non-specific van der Waal's interactions between core side chains of the substrate tail and a subset of YVG loops at the top of the ClpX axial pore.
Highlights
Cells maintain homeostasis by balancing protein synthesis and degradation with growth
Given the length of the axial pore (~35 A ; Glynn et al, 2009), we reasoned that ClpX should only interact with residues within the cassette region during green fluorescent protein (GFP) unfolding
Because the pore loops of ClpX and other AAA+ motors interact with every other substrate residue in cryo-EM structures (Monroe et al, 2017; Gates et al, 2017; Puchades et al, 2017; de la Pena et al, 2018; Majumder et al, 2019; Dong et al, 2019; White et al, 2018), we investigated whether a similar spacing of large side chains enhances grip
Summary
Cells maintain homeostasis by balancing protein synthesis and degradation with growth. Regulated degradation typically requires a protein-unfolding motor of the AAA+ family (ATPases associated with various cellular activities) that associates with a self-compartmentalized protease (e.g., ClpX with ClpP, the 19S regulatory particle with the 20S proteasome) or is genetically tethered to a protease (e.g., Lon, FtsH, Yme1) (Sauer and Baker, 2011; Olivares et al, 2016; Glynn, 2017). ClpX is a ring-shaped AAA+ homohexamer that functions autonomously in protein remodeling in bacteria and eukaryotic organelles and associates with ClpP tetradecamers to form the ATPdependent ClpXP protease (Baker and Sauer, 2012). Proteins marked with a sequence-defined degron or post-translational modification can be recruited to the AAA+ protease directly or with assistance from auxiliary adaptors (Sauer and Baker, 2011; Trentini et al, 2016). During rescue of stalled ribosomes in Escherichia coli, the 11-residue ssrA tag is appended to the C-terminus of abortive protein products
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