Insights into the inter-ring plasticity of caseinolytic proteases from the X-ray structure ofMycobacterium tuberculosisClpP1

Abstract

Mycobacterium tuberculosis caseinolytic protease ClpP1 (Mt ClpP1) is a self-compartmentalized protease consisting of two heptameric rings stacked on top of each other, thus enclosing a catalytic chamber. Within the chamber, which can be reached through two axial pores, each of the 14 identical monomers possesses a serine protease active site. The unfolding and translocation of substrates into the chamber are mediated by associated hexameric ATPases covering the axial pores. Three crystal structures of Mt ClpP1, determined by molecular replacement, are presented in this study. Two of the models were refined to a resolution of 2.6 A and the third to 3.0 A. It was found that disorder in the handle domain affects the formation and configuration of the tetradecamer and results in condensed structures with larger equatorial pores when compared with ClpPs from other species. Additionally, this disorder accompanies conformational changes of the residues in the catalytic triad. The models also reveal structural differences within the N-terminal hairpin-loop domain, which possibly reflect the significant differences in amino-acid sequence between Mt ClpP1 and other ClpP homologues in this region.

Structural Insights into the Conformational Diversity of ClpP from Bacillus subtilis

“…4A) (Lee et al, 2010a). These different features of the axial pore region are also found in MtClpP1, in which 15 residues at the aminoterminus of MtClpP1 are also disordered (Ingvarsson et al, 2007). Furthermore, the same N-terminal segments in ADEPbound BsClpP were completely disordered, whereas those in ADEP-bound EcClpP were ordered (Lee et al, 2010a;Li et al, 2010).…”

“…The phases of compressed-BsClpP and DFP-inhibited BsClpP were obtained using ClpP from Mycobacterium tuberculosis (PDB ID: 2CE3) and the previously extended-BsClpP structure (PDB ID: 3KTG) as search models, respectively (Ingvarsson et al, 2007;Lee et al, 2010a). The model was rebuilt using the program COOT (Emsley et al, 2010).…”

“…Using the previous BsClpP model (PDB ID: 3KTG), we unsuccessfully attempted to solve the phase problem by molecular replacement. However, using a more distantly related starting model, a structure of ClpP1 from M. tuberculosis (MtClpP1, PDB ID: 2CE3), which possesses an unusual ClpP structure (Ingvarsson et al, 2007), gave a successful solution. Following extensive model rebuilding and refinement, we obtained the final model refined at 2.6 Å resolution with disordered residues at the axial loop and handle region in each monomer (residues 1-18 and 125-137), as in the case of MtClpP1 (Ingvarsson et al, 2007).…”

“…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). An elegant quantitative NMR study of E. coli ClpP (EcClpP) suggested the presence of dynamic pores for peptide release, although clear structural information on this pore remains to be determined (Sprangers et al, 2005).…”

Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad

“…1A, Fig. S1B) (10)(11)(12)(13)(14)(15)(16)(17)(18). Cocrystallization of E. coli ClpP with an irreversible dipeptide chloromethylketone inhibitor confirmed the reactivity of the catalytic triad residues Ser98 and His123 and illustrate a binding site for the dipeptide within the Gly-rich loop region that adopts an antiparallel betastrand (19) (Fig.…”