Structural and motional contributions of the Bacillus subtilis ClpC N-domain to adaptor protein interactions

Abstract

The AAA(+) (ATPases associated with a variety of cellular activities) superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities. The NMR solution structure of the N-terminal repeat domain of ClpC (N-ClpCR) comprises two structural repeats of a four-helix motif. NMR experiments used to map the MecA adaptor protein interaction surface of N-ClpCR reveal that regions involved in the interaction possess conformational flexibility and conformational exchange on the microsecond-to-millisecond timescale. The electrostatic surface of N-ClpCR differs substantially from the N-domain of Escherichia coli ClpA and ClpB, suggesting that the electrostatic surface characteristics of HSP100/Clp N-domains may play a role in adaptor protein and substrate interaction specificity, and perhaps contribute to the unique adaptor protein requirement of ClpC.

Figure 1. NMR solution structure of N-ClpCR

(a) Stereo backbone trace of the thirty lowest-energy NMR solution structures colored using the chainbow feature of pymol (N-terminus, blue; C-terminus, red). (b) Cartoon diagram of the thirty lowest-energy NMR solution structures colored using the chainbow feature of pymol (N-terminus, blue; C-terminus, red). (c) Putty/sausage diagram illustrating regions of structural variation within the NMR ensemble colored using the chainbow feature of pymol from small (blue) to large (red). (d) Cartoon diagram illustrating the sequentially similar/identical regions and hydrophobic contacts between the α2 (red) and α6 (blue) giving rise to a pseudo two-fold axis of symmetry. Color scheme according to the N-ClpCR repeat sequence in Figure 2A.

Figure 2. Secondary structure and sequence alignment of N-ClpCR to N-ClpA and N-ClpB

dssp calculated secondary structure of N-ClpCR and tcoffee sequence alignment to E. coli N-ClpA and N-ClpB. Secondary structure elements comprising the N-ClpCR NMR structure are noted in dssp code ((H, α-helix; B or E, β-strand; T, hydrogen-bonded turn) and diagram form (top). Conservation notation is as follows: identical (*), highly conserved (:) and somewhat similar (.). Repeat regions (bold font and underlined) are colored as follows: similar (green or orange) and identical (blue or red). See text for a description of colored box regions. Organism notations is as follows: B, B. subtilis; E, E. coli; S, S. elongates.

Figure 3. Structures of HSP100 N-domain proteins

(a) Cartoon diagram of the N-ClpA crystal structure [PDB code: 1r6c]. (b) Cartoon diagram of the N-ClpB crystal structure [PDB code: 1khy; chain A]. (c) Cartoon diagram of the N-ClpCR NMR solution structure closest to the mean calculated ML superposition structure reported by theseus.

Figure 4. Structural superposition of N-ClpA, N-ClpB, and N-ClpCR

(a) Ribbon diagram of the ML superposition of N-ClpA, N-ClpB, and N-ClpCR. (b) Putty/sausage diagram illustrating regions of structural variation in the theseus ML superposition of N-ClpA, N-ClpB, and N-ClpCR.

Figure 5. Mapping of the MecA-binding surface of N-ClpCR

(a) Examples of MecA-induced peak intensity decreases in the [¹H,¹⁵N]-TROSY-HSQC spectra spectra of N-ClpCR at 30 μM (black) and 50 μM (blue) MecA, as compared to the N-ClpCR base spectrum (red). (b) N-ClpCR residues that display larger initial changes in chemical shift perturbation (green), line broadening (peak intensity decrease; red), or both (yellow). Residues highlighted correspond to values that differ from the mean value by (1.2 ×σ). (c) N-ClpA (white) and ClpS (orange) co-crystal structure [PDB code: 1r6q]. (d) N-ClpA residues involved in the interaction with ClpS via interface A/acid loop surface (orange) or the non-specific binding interface C surface (blue).

Figure 6. N-ClpCR backbone dynamics and PCA correlations

(a) Backbone order parameters (S²; black circles) and ML Cα R.M.S.D. (red line) vs. residue number. (b) S² and R_ex values plotted on the N-ClpCR structure. The radius of the backbone is directly proportional to 1-S² (green regions); a larger radius indicates increased flexibility. White regions indicate the lack of experimental data, and the backbone radii of these residues were interpolated between neighboring residues. Residues with R_ex contributions to the ¹⁵N R₂ relaxation rates (red spheres) are indicative of motions on the μs-ms timescale. (c) The first principal component from the maximum likelihood correlation matrix for the ML superposition of the N-ClpCR structure ensemble. Regions colored similarly (cyan or magenta) are self-correlated, whereas regions colored differently (cyan vs. magenta) are anti-correlated.

Figure 7

N-domain electrostatic surface characteristics. apbs-calculated electrostatic potential displayed on the solvent accessible surfaces of (a) N-ClpA, (b) N-ClpB, and (c) N-ClpCR. The structures displayed in the left panels are oriented as shown in Figure 3A, 3B, and 3C, respectively.

Figure 8. Model of ClpC N-domain and Linker domain interactions and N-ClpCR hexameric ring structure based on the cryo-EM Hsp104 model

(a) Structural superposition of N-ClpCR to the cryo-EM model of Hsp104. The Hsp104 model is shown as a Cα/sphere trace colored according to the domain identity (yellow, N-domain; red/ orange, NBD1; green, linker domain; blue/light blue NBD2), whereas the N-ClpCR structure is shown with cylindrical helices colored as in Figure 1A. (b) apbs-calculated electrostatic potential of the hexameric model of N-ClpCR viewed looking down the C6 axis of symmetry. Six N-ClpCR molecules were super-positioned to a monomer within the Hsp104 cryo-EM model. (c) N-ClpCR residues that display a large initial line broadening and/or chemical shift changes in the MecA NMR titration mapped onto the hexameric model of N-ClpCR. Color coding is the same as that shown in Figure 5B. (d) S² values mapped onto the hexameric model of N-ClpCR. Amino acid side-chains are colored white-to-brown as represented in Figure 6A and 6B and proportional to 1-S². One monomer is removed from the hexamer to illustrate the monomeric contribution to the hexameric assembly.