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.     

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.     

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.     

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.     

Proteasome-Related HslU and HslV Genes Typical of Eubacteria Are Widespread in Eukaryotes

Many eubacteria contain an ATP-dependent protease complex, which is built by multiple copies of the HslV and HslU proteins and is therefore called HslVU. HslU proteins are AAA + ATPases, while HslV proteins are proteases that show highly significant similarity to β subunits of proteasomes. Therefore, the HslVU complex has been envisaged as a precursor or ancestral type of proteasome. Here we show that species of most of the main eukaryotic lineages have HslU and HslV genes very similar to those found in proteobacteria. We have detected them in amoebozoa, plantae, chromoalveolata, rhizaria, and excavata species. Phylogenetic analyses suggest that these genes have been obtained by endosymbiosis from the proteobacterial ancestor that gave rise to eukaryotic mitochondria. The products encoded by these eukaryotic genes adopt, according to modeling based on the known crystal structures of prokaryotic HslU and HslV proteins, conformations that are compatible with their being fully active, suggesting that functional HslVU complexes may be present in many eukaryotic species. [Reviewing Editor: Dr. Yves Van de Peer]     

The Quaternary Arrangement of HslU and HslV in a Cocrystal: A Response to Wang, Yale

Protease HslV and ATPase HslU form an ATP-dependent protease in bacteria. We have previously determined the structure of the components of this protease. In the case of HslU, the structure was derived from HslU-HslV cocrystals, combining phase information from MAD and the previously determined HslV model. Whereas the structures of the components were confirmed in detail by later structures, the quaternary arrangement of HslV and HslU was not reproduced in later crystal forms. In a recent communication to this journal, Wang attempted a reinterpretation of our original data to account for this difference. In response, we demonstrate that difference Pattersons, difference Fouriers, molecular replacement calculations, R factors, and omit maps all support our original analysis and prove that the suggested reinterpretation is false by these criteria. In particular, we show that our crystals are essentially untwinned and that only the originally reported quaternary arrangement of HslV and HslU particles is consistent with the experimental data. We finally demonstrate that Wang's newly introduced R(tpart) method to predict translational corrections for a subset of the unit cell contents is systematically flawed.     

Molecular Modeling Studies of the Interaction Between Plasmodium falciparum HslU and HslV Subunits

Abstract

The PfHslUV, a Plasmodium falciparum homolog of prokaryotic HslUV systems, is a newly identified drug target. The HslUV complex is an assembly of Heat Shock Locus gene products U and V. The formation of complete complex is essential for the proteasome to carry out its biochemical and physiological role in the parasite, namely to degrade specific target proteins in an ATP-dependent chaperone assisted manner. PfHslV subunit, a protease, exhibits increased proteolytic activity in the presence of PfHslU, the subunit believed to be responsible for allosteric activation of PfHslV. In the present work, we have employed computational methods to simulate the interaction of PfHslU and PfHslV subunits. We have used three methods—namely homology modeling, molecular docking and computational alanine scanning to model the complex, to predict the binding mode of PfHslU-V interaction and to predict the binding-energy hot-spots in protein-protein interface, respectively. The three dimensional models of PfHslV and PfHslU have been generated using MODELLER, based on the crystal structures of prokaryotic HslUV complex as templates. The modeled structures were docked using PatchDock, a geometry-based molecular docking algorithm. Finally, a three-dimensional PfHslUV complex model was generated that helped in comparing protein-protein interface characteristics with that of crystal structures of prokaryotic HslUV. Further, computational alanine scanning analysis of the generated complex was performed to calculate the binding free energy changes (ΔΔG_bind), which helped in identifying residues crucial for PfHslU and PfHslV interactions.     

Crystal structure of HslUV complexed with a vinyl sulfone inhibitor: corroboration of a proposed mechanism of allosteric activation of HslV by HslU

On the basis of the structure of a HslUV complex, a mechanism of allosteric activation of the HslV protease, wherein binding of the HslU chaperone propagates a conformational change to the active site cleft of the protease, has been proposed. Here, the 3.1 A X-ray crystallographic structure of Haemophilus influenzae HslUV complexed with a vinyl sulfone inhibitor is described. The inhibitor, which reacts to form a covalent linkage to Thr1 of HslV, binds in an "antiparallel beta" manner, with hydrogen-bond interactions between the peptide backbone of the protease and that of the inhibitor, and with two leucinyl side chains of the inhibitor binding in the S1 and S3 specificity pockets of the protease. Comparison of the structure of the HslUV-inhibitor complex with that of HslV without inhibitor and in the absence of HslU reveals that backbone interactions would correctly position a substrate for cleavage in the HslUV complex, but not in the HslV protease alone, corroborating the proposed mechanism of allosteric activation. This activation mechanism differs from that of the eukaryotic proteasome, for which binding of activators opens a gated channel that controls access of substrates to the protease, but does not perturb the active site environment.     

