Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase

HslVU is an ATP-dependent protease consisting of HslU ATPase and HslV peptidase. In an HslVU complex, the central pores of HslU hexamer and HslV dodecamer are aligned and the proteolytic active sites are sequestered in the inner chamber of HslV. Thus, the degradation of natively folded proteins requires unfolding and translocation processes for their access into the proteolytic chamber of HslV. A highly conserved GYVG(93) sequence constitutes the central pore of HslU ATPase. To determine the role of the pore motif on protein unfolding and translocation, we generated various mutations in the motif and examined their effects on the ability of HslU in supporting the proteolytic activity of HslV against three different substrates: SulA as a natively folded protein, casein as an unfolded polypeptide, and a small peptide. Flexibility provided by Gly residues and aromatic ring structures of the 91st amino acid were essential for degradation of SulA. The same structural features of the GYVG motif were highly preferred, although not essential, for degradation of casein. In contrast, none of the features were required for peptide hydrolysis. Mutations in the GYVG motif of HslU also showed marked influence on its ATPase activity, affinity to ADP, and interaction with HslV. These results suggest that the GYVG motif of HslU plays important roles in unfolding of natively folded proteins as well as in translocation of unfolded proteins for degradation by HslV. These results also implicate a role of the pore motif in ATP cleavage and in the assembly of HslVU complex.     

HslVU ATP-dependent Protease Utilizes Maximally Six among Twelve Threonine Active Sites during Proteolysis

HslVU is a bacterial ATP-dependent protease distantly related to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecameric HslV protease. As a homolog of the 20 S proteasome β-subunits, HslV also uses the N-terminal threonine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14 β-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activity. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only ∼6 active sites is sufficient to support full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activity. Using proteasome inhibitors, we also demonstrate that substrate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites is decreased to ∼6 but is gradually compromised upon further reduction. These results with a mathematical model suggest HslVU utilizes no more than 6 active sites at any given time, presumably because of the action of HslU. These results also suggest that each ATP-bound HslU subunit activates one HslV subunit and that substrate bound to the HslV active site stimulates the HslU ATPase activity by stabilizing the HslV-HslU interaction. We propose this mechanism plays an important role in supporting complete degradation of substrates while preventing wasteful ATP hydrolysis in the resting state by controlling the interaction between HslV and HslU through the catalytic engagement of the proteolytic active sites.     

The C-terminal tails of HslU ATPase act as a molecular switch for activation of HslV peptidase

The bacterial HslVU ATP-dependent protease is a homolog of the eukaryotic 26 S proteasome. HslU ATPase forms a hexameric ring, and HslV peptidase is a dodecamer consisting of two stacked hexameric rings. In HslVU complex, the HslU and HslV central pores are aligned, and the proteolytic active sites are sequestered in an internal chamber of HslV, with access to this chamber restricted to small axial pores. Here we show that the C-terminal tails of HslU play a critical role in the interaction with and activation of HslV peptidase. A synthetic tail peptide of 10 amino acids could replace HslU in supporting the HslV-mediated hydrolysis of unfolded polypeptide substrates such as alpha-casein, as well as of small peptides, suggesting that the HslU C terminus is involved in the opening of the HslV pore for substrate entry. Moreover, deletion of 7 amino acids from the C terminus prevented the ability of HslU to form an HslVU complex with HslV. In addition, deletion of the C-terminal 10 residues prevented the formation of an HslU hexamer, indicating that the C terminus is required for HslU oligomerization. These results suggest that the HslU C-terminal tails act as a molecular switch for the assembly of HslVU complex and the activation of HslV peptidase.     

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.     

Binding of MG132 or deletion of the Thr active sites in HslV subunits increases the affinity of HslV protease for HslU ATPase and makes this interaction nucleotide-independent

