Recent structural insights into the mechanism of ClpP protease regulation by AAA+ chaperones and small molecules.

ClpP is a highly conserved serine protease that is a critical enzyme in maintaining protein homeostasis and is an important drug target in pathogenic bacteria and various cancers. In its functional form, ClpP is a self-compartmentalizing protease composed of two stacked heptameric rings that allow protein degradation to occur within the catalytic chamber. ATPase chaperones such as ClpX and ClpA are hexameric ATPases that form larger complexes with ClpP and are responsible for the selection and unfolding of protein substrates prior to their degradation by ClpP. Although individual structures of ClpP and ATPase chaperones have offered mechanistic insights into their function and regulation, their structures together as a complex have only been recently determined to high resolution. Here, we discuss the cryoelectron microscopy structures of ClpP-ATPase complexes and describe findings previously inaccessible from individual Clp structures, including how a hexameric ATPase and a tetradecameric ClpP protease work together in a functional complex. We then discuss the consensus mechanism for substrate unfolding and translocation derived from these structures, consider alternative mechanisms, and present their strengths and limitations. Finally, new insights into the allosteric control of ClpP gained from studies using small molecules and gain or loss-of-function mutations are explored. Overall, this review aims to underscore the multilayered regulation of ClpP that may present novel ideas for structure-based drug design.

Human ClpP protease, a promising therapy target for diseases of mitochondrial dysfunction.

Human caseinolytic protease P (HsClpP), an ATP-dependent unfolding peptidase protein in the mitochondrial matrix, controls protein quality, regulates mitochondrial metabolism, and maintains the integrity and enzyme activity of the mitochondrial respiratory chain (RC). Studies show that abnormalities in HsClpP lead to mitochondrial dysfunction and various human diseases. In this review, we provide a comprehensive overview of the structure and biological function of HsClpP, and the involvement of its dysexpression or mutation in mitochondria for a panel of important human diseases. We also summarize the structural types and binding modes of known HsClpP modulators. Finally, we discuss the challenges and future directions of HsClpP targeting as promising approach for the treatment of human diseases of mitochondrial origin.

Molecular and structural insights into an asymmetric proteolytic complex (ClpP1P2) from Mycobacterium smegmatis.

The ClpP protease is found in all kingdoms of life, from bacteria to humans. In general, this protease forms a homo-oligomeric complex composed of 14 identical subunits, which associates with its cognate ATPase in a symmetrical manner. Here we show that, in contrast to this general architecture, the Clp protease from Mycobacterium smegmatis (Msm) forms an asymmetric hetero-oligomeric complex ClpP1P2, which only associates with its cognate ATPase through the ClpP2 ring. Our structural and functional characterisation of this complex demonstrates that asymmetric docking of the ATPase component is controlled by both the composition of the ClpP1 hydrophobic pocket (Hp) and the presence of a unique C-terminal extension in ClpP1 that guards this Hp. Our structural analysis of MsmClpP1 also revealed openings in the side-walls of the inactive tetradecamer, which may represent sites for product egress.

Cryo-EM structure of the ClpXP protein degradation machinery.

The ClpXP machinery is a two-component protease complex that performs targeted protein degradation in bacteria and mitochondria. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP, where they are degraded. Formation of the complex involves a symmetry mismatch, because hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insight into the ClpX-ClpP cross-talk and activation mechanism. Comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of the complex mode of action of ClpXP and provides a structural framework for future pharmacological applications.

Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase.

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.

Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor.

AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.

Dual stoichiometry and subunit organization in the ClpP1/P2 protease from the cyanobacterium Synechococcus elongatus.