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.     

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.     

Nucleotide triphosphates inhibit the degradation of unfolded proteins by HslV peptidase

Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.     

The crystal structure of the chemokine domain of fractalkine shows a novel quaternary arrangement

Fractalkine, or neurotactin, is a chemokine that is present in endothelial cells from several tissues, including brain, liver, and kidney. It is the only member of the CX(3)C class of chemokines. Fractalkine contains a chemokine domain (CDF) attached to a membrane-spanning domain via a mucin-like stalk. However, fractalkine can also be proteolytically cleaved from its membrane-spanning domain to release a freely diffusible form. Fractalkine attracts and immobilizes leukocytes by binding to its receptor, CX(3)CR1. The x-ray crystal structure of CDF has been solved and refined to 2.0 A resolution. The CDF monomers form a dimer through an intermolecular beta-sheet. This interaction is somewhat similar to that seen in other dimeric CC chemokine crystal structures. However, the displacement of the first disulfide in CDF causes the dimer to assume a more compact quaternary structure relative to CC chemokines, which is unique to CX(3)C chemokines. Although fractalkine can bind to heparin in vitro, as shown by comparison of electrostatic surface plots with other chemokines and by heparin chromatography, the role of this property in vivo is not well understood.     

The first crystal structure of a Mimosoideae lectin reveals a novel quaternary arrangement of a widespread domain

The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of beta-prism domains, and highlight the adaptability of the beta-prism fold as a building block in the evolution of plant lectins. The P.platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroid-shaped structure of the P.platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P.platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms.     

Effects of hydrostatic pressure on the quaternary structure and enzymatic activity of a large peptidase complex from Pyrococcus horikoshii

While molecular adaptation to high temperature has been extensively studied, the effect of hydrostatic pressure on protein structure and enzymatic activity is still poorly understood. We have studied the influence of pressure on both the quaternary structure and enzymatic activity of the dodecameric TET3 peptidase from Pyrococcus horikoshii. Small angle X-ray scattering (SAXS) revealed a high robustness of the oligomer under high pressure of up to 300 MPa at 25°C as well as at 90°C. The enzymatic activity of TET3 was enhanced by pressure up to 180 MPa. From the pressure behavior of the different rate-constants we have determined the volume changes associated with substrate binding and catalysis. Based on these results we propose that a change in the rate-limiting step occurs around 180 MPa.     

The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold

The high number of quaternary structures observed for lectins highlights the important role of these oligomeric assemblies during carbohydrate recognition events. Although a large diversity in the mode of association of lectin subunits is frequently observed, the oligomeric assemblies of plant lectins display small variations within a single family. The crystal structure of the mannose-binding jacalin-related lectin from Calystegia sepium (Calsepa) has been determined at 1.37-A resolution. Calsepa exhibits the same beta-prism fold as identified previously for other members of the family, but the shape and the hydrophobic character of its carbohydrate-binding site is unlike that of other members, consistent with surface plasmon resonance analysis showing a preference for methylated sugars. Calsepa reveals a novel dimeric assembly markedly dissimilar to those described earlier for Heltuba and jacalin but mimics the canonical 12-stranded beta-sandwich dimer found in legume lectins. The present structure exemplifies the adaptability of the beta-prism building block in the evolution of plant lectins and highlights the biological role of these quaternary structures for carbohydrate recognition.     

An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron

Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids.     

Inhibition kinetics and emodin cocrystal structure of a type II polyketide ketoreductase

Type II polyketides are a class of natural products that include pharmaceutically important aromatic compounds such as the antibiotic tetracycline and antitumor compound doxorubicin. The type II polyketide synthase (PKS) is a complex consisting of 5-10 standalone domains homologous to fatty acid synthase (FAS). Polyketide ketoreductase (KR) provides regio- and stereochemical diversity during the reduction. How the type II polyketide KR specifically reduces only the C9 carbonyl group is not well understood. The cocrystal structures of actinorhodin polyketide ketoreductase (actKR) bound with NADPH or NADP+ and the inhibitor emodin were solved with the wild type and P94L mutant of actKR, revealing the first observation of a bent p-quinone in an enzyme active site. Molecular dynamics simulation help explain the origin of the bent geometry. Extensive screening for in vitro substrates shows that unlike FAS KR, the actKR prefers bicyclic substrates. Inhibition kinetics indicate that actKR follows an ordered Bi Bi mechanism. Together with docking simulations that identified a potential phosphopantetheine binding groove, the structural and functional studies reveal that the C9 specificity is a result of active site geometry and substrate ring constraints. The results lay the foundation for the design of novel aromatic polyketide natural products with different reduction patterns.     

Evolution of quaternary structure in a homotetrameric enzyme

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits.