Metal-catalyzed oxidation of Escherichia coli glutamine synthetase: correlation of structural and functional changes

Metal-catalyzed oxidation of proteins has been implicated in a variety of biological processes, particularly in the marking of proteins for subsequent proteolytic degradation. The metal-catalyzed oxidation of bacterial glutamine synthetase causes conformational, covalent, and functional alterations in the protein. To understand the structural basis of the functional changes, the time course of oxidative modification of glutamine synthetase was studied utilizing a nonenzymic model oxidation system consisting of ascorbate, oxygen, and iron. The structural modifications induced included: decreased thermal stability; weakening of subunit interactions; decrease in isoelectric point; introduction of carbonyl groups into amino acid side chains; and loss of two histidine residues. These changes did not denature the protein, but instead induced relatively subtle changes. Indeed, even the most extensively modified protein had a sedimentation velocity which was identical to that of the native enzyme. Comparison of the time courses of the structural and functional changes established that: (i) Loss of the metal binding site and of catalytic activity occurred with loss of one histidine per subunit; (ii) increased susceptibility to proteolysis occurred with loss of two histidine residues per subunit. Thus, oxidation at one site suffices to inactivate the enzyme, but two sites must be modified to induce susceptibility to proteolysis. The limited and specific changes induced by metal-catalyzed oxidation are consistent with a site-specific free radical mechanism.     

Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting directly with attached fluorophores at the same site. Here we show that ligands binding to the β2-α1 interface GABA binding site produce conformational changes at the adjacent subunit interface. This is most likely due to agonist-induced loop C closure directly altering loop F conformation at the adjacent α1-β2 subunit interface. However, as antagonists and agonists produce identical α1 subunit loop F conformational changes, these conformational changes appear unimportant for gating. Finally, we demonstrate that TM2-TM3 loops from adjacent β2 subunits in α1 β2 receptors can dimerize via K24'C disulfides in the closed state. This result implies unexpected conformational mobility in this crucial part of the gating machinery. Together, this information provides new insights into the activation mechanisms of Cys-loop receptors.     

Nucleotide-induced conformational change in the catalytic subunit of the phosphate-specific transporter from M. tuberculosis: implications for the ATPase structure

The nucleotide binding subunit of the phosphate-specific transporter (PstB) from Mycobacterium tuberculosis is a member of the ABC family of permeases, which provides energy for transport through ATP hydrolysis. We utilized the intrinsic fluorescence of the single tryptophan containing protein to study the structural and conformational changes that occur upon nucleotide binding. ATP binding appeared to lead to a conformation in which the tryptophan residue had a higher degree of solvent exposure and fluorescence quenching. Substantial alteration in the proteolysis profile of PstB owing to nucleotide binding was used to decipher conformational change in the protein. In limited proteolysis experiments, we found that ATP or its nonhydrolyzable analog provided significant protection of the native protein, indicating that the effect of nucleotide on PstB conformation is directly associated with nucleotide binding. Taken together, these results indicate that nucleotide binding to PstB is accompanied by a global conformational change of the protein, which involves the helical domain from Arg137 to Trp150. Results reported here provide evidence that the putative movement of the alpha-helical sub-domain relative to the core sub-domain, until now only inferred from X-ray structures and modeling, is indeed a physiological phenomenon and is nucleotide dependent.     

Solution structure, conformational dynamics, and CD4-induced activation in full-length, glycosylated, monomeric HIV gp120

The gp120 subunit of the HIV Env glycoprotein is responsible for receptor interactions leading to viral entry and is a primary target for neutralizing antibodies. Most structural studies have focused on the heavily truncated, deglycosylated gp120 core, leaving fundamental aspects of the glycoprotein that are responsible for immune evasion and receptor-induced activation unresolved. Here we investigate full-length, glycosylated HIV gp120 in unliganded and CD4-bound forms by using small-angle X-ray scattering to visualize global structural reorganization and hydrogen/deuterium exchange to track changes in local conformational dynamics. The studies revealed unliganded full-length gp120 to be considerably more dynamic, particularly at the CD4 binding site, than suggested by previous studies of the subunit core alone. The large V1/V2 loops, previously unmapped, are positioned to mask the coreceptor binding site in an orientation that recapitulates that observed in the Env trimer. CD4 binding shifts V1/V2 to unmask the coreceptor binding site and triggers profound dynamic changes in gp120 spanning from the binding site to the gp41-interactive face of gp120. These findings provide further insights on the structural basis of Env antigenicity and immunogenicity and of allosteric effects upon receptor binding.     