HslVU is an ATP-dependent protease in bacteria consisting of HslV dodecamer and HslU hexamer. Upon ATP binding, HslU ATPase allosterically activates the catalytic function of HslV protease by 1-2 orders of magnitude. However, relatively little is known about the role of HslV in the control of HslU function. Here we describe the involvement of the N-terminal Thr active sites (Thr-1) of HslV in the communication between HslV and HslU. Binding of proteasome inhibitors to Thr-1 led to a dramatic increase in the interaction between HslV and HslU with a marked increase in ATP hydrolysis by HslU. Moreover, carbobenzoxy-leucyl-leucyl-leucinal (MG132) could bind to Thr-1 of free HslV, and this binding induced a tight interaction between HslV and HslU with the activation of HslU ATPase, suggesting that substrate-bound HslV can allosterically regulate HslU function. Unexpectedly, the deletion of Thr-1 also caused a dramatic increase in the affinity between HslV and HslU even in the absence of ATP. Furthermore, the increase in the number of the Thr-1 deletion mutant subunit in place of HslV subunit in a dodecamer led to a proportional increase in the affinity between HslV and HslU with gradual activation of HslU ATPase. Although the molecular mechanism elucidating how the Thr-1 deletion influences the interaction between HslV and HslU remains unknown, these results suggest an additional allosteric mechanism for the control of HslU function by HslV. Taken together, our findings indicate a critical involvement of Thr-1 of HslV in the reciprocal control of HslU function and, thus, for their communication.     

The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel-shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I-domain loops increase K(M) and decrease V(max) for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine.     

Insights into the Molecular Evolution of HslU ATPase through Biochemical and Mutational Analyses

The ATP-dependent HslVU complexes are found in all three biological kingdoms. A single HslV protease exists in each species of prokaryotes, archaea, and eukaryotes, but two HslUs (HslU1 and HslU2) are present in the mitochondria of eukaryotes. Previously, a tyrosine residue at the C-terminal tail of HslU2 has been identified as a key determinant of HslV activation in Trypanosoma brucei and a phenylalanine at the equivalent position to E. coli HslU is found in T. brucei HslU1. Unexpectedly, we found that an F441Y mutation in HslU enhanced the peptidase and caseinolytic activity of HslV in E. coli but it showed partially reduced ATPase and SulA degradation activity. Previously, only the C-terminal tail of HslU has been the focus of HslV activation studies. However, the Pro315 residue interacting with Phe441 in free HslU has also been found to be critical for HslV activation. Hence, our current biochemical analyses explore the importance of the loop region just before Pro315 for HslVU complex functionality. The proline and phenylalanine pair in prokaryotic HslU was replaced with the threonine and tyrosine pair from the functional eukaryotic HslU2. Sequence comparisons between multiple HslUs from three different biological kingdoms in combination with biochemical analysis of E. coli mutants have uncovered important new insights into the molecular evolutionary pathway of HslU.     

Small molecule activators of proteasome-related HslV peptidase

The HslVU is the proteasome-related two component system composed of HslV peptidase and HslU chaperone. It is involved in the degradation of an array of intracellular proteins. The presence of HslVU homologs in pathogenic microbes and its absence in human makes it an antimicrobial drug target. The functional HslVU complex forms when HslV dodecamer is flanked at both ends by HslU hexamers. In the HslVU complex, eight residues at the carboxy termini of HslU subunits intercalate into a clefts between two adjacent HslV subunits causing a conformational change in the active site of HslV which in turn results in the allosteric activation of HslV peptidase. Here, we report small molecules capable of activating HslV peptidase in the absence of its natural activator HslU ATPase. For this purpose, virtual screening of an in-house library of synthetic and natural compounds was performed to find out ligands mimicking the interaction of HslU carboxy terminus with HslV dodecamer. The benzimidazole, quinazoline and chromone derivatives were suggested by ligand docking to bind at the HslU carboxy termini intercalation pockets in the HslV dodecamer. This was confirmed by HslV activation and isothermal titration calorimetry assays with these compounds that gave ED₅₀ in sub-micromolar range (0.6–1.5 μM). The results showed for the first time that small, extracellular non-peptidic molecules can allosterically activate the peptide hydrolytic activity of HslV which in turn would initiate intracellular proteolysis.     

Structure and reactivity of an asymmetric complex between HslV and I-domain deleted HslU,a prokaryotic homolog of the eukaryotic proteasome

