The ClpA chaperone and the two adaptor proteins modulate the fate of the model substrate tagged with a SsrA-degron of Leptospira.

Abstract

Bacterial caseinolytic protease (Clp) chaperone-protease complexes are essential for the degradation of misfolded and aggregated protein substrates. The spirochaete Leptospira interrogans possesses a set of Clp adaptor proteins (ClpS1 and ClpS2) and chaperones (ClpX, ClpA and ClpC), which are believed to associate with two distinct isoforms of ClpP (ClpP1 and ClpP2). This study explores the structural and functional properties of LinClpA, LinClpS1 and LinClpS2 derived from L. interrogans. LinClpA, a 740-amino acid protein, features an N-terminal domain and two AAA+ ATPase domains (D-I and D-II), containing conserved motifs critical for ATP binding and hydrolysis. LinClpS1 and LinClpS2 exhibit similar structures, yet they possess distinct binding pockets for N-degron substrates. Biochemical assays indicate that the N-domain-deleted variant of LinClpA (LinClpAΔN) exhibits a nucleotide-induced oligomerization tendency similar to LinClpA's but demonstrates higher ATPase activity. Interaction studies have shown that LinClpA's ATPase activity is enhanced in the presence of LinClpP isoforms and inhibited by LinClpS isoforms. In contrast, the activity of LinClpAΔN remained unaffected by LinClpS1 and LinClpS2, highlighting the significance of the N-domain of LinClpA in adaptor protein interactions. Furthermore, the study predicted and evaluated the role of the C-degron tag called small stable RNA A in facilitating protein degradation by the L. interrogans ClpAP1P2 machinery.