Generation of hydrophobic patch variants of ClpP1 and ClpP2.

<p>A. Cartoon representation of the LGF-loops (dark blue) of the chaperone binding to hydrophobic surface patches (green) on the protease core (grey). B. Top view of a heptameric protease ring. The hydrophobic patches (green) are formed by residues of two adjacent protease subunits (grey). C. Mutations introduced in ClpP1 and ClpP2 to create the hydrophobic patch variants hpClpP1 (ClpP1^{S61A, Y63V, L83A, Y91V}) and hpClpP2 (ClpP2^{Y75V, Y95V}). D. Alignment of Mtb ClpP1 and ClpP2 with EcClpP. Conservation is colored from white (not conserved) to black (identical). The identity between ClpP1/ClpP2 is 39.5%, between EcClpP/ClpP1 46% and EcClpP/ClpP2 44.4%. Red arrows highlight the residue positions of the EcClpP hydrophobic patch residues. Residues depicted in green in panel E are marked with a green box. E. Surface representation of ClpP1, ClpP2 (4U0G.pdb) and EcClpP (3MT6.pdb) ring faces. Individual subunits are colored alternatingly in light and dark grey. Hydrophobic patch residues are colored in green and labelled accordingly. For ClpP1 and ClpP2 the hydrophobic patch residues used for mutation are shown, for EcClpP reference residues are shown as described in the literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125345#pone.0125345.ref014" target="_blank">14</a>]. F. Creation of a set of mature mixed wild-type ClpP1 and ClpP2 (wtP1, wtP2) and hydrophobic patch variants (hpP1, hpP2). Large-scale processing of ClpP1 and ClpP2 (70 μM protomer each) containing the N-terminal propeptide (proClpP1, proClpP2) to mature ClpP1 and ClpP2 (mClpP1, mClpP2) in the presence of 1 mM activator. The reaction was performed in Buffer A. All samples were run on the same gel. The lane containing the size marker was removed for better visual representation (white line). G. Analytical gel filtration was performed with the mature ClpP1P2 complexes created in panel F. The peak at 11 ml shows that all complexes have assembled into double-rings.