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

The interaction of the nucleotide-binding subunit B with subunit F is essential in coupling of ion pumping and ATP synthesis in A₁A_O ATP synthases. Here we provide structural and thermodynamic insights on the nucleotide binding to the surface of subunits B and F of Methanosarcina mazei Gö1 A₁A_O ATP synthase, which initiated migration to its final binding pocket via two transitional intermediates on the surface of subunit B. NMR- and fluorescence spectroscopy as well as ITC data combined with molecular dynamics simulations of the nucleotide bound subunit B and nucleotide bound B-F complex in explicit solvent, suggests that subunit F is critical for the migration to and eventual occupancy of the final binding site by the nucleotide of subunit B. Rotation of the C-terminus and conformational changes in subunit B are initiated upon binding with subunit F causing a perturbation that leads to the migration of ATP from the transition site 1 through an intermediate transition site 2 to the final binding site 3. This mechanism is elucidated on the basis of change in binding affinity for the nucleotide at the specific sites on subunit B upon complexation with subunit F. The change in enthalpy is further explained based on the fluctuating local environment around the binding sites.     

Phosphorylase kinase conformers. Detection by proteases

A variety of proteases have been evaluated as potential structural and conformational probes of nonphosphorylated and phosphorylated phosphorylase kinase. In general, the enzyme's alpha subunit is rapidly degraded, followed in most cases by hydrolysis of the beta subunit; the gamma subunit is resistant to most proteases. Trypsin clearly distinguishes between the nonactivated and activated conformers of phosphorylase kinase, in that the beta subunit in phosphorylated enzyme, as opposed to nonphosphorylated enzyme, is markedly protected from tryptic attack. In contrast, only a small difference in the rates of proteolysis of the alpha subunit in phosphorylated and nonphosphorylated enzyme is seen, even when a protease is used that is highly selective for the alpha subunit, such as chymotrypsin or endoproteinase Arg C. Incubation of nonphosphorylated phosphorylase kinase with either Mg2+ or Ca2+, which are activating cations, also protects the beta subunit from tryptic hydrolysis, whereas Mn2+, which inhibits the kinase activity, has little effect on proteolysis. The allosteric activator ADP also causes the beta subunit to become refractory to trypsin and mimics the effects of phosphorylation. Similar effector-induced conformational changes in the beta subunit are also observed with enzyme in which the alpha subunit has previously been selectively destroyed. These data indicate that activation of phosphorylase kinase by dissimilar mechanisms is associated with a conformational change in the enzyme's beta subunit that is detectable by trypsin and confirm earlier studies from this laboratory employing a chemical cross-linker as a conformational probe for activated and nonactivated conformers of the enzyme (Fitzgerald, T. J., and Carlson, G. M. (1984) J. Biol. Chem. 259, 3266-3274).     

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.     

Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation

BACKGROUND: Base excision repair initiated by human thymine-DNA glycosylase (TDG) results in the generation of abasic sites (AP sites) in DNA. TDG remains bound to this unstable repair intermediate, indicating that its transmission to the downstream-acting AP endonuclease is a coordinated process. Previously, we established that posttranslational modification of TDG with Small Ubiquitin-like MOdifiers (SUMOs) facilitates the dissociation of the DNA glycosylase from the product AP site, but the underlying molecular mechanism remained unclear. RESULTS: We now show that upon DNA interaction, TDG undergoes a dramatic conformational change, which involves its flexible N-terminal domain and accounts for the nonspecific DNA binding ability of the enzyme. This function is required for efficient processing of the G.T mismatch but then cooperates with the specific DNA contacts established in the active site pocket of TDG to prevent its dissociation from the product AP site after base release. SUMO1 conjugation to the C-terminal K330 of TDG modulates the DNA binding function of the N terminus to induce dissociation of the glycosylase from the AP site while it leaves the catalytic properties of base release in the active site pocket of the enzyme unaffected. CONCLUSION: Our data provide insight into the molecular mechanism of SUMO modification mediated modulation of enzymatic properties of TDG. A conformational change, involving the N-terminal domain of TDG, provides unspecific DNA interactions that facilitate processing of a wider spectrum of substrates at the expense of enzymatic turnover. SUMOylation then reverses this structural change in the product bound TDG.     