In the prokaryotic homolog of the eukaryotic proteasome, HslUV, the "double donut" HslV protease is allosterically activated by HslU, an AAA protein of the Clp/Hsp100 family consisting of three (amino-terminal, carboxy-terminal, and intermediate) domains. The intermediate domains of HslU, which extend like tentacles from the hexameric ring formed by the amino-terminal and carboxy-terminal domains, have been deleted; an asymmetric HslU(DeltaI)(6)HslV(12) complex has been crystallized; and the structure has been solved to 2.5A resolution, revealing an assembly in which a HslU(DeltaI) hexamer binds one end of the HslV dodecamer. The conformation of the protomers of the HslU(DeltaI)-complexed HslV hexamer is similar to that in the symmetric wild-type HslUV complex, while the protomer conformation of the uncomplexed HslV hexamer is similar to that of HslV alone. Reaction in the crystals with a vinyl sulfone inhibitor reveals that the HslU(DeltaI)-complexed HslV hexamer is active, while the uncomplexed HslV hexamer is inactive. These results confirm that HslV can be activated by binding of a hexameric HslU(DeltaI)(6) ring lacking the I domains, that activation is effected through a conformational change in HslV rather than through alteration of the size of the entry channel into the protease catalytic cavity, and that the two HslV(6) rings in the protease dodecamer are activated independently rather than cooperatively.     

Characterization of Protomer Interfaces in HslV Protease; the Bacterial Homologue of 20S Proteasome

HslVU, a two-component proteasome-related prokaryotic system is composed of HslV protease and HslU ATPase. HslV protomers assemble in a dodecamer of two-stacked hexameric rings that form a complex with HslU hexamers. The intra- and inter-ring protomer interfaces in the HslV dodecamer underpin the integrity and functionality of HslVU. Structural characterization of HslV from different bacteria illustrated considerable differences in interacting residues, accessible surface and gap volumes at the intra-ring interface that is primarily stabilized by polar interactions. Amino acid residues Lys28, Arg83 and Asp111 have envisaged as hot spots at this HslU-interacting interface. The inter-ring interfaces that are made up of side chain packing of hydrophobic residues are structurally conserved. Hyperthermostable bacterium T. maritima HslV has extensively networked polar/nonpolar interactions and highly packed environment at all interfaces. Present data demonstrates that HslV protomer interfaces perform distinct functions; whereas intra-ring interface participates in HslV:HslU interaction resulting in allosteric activation of HslV protease by HslU, the inter-ring interfaces uphold the oligomeric form of HslV.     

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism

BACKGROUND: The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS: We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS: Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.     

Isolation and characterization of the prokaryotic proteasome homolog HslVU (ClpQY) from Thermotoga maritima and the crystal structure of HslV

Heat-shock locus VU (HslVU) is an ATP-dependent proteolytic system and a prokaryotic homolog of the proteasome. It consists of HslV, the protease, and HslU, the ATPase and chaperone. We have cloned, sequenced and expressed both protein components from the hyperthermophile Thermotoga maritima. T. maritima HslU hydrolyzes a variety of nucleotides in a temperature-dependent manner, with the optimum lying between 75 and 80 °C. It is also nucleotide-unspecific for activation of HslV against amidolytic and caseinolytic activity. The Escherichia coli and T. maritima HslU proteins mutually stimulate HslV proteins from both sources, suggesting a conserved activation mechanism. The crystal structure of T. maritima HslV was determined and refined to 2.1-Å resolution. The structure of the dodecameric enzyme is well conserved compared to those from E. coli and Haemophilus influenzae. A comparison of known HslV structures confirms the presence of a cation-binding site, although its exact role in the proteolytic mechanism of HslV remains unclear. Amongst factors responsible for the thermostability of T. maritima HslV, extensive ionic interactions/salt-bridge networks, which occur specifically in the T. maritima enzyme in comparison to its mesophilic counterparts, seem to play an important role.     

Isolation and characterization of the prokaryotic proteasome homolog HslVU (ClpQY) from Thermotoga maritima and the crystal structure of HslV

Heat-shock locus VU (HslVU) is an ATP-dependent proteolytic system and a prokaryotic homolog of the proteasome. It consists of HslV, the protease, and HslU, the ATPase and chaperone. We have cloned, sequenced and expressed both protein components from the hyperthermophile Thermotoga maritima. T. maritima HslU hydrolyzes a variety of nucleotides in a temperature-dependent manner, with the optimum lying between 75 and 80 °C. It is also nucleotide-unspecific for activation of HslV against amidolytic and caseinolytic activity. The Escherichia coli and T. maritima HslU proteins mutually stimulate HslV proteins from both sources, suggesting a conserved activation mechanism. The crystal structure of T. maritima HslV was determined and refined to 2.1-Å resolution. The structure of the dodecameric enzyme is well conserved compared to those from E. coli and Haemophilus influenzae. A comparison of known HslV structures confirms the presence of a cation-binding site, although its exact role in the proteolytic mechanism of HslV remains unclear. Amongst factors responsible for the thermostability of T. maritima HslV, extensive ionic interactions/salt-bridge networks, which occur specifically in the T. maritima enzyme in comparison to its mesophilic counterparts, seem to play an important role.     

Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.

Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.     

Nucleotide-dependent substrate recognition by the AAA+ HslUV protease

ATP-dependent protein degradation is controlled principally by substrate recognition. The AAA+ HslU ATPase is thought to bind protein substrates, denature them, and translocate the unfolded polypeptide into the HslV peptidase. The lack of well-behaved high-affinity substrates for HslUV (ClpYQ) has hampered understanding of the rules and mechanism of substrate engagement. We show that HslUV efficiently degrades Arc repressor, especially at heat-shock temperatures. Degradation depends on sequences near the N terminus of Arc. Fusion protein and peptide-binding experiments demonstrate that this sequence is a degradation tag that binds directly to HslU. Strong binding of this tag to the enzyme requires ATP and Mg(2+). Furthermore, fusion of this sequence to a protein with marked mechanical stability leads to complete degradation. Thus, these experiments demonstrate that HslUV is a powerful protein unfoldase and that initial substrate engagement by the HslU ATPase must occur after ATP binding.     

Characterization of the HslU chaperone affinity for HslV protease

The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd = 1 microM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd approximately 0.2 microM) after binding with the MBP approximately SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP approximately SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP approximately SulA with 10(-9) M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd = 0.2 microM) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP approximately SulA stimulates a conformational change in HslU to a high-affinity form for HslV.     

Crystal and solution structures of an HslUV protease-chaperone complex

HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.     

Asymmetric nucleotide transactions of the HslUV protease

ATP binding and hydrolysis are critical for protein degradation by HslUV, a AAA ⁺ machine containing one or two HslU₆ ATPases and the HslV₁₂ peptidase. Although each HslU homohexamer has six potential ATP-binding sites, we show that only three or four ATP molecules bind at saturation and present evidence for three functional subunit classes. These results imply that only a subset of HslU and HslUV crystal structures represents functional enzyme conformations. Our results support an asymmetric mechanism of ATP binding and hydrolysis, and suggest that molecular contacts between HslU and HslV vary dynamically throughout the ATPase cycle. Nucleotide binding controls HslUV assembly and activity. Binding of a single ATP allows HslU to bind HslV, whereas additional ATPs must bind HslU to support substrate recognition and to activate ATP hydrolysis, which powers substrate unfolding and translocation. Thus, a simple thermodynamic hierarchy ensures that substrates bind to functional HslUV complexes, that ATP hydrolysis is efficiently coupled to protein degradation, and that working HslUV does not dissociate, allowing highly processive degradation.     

Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU.

BACKGROUND: The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease. RESULTS: We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism. CONCLUSIONS: The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.     

Eubacterial HslV and HslU subunits homologs in primordial eukaryotes

ATP-dependent protease complexes are present in all three kingdoms of life, where they rid the cell of misfolded or damaged proteins and control the level of certain regulatory proteins. They include the proteasome in Eukaryotes, Archea, and Actinomycetales and the HslVU (ClpQY) complex in other eubacteria. We showed that genes homologous to eubacterial HslV (ClpQ) and HslU (ClpY) are present in the genome of trypanosomatid protozoa and are expressed. The features of the cDNAs indicated that bona fide trypanosomatid messengers had been cloned and ruled out bacterial contamination as the source of the material. The N-terminal microsequence of HslV from Leishmania infantum (Protozoa: Kinetoplastida) permitted the identification of the propeptide cleavage site and indicated that an active protease is present. High similarities (> or =57.5%) with the prototypical HslV and HslU from Escherichia coli and conservation of residues essential for biochemical activity suggested that a functional HslVU complex is present in trypanosomatid protozoa. The structure of the N-termini of HslV and HslU further suggested mitochondrial localization. Phylogenetic analysis indicated that HslV and HslU from trypanosomatids clustered with eubacterial homologs but did not point to any particular bacterial lineage. Because typical eukaryotic 20S proteasomes are present in trypanosomatids, we concluded that the eubacterial HslVU and the eukaryotic multicatalytic protease are simultaneously present in these organisms. To our knowledge this is the first report of a eubacterial HslVU complex in eukaryotes and, consequently, of the simultaneous occurrence of both a proteasome and HslVU in living cells.