Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions

Abstract

Cellular proteostasis involves not only the expression of proteins in response to environmental needs, but also the timely repair or removal of damaged or unneeded proteins. AAA+ motor proteins are critically involved in these pathways. Here, we review the structure and function of AAA+ proteins ClpA, ClpB, and Hsp104. ClpB and Hsp104 rescue damaged proteins from toxic aggregates and do not partner with any protease. ClpA functions as the regulatory component of the ATP dependent protease complex ClpAP, and also remodels inactive RepA dimers into active monomers in the absence of the protease. Because ClpA functions both with and without a proteolytic component, it is an ideal system for developing strategies that address one of the major challenges in the study of protein remodeling machines: how do we observe a reaction in which the substrate protein does not undergo covalent modification? Here, we review experimental designs developed for the examination of polypeptide translocation catalyzed by the AAA+ motors in the absence of proteolytic degradation. We propose that transient state kinetic methods are essential for the examination of elementary kinetic mechanisms of these motor proteins. Furthermore, rigorous kinetic analysis must also account for the thermodynamic properties of these complicated systems that reside in a dynamic equilibrium of oligomeric states, including the biologically active hexamer.

Keywords: ClpA; ClpB; Hsp104; kinetics; thermodynamics; translocation mechanism.

Figure 1

Structural comparison of ClpA, ClpB, and Hsp104. (A) Sequence alignment showing relative organization of N domain, NBD1, NBD2, and M domain in the AAA+ protomers compared. (B) Protomer crystal structures of E. coli ClpA (PDB ID code 1ksf) (Guo et al., 2002b), T. thermophilus ClpB (PDB ID code 1qvr– chain C) (Lee et al., 2003), and C. thermophilum Hsp104 (PDB ID 5d4w – chain A) (Heuck et al., 2016). E. coli ClpA and T. thermophilus ClpB N domains are shown in pink. C. thermophilum Hsp104 also has an N terminal domain, however its electron density was not resolved, likely due to flexibility. Nucleotide Binding Domain 1 (NBD1) is shown in blue for each protomer. In ClpB and Hsp104, the Middle Domain (M Domain) is shown in gold, extending in a coiled-coil from within NBD1. Nucleotide Binding Domain 2 (NBD2) is shown in purple. Bound nucleotide is shown as black spheres. These images were prepared using PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC (Schrodinger, 2015a,b,c). Adaptation of structural comparison presented in Doyle and Wickner (2009). (B) Single particle reconstructions of E. coli ClpA (EMD-1673) (Effantin et al., 2010), E. coli ClpB (EMD-2563) and S. cerevisiae Hsp104 (EMD-2561) (Carroni et al., 2014), hexameric rings from cryo-electron microscopy. ClpA and Hsp104 models were built from images of the motor protein bound to ClpP. For Hsp104, this required use of HAP, the variant designed by the Bukau group to interact with ClpP. Top row shows views from the side. Note that the N terminal domain of ClpA was not defined in the electron density map, likely due to flexibility, similar to the observation from the crystallographic study of Hsp104. Bottom row shows views from the top, looking down through the axial channel. These images were prepared using UCSF Chimera (Computer Graphics Laboratory, University of California, San Francisco).

Figure 2

Species fraction plot as a function to total [ClpB] in monomer units. Species fractions were simulated using apparent equilibrium constants for the oligomerization of each ClpB n-mer (L_{n, app}) predicted in the presence of (A) 100 μM and (B) 2 mM ATPγS, as well as ClpB n-mer nucleotide binding constants, and stoichiometries reported in Lin and Lucius (2016). The equilibrium constants for the ClpB n-mer oligomerization in the absence of nucleotide (L_n,0) were used as reported in Lin and Lucius (2015b). The solid lines represent the fraction of monomer (red), dimer (green), tetramer (blue), and hexamer (black) present as a function of total [ClpB] in μM monomer.

Figure 3

Schematic of single turnover fluorescence stopped-flow experiment. ATPγS-bound ClpA is pre-assembled with a fluorescently labeled, unstructured polypeptide substrate, fluor-peptide. The fluor-peptide bound ClpA complex (left) is then rapidly mixed with a solution of ATP and a non-fluorescent peptide (protein trap, right) held in large excess over the fluorescently modified peptide concentration. Upon mixing, any ClpA hexamers that dissociate from the fluorescently modified peptide will be swiftly bound by protein trap, ensuring the reaction monitored is single-turnover with respect to the fluor-peptide bound ClpA complex. The mixture is excited at a specified fluorophore excitation wavelength (λ_EX), and fluorescence emission at an indicated fluorophore emission wavelength (λ_EM) is monitored as a function of time.

Figure 4

General scheme of a translocating enzyme mechanism. Translocating enzyme (E) in complex with a peptide (P) of length L, (E·P)_L, will either translocate the peptide through a translocation rate constant (k_t) to form an peptide intermediate translocated by a some distance m, I_(L−m), or dissociate from the peptide through a dissociation rate constant (k_d). The translocase proceeds through multiple translocation steps of a given step-size (m) until the peptide is fully translocated.

Figure 5

Structural models of ClpA and ClpAP complex in various states of peptide ligation. Models of (A) hexameric ClpA, (B) 1:1 ClpAP, and (C) 2:1 ClpAP bound by one peptide or (D) bound by two peptides. Structures are shown as side views in complex with a cartoon of an unstructured polypeptide substrate (black). The single particle reconstruction of E. coli ClpA hexamer (EMD-1673) (Effantin et al., 2010) is shown with monomers colored in alternating red shades. In the ClpAP complexes, a molecular surface from the crystal structure of E. coli ClpP tetradecamer (PDB-2FZS) (Szyk and Maurizi, 2006) is shown with protomers in the each heptameric ring colored in alternating shades of either light blue (top) or dark blue (bottom). The models shown here are not energy minimized. Images were prepared using UCSF Chimera (Computer Graphics Laboratory, University of California, San Francisco).

Figure 6

Proposed model of the movement of pore loops in NBD1 and NBD2 of ClpA in polypeptide translocation. (A) In the absence of ClpP, conformational changes in the pore loops of both NBD1 and NBD2 contribute to the translocation of polypeptide substrate through the ClpA axial channel. ATP binding and hydrolysis at NBD1 results in a pore loop conformational change that moves the incoming polypeptide substrate ~14 amino acids down the axial channel toward NBD2. This results in the formation of a polypeptide substrate loop in the axial space between NBD1 and NBD2. This loop is moved through the axial channel by multiple rounds of ATP hydrolysis cycles at NBD2 that lead to the translocation of 2–5 amino acids per cycle by the NBD2 pore loops. (B) In the presence of ClpP, polypeptide translocation is driven by NBD2 ATP hydrolysis induced conformational changes. Cycles of ATP binding and hydrolysis at NBD1, do not result in conformational changes that limit the observed ClpAP catalyzed translocation rate.