The metal ion binding properties of calreticulin modulate its conformational flexibility and thermal stability

Calreticulin (CRT) is a soluble chaperone involved in the conformational maturation of glycoproteins in the endoplasmic reticulum. Using biochemical and biophysical techniques including circular dichroism, proteolysis, and analytical ultracentrifugation, we have determined the effects of calcium and zinc ions on the structural properties of human CRT. Circular dichroism analysis has shown that the binding of calcium and zinc ions to CRT induces no significant changes in the secondary structure of the protein but affects in very distinct ways the local tertiary packing of these elements. More specifically, these studies have revealed that CRT adopts a more rigid and thermally stable structure upon binding calcium ions and a more loosely packed and thermally destabilized structure upon binding zinc ions. Consistent with these results, proteolysis experiments demonstrated that the intrinsic conformational flexibility of CRT can be modulated toward either a decrease or an increase in susceptibility to cleavage by chymotrypsin upon binding calcium or zinc ions, respectively. Results from sedimentation analysis indicated that the global three-dimensional structure of CRT is essentially unchanged upon binding calcium ions. In marked contrast, CRT self-associates reversibly to form dimers upon binding zinc ions. Collectively, our results provide evidence that calcium and zinc ions induce strikingly different changes in the biochemical and structural properties of CRT.     

Structural analysis of the human Rad51 protein-DNA complex filament by tryptophan fluorescence scanning analysis: transmission of allosteric effects between ATP binding and DNA binding

Human Rad51 (HsRad51) catalyzes the strand exchange reaction, a crucial step in homologous recombination, by forming a filamentous complex with DNA. The structure of this filament is modified by ATP, which is required and hydrolyzed for the reaction. We analyzed the structure and the ATP-promoted conformational change of this filament. We systematically replaced aromatic residues in the protein, one at a time, with tryptophan, a fluorescent probe, and examined its effect on the activities (DNA binding, ATPase, ATP-promoted conformational change, and strand exchange reaction) and the fluorescence changes upon binding of ATP and DNA. Some residues were also replaced with alanine. We thus obtained structural information about various positions of the protein in solution. All the proteins conserved, at least partially, their activities. However, the replacement of histidine at position 294 (H294) and phenylalanine at 129 (F129) affected the ATP-induced conformational change of the DNA-HsRad51 filament, although it did not prevent DNA binding. F129 is considered to be close to the ATP-binding site and to H294 of a neighboring subunit. ATP probably modifies the structure around F129 and affects the subunit/subunit contact around H294 and the structure of the DNA-binding site. The replacement also reduced the DNA-dependent ATPase activity, suggesting that these residues are also involved in the transmission of the allosteric effect of DNA to the ATP-binding site, which is required for the stimulation of ATPase activity by DNA. The fluorescence analyses supported the structural change of the DNA-binding site by ATP and that of the ATP-binding site by DNA. This information will be useful to build a molecular model of the Rad51-DNA complex and to understand the mechanism of activation of Rad51 by ATP and that of the Rad51-promoted strand exchange reaction.     

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.     

Protease-dependent cell entry mechanism of coronaviruses

Previous studies have demonstrated that the SARS-CoV S protein requires proteolytic cleavage by elastase, cathepsin or TMPRSS2 for S-mediated cell-cell or virus-cell membrane fusion. Activation of viral glycoprotein (GP) by protease also has been reported for influenza virus. The most distinctive difference between influenza virus and SARS-CoV is the stage during virus replication in which viral glycoproteins are cleaved by proteases. In influenza virus, the protease makes a simple cut in the GP during maturation. In contrast, SARS-CoV S protein is cleaved by the protease following receptor-induced conformational changes. The protease cleavage site in S protein is thought to be exposed only after receptor binding. In support of this model, we reported that the S protein of mouse hepatitis virus type 2 (MHV-2), which is highly similar to the S protein of SARS-CoV, requires two-step conformational changes mediated by sequential receptor binding and proteolysis to be activated for membrane fusion. Such a mechanism allows for tight temporal control over fusion by protecting the activating cleavage site from premature proteolysis yet allowing efficient cleavage upon binding to the receptor on target cells.     

The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA

The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.     

Relationship between the affinity and proteolysis of the insulin receptor. Evidence that higher affinity receptors are preferentially degraded

125I-Insulin binding to rat liver plasma membranes initiated two processes that occurred with similar time courses: an increase of receptor affinity for hormone and degradation of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000. Inhibitors of serine proteinases prevented alpha subunit degradation without affecting the affinity change. This shows that the change of affinity is not produced by receptor proteolysis and that the intact alpha subunit of the insulin receptor can exist as a higher or lower affinity species. Hormone binding was much more rapid than receptor proteolysis and the initial rate of alpha subunit degradation was independent of the concentration of occupied lower affinity receptors. Only persistent hormone binding and the accumulation of higher affinity insulin-receptor complexes led to significant receptor proteolysis. As the incubation time between 125I-insulin and membranes increased, the rate at which hormone dissociated from Mr 135,000 complexes diminished, whereas hormone dissociated from Mr 120,000 complexes slowly after brief or extended incubations. These observations suggest that 125I-insulin binds to membranes to form low affinity complexes that are not substrates for proteolysis. A slow conformational change produces higher affinity hormone-receptor complexes that are selectively degraded. Thus, the conversion between states of affinity may play a role in the regulation of receptor proteolysis and, consequently, insulin action in cells.     