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. To investigate the proteolytic core of the ClpXP1/P2 protease from the cyanobacterium Synechococcus elongatus we have used a non-denaturing mass spectrometry approach. We show that the proteolytic core is a double ring tetradecamer consisting of an equal number of ClpP1 and ClpP2 subunits with masses of 21.70 and 23.44 kDa, respectively. Two stoichiometries are revealed for the heptameric rings: 4ClpP1+3ClpP2 and 3ClpP1+4ClpP2. When combined in the double ring the stoichiometries are (4ClpP1+3ClpP2)+(3ClpP1+4ClpP2) and 2×(3ClpP1+4ClpP2) with a low population of a 2×(4ClpP1+3ClpP2) tetradecamer. The assignment of the stoichiometries is confirmed by collision-induced dissociation of selected charge states of the intact heptamer and tetradecamer. Presence of the heterodimers, heterotetramers and heterohexamers, and absence of the mono-oligomers, in the mass spectra of the partially denatured protease indicates that the ring complex consists of a chain of ClpP1/ClpP2 heterodimers with the ring completed by an additional ClpP1 or ClpP2 subunit.

The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features.

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits.

Optimal efficiency of ClpAP and ClpXP chaperone-proteases is achieved by architectural symmetry.

A common feature of chaperone-proteases is architectural two-fold symmetry across the proteolytic cylinder. Here we investigate the role of symmetry for the function of ClpAP and ClpXP assemblies. We generated asymmetric ClpP particles in which the two rings differ in ClpA and ClpX binding capability and/or in proteolytic activity. Rapid-kinetic fluorescence measurements and steady-state experiments indicate that single 2:1 ClpAP or ClpXP complexes are as efficient in substrate degradation as two 1:1 ClpAP or ClpXP assemblies. This implies that the two chaperone components work independently. However, an asymmetric ClpP particle composed of one active and one inactive ring can stimulate ATPase activity of ClpA regardless of whether ClpA binds to the active ring or to the opposite side of ClpP, across the ring of inactivated protease. Thus, we propose that conformational transitions in ClpP are concerted and allosteric effects are transferred simultaneously to both associated chaperones, leading to synchronized activation.

Computer simulation of I27 translocation through ClpY reveals a critical role of protein mechanical strength and local stability.

Clp family is one type of AAA+ proteases, which catalyze protein degradation and translocation. Because of the steric restriction of the complex structure, the substrates have to be denaturated before accessing the active sites of the peptidases. This type of translocation-induced protein unfolding has been studied in bulk biochemical experiments, but the detailed dynamic process is still unknown. Two models are proposed: the target protein somehow unfolds before it is pulled through a protease or the target protein is unfolded by pulling force during the translocation. We performed steered molecular dynamics (SMD) simulations to pull a model protein I27 and its variants (V11P, V13P and V15P) through ClpY, which is a member of Clp family with the available crystal structure. Resulting force-position profiles showed that the protein translocation needs a large initial force to break it open, and further unfolding needs relatively weaker forces. Comparison of the unfolding forces among translocation of I27 and its variants showed that the local mechanical stability of the protein determines the unfolding force. We also simulated the I27 translocation starting with different orientations and found that the unfolding dynamics are similar. The simulations presented here, combined with published experimental data, support the model that the target protein is pulled apart during translocation, and the force needed to unfold a protein follows the local stability model. This model does not only give a close insight into the process of force-driven protein unfolding in translocation, but also is instructive to design protein in protein degradation, which is one of the most important steps in cellular cycles.

Modeling AAA+ ring complexes from monomeric structures.

AAA+ proteins form large, ring-shaped complexes, which act as energy-dependent unfoldases of macromolecules. Many crystal structures of proteins in this superfamily have been determined, but mostly in monomeric or non-physiological oligomeric forms. The assembly of ring-shaped complexes from monomer coordinates is, therefore, of considerable interest. We have extracted structural features of complex formation relating to the distance of monomers from the central axis, their relative orientation and the molecular contacts at their interfaces from experimentally determined oligomers and have implemented a semi-automated modeling procedure based on RosettaDock into the iMolTalk server (http://protevo.eb.tuebingen.mpg.de/iMolTalk). As examples of this procedure, we present here models of Apaf-1, MalT and ClpB. We show that the recent EM-based model of the apoptosome is not compatible with the conserved structural features of AAA+ complexes and that the D1 and D2 rings of ClpB are most likely offset by one subunit, in agreement with the structure proposed for ClpA.