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Comparative Study
. 2006 Feb;153(2):113-28.
doi: 10.1016/j.jsb.2005.09.011. Epub 2005 Dec 1.

The asymmetry in the mature amino-terminus of ClpP facilitates a local symmetry match in ClpAP and ClpXP complexes

Maria C Bewley  1 , Vito Graziano, Kathleen Griffin, John M Flanagan
Affiliations
Comparative Study

The asymmetry in the mature amino-terminus of ClpP facilitates a local symmetry match in ClpAP and ClpXP complexes

Maria C Bewley et al. J Struct Biol. 2006 Feb.

Abstract

ClpP is a self-compartmentalized proteolytic assembly comprised of two, stacked, heptameric rings that, when associated with its cognate hexameric ATPase (ClpA or ClpX), form the ClpAP and ClpXP ATP-dependent protease, respectively. The symmetry mismatch is an absolute feature of this large energy-dependent protease and also of the proteasome, which shares a similar barrel-shaped architecture, but how it is accommodated within the complex has yet to be understood, despite recent structural investigations, due in part to the conformational lability of the N-termini. We present the structures of Escherichia coli ClpP to 1.9A and an inactive variant that provide some clues for how this might be achieved. In the wild type protein, the highly conserved N-terminal 20 residues can be grouped into two major structural classes. In the first, a loop formed by residues 10-15 protrudes out of the central access channel extending approximately 12-15A from the surface of the oligomer resulting in the closing of the access channel observed in one ring. Similar loops are implied to be exclusively observed in human ClpP and a variant of ClpP from Streptococcus pneumoniae. In the other ring, a second class of loop is visible in the structure of wt ClpP from E. coli that forms closer to residue 16 and faces toward the interior of the molecule creating an open conformation of the access channel. In both classes, residues 18-20 provide a conserved interaction surface. In the inactive variant, a third class of N-terminal conformation is observed, which arises from a conformational change in the position of F17. We have performed a detailed functional analysis on each of the first 20 amino acid residues of ClpP. Residues that extend beyond the plane of the molecule (10-15) have a lesser effect on ATPase interaction than those lining the pore (1-7 and 16-20). Based upon our structure-function analysis, we present a model to explain the widely disparate effects of individual residues on ClpP-ATPase complex formation and also a possible functional reason for this mismatch.

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Figures

Fig. 1
Fig. 1
The amino-terminal residues of ClpP are conserved across species, yet adopt distinct conformations in each monomer. (A) Amino acid sequence alignment of the N-terminal residues of mature ClpP sequences from a representative set of species: E. coli, Yersinia pestis, Haemophilus influenzae, Bacillus subtilis, Synechocystis, Treponema pallidum, Aquifex aeolicus, Thermotoga maritima, human, and mouse. The amino acids are colored as follows: M, A, L, I, and V: gray; P and G: brown; E and D: red; H, R, and K: blue; Q, N, T, and S: green; F, W, and Y: purple. (B) The amino-terminal residues of ring-1 extend out of the pore. Monomers A–D are shown as ribbon drawings: residues 21–193 are colored white. Residues upto 20, when observed in the electron density, are colored uniquely for each chain: red, yellow, cyan, and blue, respectively. (C) The ordered amino-terminal residues of ring-2 do not extend out of the pore. Monomers H, I, J, and N are shown as ribbon drawings: residues 21–193 are colored white. Residues upto 20, where observed in the electron density, in chains H, I, J, and N are colored green, yellow, magenta, and black, respectively. (D) Molecular surface of the X-ray structure of ring-1. Each monomer is shown in a distinct color, with observed residues upto residue 20 in a deeper hue. (E) Molecular surface of the X-ray structure of ring-2 colored in the same manner as (D). Panels (B) and (C) were drawn using the program Molscript (Kraulis, 1991), and panels (D) and (E) were drawn using the program GRASP (Nicholls et al., 1991).
Fig. 2
Fig. 2
Section of |Fo| – |Fc| electron density simulated annealing omit map corresponding to (A) one loop in the up conformation and (B) one loop in the down conformation. The electron density is shown as a blue chicken wire and contoured at a sigma level of 0.7. The residues are shown in ball-and-stick representation, colored according to their atom type: carbon, yellow; oxygen, red; nitrogen, blue; sulfur, green. Hydrogen bonds are shown as a broken magenta line. The figure was produced from a snapshot image taken directly from the screen, using the program TURBO to display the electron density map. Labeling was performed in Adobe Photoshop. Electron density corresponding to F17 is visible in both conformations, however, in (B), the side chain electron density lies outside of the plane of the section.
Fig. 3
Fig. 3
The effects of residues in the mature amino-terminus on propeptide processing. (A) SDS gel of wt protein, each of the amino terminal mutants, the D171 active site mutant and the double mutant V6A + D171A showing the degree of propeptide processing. (B) Surface representation residues 12–193 of chains L and M and residues 13–193 of chain M in a ClpP heptamer, as observed in the electron density (colored red, gray, and purple, respectively). Residues 1–12 of chain M have been modeled by eye along the internal groove that runs approximately parallel to the subunit interface. It is drawn as a ball-and-stick representation and colored according to atom type: blue, nitrogen; red, oxygen; yellow, carbon; green, sulfur. The position of the competitive inhibitor, acetyl-LLM, is also shown in ball-and stick representation.
Fig. 4
Fig. 4
Kinetic analysis of the ATP-dependent degradation of α-casein by ClpAP, using either wild type or variant ClpP oligomers. (A) The effects of representative N-terminal alanine substitutions in ClpP on the ATP and ClpA-dependent hydrolysis of α-casein, WT (●), R15A (▲), G13A (▽), and V6A (◆). (B) Effects of increasing concentrations of inactive three ClpP variants on the ATP-dependent degradation of α-casein by wt ClpAP (ClpP-D171A (●), ClpP-V6A (■), and ClpP-V6A + D171A (▼).
Fig. 5
Fig. 5
The most deleterious mutations lie within the inner surface of the pore of ring-1. Symmetrized model of ClpP where the monomer of chain B was rotated onto the positions of chain A, D–G, respectively. Chain C is shown as it exists in the crystal structure. Space filling representation of ClpP with residues 1–20 colored according to the kinetic data in Table 2. Red represents inactive variants, orange represents a 5000- to 10 000-fold decrease in Kapp values, yellow represents a 400- to 1500-fold reduction in ClpA binding, and green represents wild type-like activity. Residues 21–193 are colored white. (A) A top-view of the ClpP heptamer. The top of the pore is lined with residues (T10, R12, and R15) that have significant effects on ClpA binding. (B) Side view of four of the ClpP monomers. The central pore contains a ring of residues that abrogate ClpA binding, protected from solution by a layer of residues that have little or no effect upon ClpA binding.
Fig. 6
Fig. 6
Phe17 has a different rotomer conformation in the structure of the V6A variant compared to that of the wild type. Ribbon diagram for the ClpP monomer. Residues 25–193 are colored in gray and the amino terminal 24 residues, where visible in the electron density, are colored according to their structure. Representative conformations of the wild type protein, corresponding to the up and down conformations, are colored green and blue, respectively. The conformations of the loops in the structures of V6A and V6A + D171A superimpose and are colored in red. In each case, the location of F17 is shown in a ball-and-stick representation. This figure was drawn using Bobscript (Esnouf, 1991) and Raster3D (Merritt and Bacon, 1997).
Fig. 7
Fig. 7
Docked model of the hexameric ClpX onto the heptameric ClpP. The molecular surface of a symmetrized ClpP heptamer is shown and a worm of the ClpX hexamer. The dome surface generated by the N-termini of ClpP provides a complementary docking surface for the interaction at the pore of ClpX. The IGF loop of ClpX is colored magenta. The hydrophobic groove on the surface of ClpP is colored blue with the exception of F112, which has been characterized biochemically, is colored yellow. The inset shows this groove in more detail.
Fig. 8
Fig. 8
Schematic of ClpXP, showing how a local pseudo-6-fold interaction in ClpP allows ClpX to dock. A local pseudo-6-fold symmetric interaction at the central pore allows for a tethering of the molecule. Small changes in the local conformation of the loop, including the IGF motif, are driven by ATP hydrolysis. These movements can be “sensed” by ClpP through the interactions of some of the IGF motifs with the corresponding hydrophobic depressions on the ClpP surface. ClpP is represented as a blue heptamer. The hydrophobic groove, including F112, is represented as a yellow circle and the amino-terminal 20 residues are drawn as green ovals. The ATPase component is drawn as a red hexagon and the IGF residues within the motif are shown as a cyan circle.
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References

    1. Beuron F, Maurizi MR, et al. At sixes and sevens: characterization of the symmetry mismatch of the ClpAP chaperone-assisted protease. J Struct Biol. 1998;123(3):248–259. - PubMed
    1. Bochtler M, Ditzel L, et al. Crystal structure of heat shock locus V (HslV) from Escherichia coli. Proc Natl Acad Sci USA. 1997;94(12):6070–6074. - PMC - PubMed
    1. Bochtler M, Hartmann C, et al. The structures of HsIU and the ATP-dependent protease HsIU-HsIV. Nature. 2000;403(6771):800–805. - PubMed
    1. Dodson E. Report of a workshop on the use of statistical validators in protein X-ray crystallography. Acta Crystallogr D Biol Crystallogr. 1996;52(Pt. 1):228–234. - PubMed
    1. Esnouf RM. J Mol Graph. 1991;15:132–134. - PubMed

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