Structures of ClpP in complex with acyldepsipeptide antibiotics reveal its activation mechanism

Abstract

Clp-family proteins are prototypes for studying the mechanism of ATP-dependent proteases because the proteolytic activity of the ClpP core is tightly regulated by activating Clp-ATPases. Nonetheless, the proteolytic activation mechanism has remained elusive because of the lack of a complex structure. Acyldepsipeptides (ADEPs), a recently discovered class of antibiotics, activate and disregulate ClpP. Here we have elucidated the structural changes underlying the ClpP activation process by ADEPs. We present the structures of Bacillus subtilis ClpP alone and in complex with ADEP1 and ADEP2. The structures show the closed-to-open-gate transition of the ClpP N-terminal segments upon activation as well as conformational changes restricted to the upper portion of ClpP. The direction of the conformational movement and the hydrophobic clustering that stabilizes the closed structure are markedly different from those of other ATP-dependent proteases, providing unprecedented insights into the activation of ClpP.

Structural Insights into the Conformational Diversity of ClpP from Bacillus subtilis

“…BsClpP was prepared as previously reported (Lee et al, 2010a). Briefly, BsClpP was cloned into pET-26b vector containing a six-residue histidine affinity tag at the carboxyl-terminus and then transformed into BL21(DE3).…”

“…Crystals of compressed-BsClpP were obtained using reservoir conditions comprising 100 mM sodium citrate (pH 5.6), 100 mM Li 2 SO 4 and 10-12% (w/v) PEG 4000. The same crystallization produced two different crystal forms, compressed BsClpP with space group C2 (Table 1) and extended BsClpP with space group P2 1 2 1 2 (PDB ID: 3KTH) (Lee et al, 2010a). For the cryo-experiment, a single crystal was transferred to the reservoir solution containing 20% (w/v) glycerol prior to flash-freezing in a nitrogen stream at -173°C.…”

“…Furthermore, it shares functional and architectural features with other energy-dependent protease systems such as HslVU in prokaryotes and the 26S proteasome in eukaryotes (Bochtler et al, 1999;Pickart and Cohen, 2004;Rohrwild et al, 1996;Song et al, 2000;Yu and Houry, 2007). Structural and biochemical studies of these ATPdependent proteases have provided a wealth of information regarding the mechanism of substrate recognition and protease activation Lee et al, 2010a;Park and Song, 2008;Park et al, 2007;Rabl et al, 2008;Ramachandran et al, 2002;Song et al, 2000;Sousa et al, 2000;Wang et al, 2001). HslU, ClpA/ClpX and the 19S regulatory cap utilize a domain(s) for recognizing substrate proteins and delivering these in an unfolded form, generated by ATP hydrolysis, to the matching proteases (Bochtler et al, 1999;Park and Song, 2008;Park et al, 2007;Sauer and Baker, 2011;Song and Eck, 2003).…”

“…HslU, ClpA/ClpX and the 19S regulatory cap utilize a domain(s) for recognizing substrate proteins and delivering these in an unfolded form, generated by ATP hydrolysis, to the matching proteases (Bochtler et al, 1999;Park and Song, 2008;Park et al, 2007;Sauer and Baker, 2011;Song and Eck, 2003). Structural studies of HslVU complex, ClpP in complex with activator acyldepsipeptide (ADEP), the 20S proteasome in complex with the 11S proteasome activator PA28, and the Cterminal peptide of proteasome-activating nucleosidase (PAN) have revealed the activation mechanism of the protease components (Lee et al, 2010a;Li et al, 2010;Rabl et al, 2008;Sousa et al, 2000;Stadtmueller and Hill, 2011;Whitby et al, 2000). As already mentioned, the cylindrical shape of the protease component has two substrate entry pores at both ends doubly capped by two AAA+ ATPases.…”

“…Presently, the structures of ClpPs from a variety of species have been reported (El Bakkouri et al, 2010;Kang et al, 2004;Kim and Kim, 2008;Lee et al, 2010a;Wang et al, 1997;Yu and Houry, 2007). In contrast to the similar overall architectural shape, a ClpP structure from Mycobacterium tuberculosis suggests a flexible ring-ring contact (Ingvarsson et al, 2007).…”