Interactions between a subset of substrate side chains and AAA+ motor pore loops determine grip during protein unfolding

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.Proteins are the workhorses of the body, fulfilling many roles essential for life processes. These molecules are made up of hundreds or thousands of small units called amino acids, which attach to each other to form a long chain. The exact sequence of amino acids determines how the protein will then fold to acquire its final, three-dimentional shape.Enzymes called proteases can degrade unneeded or faulty proteins so that the amino acids can be recycled. For instance, in bacteria, the AAA+ protease ClpXP can recognize and ‘grab’ specific patterns of amino acids at the ends of a protein. This molecular machine then tugs on the segment and unfold the protein, the way a ball of yarn unwinds when pulled from one end. The unfurled protein is then fed into a different section of ClpXP, where it is chopped into short segments for recycling.ClpXP is the best-characterized enzyme amongst AAA+ proteases. However, it is still unclear how it can grip target proteins tightly enough to allow unfolding. To investigate, Bell et al. attached different patterns of 12 amino acids to the end of a folded protein. How well ClpXP grasped each of these proteins was then measured in bacteria and in test tubes. This revealed that ClpXP attaches to six to eight amino acids at a time, suggesting that only part of the enzyme clasps on the protein. Large amino acids are better gripped than small amino acids, similar to how a knotted string is easier to hold than a smooth rope. Amino acids that are electrically charged also interfere with ClpXP attaching to the protein. Finally, ClpXP grasps multiple amino acids at the same time, which dramatically increases grip strength.Many proteins, including some found in viruses, use ‘slippery’ patterns of amino acids to avoid being gripped and unfolded by proteases. By understanding how different patterns of amino acids are grasped, it may someday be possible to engineer enzymes able to target dangerous proteins.