Conformational Changes in the tryptophan synthase from a hyperthermophile upon alpha2beta2 complex formation: crystal structure of the complex

The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.     

OP18/stathmin binds near the C-terminus of tubulin and facilitates GTP binding.

It is has been previously suggested that the protein Op18/stathmin may interact with tubulin via the alpha-tubulin subunit [Larsson, N., Marklund, U., Melander Gradin, H., Brattsand, G. & Gullberg, M. (1997) Mol. Cell. Biol. 17, 5530-5539]. In this study we have used limited proteolysis and cross-linking analysis to localize further the stathmin-binding site on alpha-tubulin. Our results indicate that such a binding site is in a region close to the C-terminus of the molecule comprising residues 307 to the subtilisin-cleavage site on the alpha-tubulin subunit. Based on a recent model of the structure of tubulin [Nogales, E., Wolf, S.G. & Dowing, D.H. (1998) Nature (London) 391, 199-203], we found that this region contained the same areas that may be involved in longitudinal contacts of alpha-tubulin subunits within the microtubule. We also observed that the binding of stathmin to tubulin can modulate the binding of GTP to tubulin, as a consequence of a conformational change in the beta-tubulin subunit that occurs upon interaction of stathmin with tubulin.     

Fluorescence resonance energy transfer on the voltage-dependent sodium channel. Spatial relationship and site coupling between the batrachotoxin and Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin receptors

A fluorescent N- methylanthraniloyl derivative of the potent depolarizing agent batrachotoxin has been used to probe the structural and conformational properties of the neurotoxin receptor site on the voltage-dependent sodium channel. Batrachotoxin A 20-alpha-N- methylanthranilate (BTX-NMA) retains high affinity for its receptor site on the synaptosomal sodium channel with a Kd between 78 and 91 nM and an average site capacity of 2 pmol/mg of synaptosomal protein in the presence of Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin. The fluorescence emission of BTX-NMA upon binding to synaptosomes indicates a hydrophobic environment. Toxin V from L. quinquestriatus, an allosteric activator, effects a 20-nm red shift in the spectrum of bound BTX-NMA and a 4-fold enhancement in the fluorescence quantum yield disclosing a conformational change into a hydrophilic environment. Fluorescence resonance energy transfer measurements show that the distance separating the receptor sites is 37 +/- 10 A. Thus, the binding of alpha-scorpion toxin must involve conformational changes that extend over large distances from the batrachotoxin-binding locus. This information together with the distance measurements between the tetrodotoxin and alpha-scorpion toxin receptors and the conformational transition associated with this distance upon batrachotoxin addition indicate a conformationally flexible channel with coupling of sites through the polyatomic framework of individual subunits or through extensive alterations in subunit/subunit interactions.     

Structure of Cry3A delta-endotoxin within phospholipid membranes.

Interaction of delta-endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha-helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta-endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha-helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.     

Resolving individual steps in the operation of ATP-dependent proteolytic molecular machines: from conformational changes to substrate translocation and processivity

Clp, Lon, and FtsH proteases are proteolytic molecular machines that use the free energy of ATP hydrolysis to unfold protein substrates and processively present them to protease active sites. Here we review recent biochemical and structural studies relevant to the mechanism of ATP-dependent processive proteolysis. Despite the significant structural differences among the Clp, Lon, and FtsH proteases, these enzymes share important mechanistic features. In these systems, mechanistic studies have provided evidence for ATP binding and hydrolysis-driven conformational changes that drive translocation of substrates, which has significant implications for the processive mechanism of proteolysis. These studies indicate that the nucleotide (ATP, ADP, or nonhydrolyzable ATP analogues) occupancy of the ATPase binding sites can influence the binding mode and/or binding affinity for protein substrates. A general mechanism is proposed in which the communication between ATPase active sites and protein substrate binding regions coordinates a processive cycle of substrate binding, translocation, proteolysis, and product release.