Structural basis for peptide recognition by archaeal oligopeptide permease A.

Oligopeptide permease A (OppA) plays an important role in the nutrition of cells and various signaling processes. In archaea, OppA is a major protein present in membrane vesicles of Thermococcales. Because there being no crystal structures of archaeal OppAs determined to date, we report the crystal structure of archaeal OppA from Thermococcus kodakaraensis (TkOppA) at 2.3 Å resolution by the single-wavelength anomalous dispersion method. TkOppA consists of three domains similarly to bacterial OppAs, and the inserted regions not present in bacterial OppAs are at the periphery of the core region. An endogenous pentapeptide was bound in the pocket of domains I and III of TkOppA by hydrogen bonds of main-chain atoms of the peptide and hydrophobic interactions. No hydrogen bonds of side-chain atoms of the peptide were observed; thus, TkOppA may have low peptide selectivity but some preference for residues 2 and 3. TkOppA has a relatively large pocket and can bind a nonapeptide; therefore, it is suitable for the binding of large peptides similarly to OppAs of Gram-positive bacteria.

The lipid raft markers stomatin, prohibitin, flotillin, and HflK/C (SPFH)-domain proteins form an operon with NfeD proteins and function with apolar polyisoprenoid lipids.

SPFH-domain proteins are found in almost all organisms across three domains: archaea, bacteria, and eukaryotes. In eukaryotic organelles, their subfamilies exhibit overlapping distribution and functions; thus, the rationality of annotation to discriminate these subfamilies remains unclear. In this review, the binding ability of prokaryotic SPFH-domain proteins towards nonpolar polyisoprenoides such as squalene and lycopene, rather than cholesterol, is discussed. The hydrophobic region at the C-terminus of SPFH-domain proteins constitutes the main region that binds apolar polyisoprenoid lipids as well as cholesterol and substantively contributes towards lipid raft formation as these regions are self-assembled together with specific lipids. Because the scaffolding proteins caveolins show common topological properties with SPFH-domain proteins such as stomatin and flotillin, the α-helical segments of stomatin proteins can flexibly move along with the membrane surface, with such movement potentially leading to membrane bending via lipid raft clustering through the formation of high order homo-oligomeric complexes of SPFH-domain proteins. We also discuss the functional significance and ancient origin of SPFH-domain proteins and the NfeD protein (STOPP) operon, which can be traced back to the ancient living cells that diverged and evolved to archaea and bacteria. Based on the molecular mechanism whereby the STOPP-protease degrades the C-terminal hydrophobic clusters of SPFH-domain proteins, it is conceivable that STOPP-protease might control the physicochemical properties of lipid rafts.

Serial intermediates with a 1 nt 3'-flap and 5' variable-length flaps are formed by cooperative functioning of Pyrococcus horikoshii FEN-1 with either B or D DNA polymerases.

Flap endonuclease-1 (FEN-1) plays important roles with DNA polymerases in DNA replication, repair and recombination. FEN-1 activity is elevated by the presence of a 1 nucleotide expansion at the 3' end in the upstream primer of substrates called "structures with a 1 nt 3'-flap", which appear to be the most preferable substrates for FEN-1; however, it is unclear how such substrates are generated in vivo. Here, we show that substrate production occurred by the cooperative function of FEN-1(phFEN-1) and Pyrococcus horikoshii DNA polymerase B (phPol B) or D (phPol D). Using various substrates, the activities of several phFEN-1 F79 mutants were compared with those of the wild type. Analysis of the activity profiles of these mutants led us to discriminate "structures with a 1 nt 3'-flap" from substrates with a 3' -projection longer than 2 nt or from those without a 3'-projection. When phFEN-1 processed a gap substrate with phPol B or phPol D, "structures with a 1 nt 3'-flap" were assumed the reaction intermediates. Furthermore, the phFEN-1 cleavage products with phPol B or D were from 1mer to 7mer, corresponding to the sizes of the strand-displacement products of these polymerases. This suggests that a series of 1 nt 3'-flap with 5'-variable length-flap configurations were generated as transient intermediates, in which the length of the 5'-flaps depended on the displacement distance of the downstream strand by phPol B or D. Therefore, phFEN-1 might act successively on displaced 5'-variable flaps.

Structural and biochemical analysis of a thermostable membrane-bound stomatin-specific protease.

Membrane-bound proteases are involved in various regulatory functions. The N-terminal region of PH1510p (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138), and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511p. In a form of human hemolytic anemia known as hereditary stomatocytosis, the stomatin protein is deficient in the erythrocyte membrane due to mis-trafficking. In order to understand the catalytic mechanism of 1510-N in more detail, here the structural and biochemical analysis of 1510-N is reported. Two degraded products were produced via acyl-enzyme intermediates. 1510-N is a thermostable protease, and thus crystallization after heat treatment of the protease-peptide complex was attempted in order to understand the catalytic mechanism of 1510-N. The structure after heat treatment is almost identical to that with no heat treatment. According to the superposition between the structures with heat treatment and with no heat treatment, the N-terminal half of the peptide is superposed well, whereas the C-terminal half of the peptide is slightly deviated. The N-terminal half of the peptide binds to 1510-N more tightly than the C-terminal half of the peptide. The flexible L2 loops of 1510-N cover the peptide, and are involved in the protease activity.

Membrane vesicles, nanopods and/or nanotubes produced by hyperthermophilic archaea of the genus Thermococcus.

Thermococcus species produce MVs (membrane vesicles) into their culture medium. These MVs are formed by a budding process from the cell envelope, similar to ectosome formation in eukaryotic cells. The major protein present in MVs of Thermococci is a peptide-binding receptor of the OppA (oligopeptide-binding protein A) family. In addition, some of them contain a homologue of stomatin, a universal membrane protein involved in vesiculation. MVs produced by Thermococcus species can recruit endogenous or exogenous plasmids and plasmid transfer through MVs has been demonstrated in Thermococcus kodakaraensis. MVs are frequently secreted in clusters surrounded by S-layer, producing either big protuberances (nanosphere) or tubular structures (nanotubes). Thermococcus gammatolerans and T. kodakaraensis produce nanotubes containing strings of MVs, resembling the recently described nanopods in bacteria, whereas Thermococcus sp. 5-4 produces filaments whose internal membrane is continuous. These nanotubes can bridge neighbouring cells, forming cellular networks somehow resembling nanotubes recently observed in Firmicutes. As suggested for bacteria, archaeal nanopods and/or nanotubes could be used to expand the metabolic sphere around cells and/or to promote intercellular communication.

Higher-order structure formation using refined monomer structures of lipid raft markers, Stomatin, Prohibitin, Flotillin, and HflK/C-related proteins.

Currently, information on the higher-order structure of Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)-domain proteins is limited. Briefly, the coordinate information (Refined PH1511.pdb) of the stomatin ortholog, PH1511 monomer, was obtained using the artificial intelligence, ColabFold: AlphaFold2. Thereafter, the 24mer homo-oligomer structure of PH1511 was constructed using the superposing method, with HflK/C and FtsH (KCF complex) as templates. The 9mer-12mer homo-oligomer structures of PH1511 were also constructed using the ab initio docking method, with the GalaxyHomomer server for artificiality elimination. The features and functional validity of the higher-order structures were discussed. The coordinate information (Refined PH1510.pdb) of the membrane protease PH1510 monomer, which specifically cleaves the C-terminal hydrophobic region of PH1511, was obtained. Thereafter, the PH1510 12mer structure was constructed by superposing 12 molecules of the Refined PH1510.pdb monomer onto a 1510-C prism-like 12mer structure formed along the crystallographic threefold helical axis. The 12mer PH1510 (prism) structure revealed the spatial arrangement of membrane-spanning regions between the 1510-N and 1510-C domains within the membrane tube complex. Based on these refined 3D homo-oligomeric structures, the substrate recognition mechanism of the membrane protease was investigated. These refined 3D homo-oligomer structures are provided via PDB files as Supplementary data and can be used for further reference.

Crystal structure of a membrane stomatin-specific protease in complex with a substrate peptide.

Membrane-bound proteases are involved in various regulatory functions. A previous report indicated that the N-terminal region of PH1510p (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511p. In humans, an absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis. Here, the crystal structure of 1510-N K138A in complex with a peptide substrate was determined at 2.25 Å resolution. In the structure, a 1510-N dimer binds to one peptide. The six central residues (VIVLML) of the peptide are hydrophobic and in a pseudopalindromic structure and therefore favorably fit into the hydrophobic active tunnel of the 1510-N dimer, although 1510-N degrades the substrate at only one point. A comparison with unliganded 1510-N K138A revealed that the binding of the substrate causes a large rotational and translational displacement between protomers and produces a tunnel suitable for binding the peptide. When the peptide binds, the flexible L2 loop of one protomer forms ß-strands, whereas that of the other protomer remains in a loop form, indicating that one protomer binds to the peptide more tightly than the other protomer. The Ala138 residues of the two protomers are located very close together (the distance between the two Cß atoms is 3.6 Å). Thus, in wild-type 1510-N, the close positioning of the catalytic Ser97 and Lys138 residues may be induced by electrostatic repulsion of the two Lys138 side chains of the protomers.

Structural and mutational studies suggest key residues to determine whether stomatin SPFH domains form dimers or trimers.

Stomatin is a major integral membrane protein in human erythrocytes. In a form of hemolytic anemia known as hereditary stomatocytosis, stomatin is deficient in the erythrocyte membrane due to mis-trafficking. It is a member of stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins, and SPFH proteins could function as membrane-bound oligomeric scaffolding proteins in lipid rafts. The previously determined structure of the SPFH domain of Pyrococcus horikoshii (Ph) stomatin formed a trimer, whereas that of mouse stomatin formed a dimer. To elucidate the difference of oligomerization state, structural and chromatographic analyses using Ph stomatin were performed, and the key residues were suggested to determine whether SPFH domains form dimers or trimers. From gel-filtration analyses, PhStom (56-234) formed a trimer or tetramer, whereas PhStom (63-234) and PhStom (56-234) K59S formed a dimer. The residues 56-62, particularly Lys59, were involved in trimerization. Based on the crystal structure of PhStom (63-234), it formed a banana-shaped dimer, as observed in mouse stomatin. Thus, residues 162-168 are involved in dimerization. This study provides important insight into the molecular function and oligomerization state of stomatin.

Inactive dimeric structure of the protease domain of stomatin operon partner protein.

The N-terminal region of the stomatin operon partner protein (STOPP) PH1510 (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511. In a form of human hemolytic anemia known as hereditary stomatocytosis, stomatin is deficient in the erythrocyte membrane owing to mis-trafficking. Stomatin is thought to act as an oligomeric scaffolding protein to support cell membranes. The cleavage of stomatin by STOPP might be involved in a regulatory system. Several crystal structures of 1510-N have previously been determined: the wild type, the K138A mutant and its complex with a substrate peptide. Here, the crystal structure of the S97A mutant of 1510-N (1510-N S97A) was determined at 2.25 Šresolution. The structure contained two 1510-N S97A molecules in the asymmetric unit. On the superposition of one monomer of the 1510-N S97A and wild-type dimers, the S97A Cα atom of the other monomer of 1510-N S97A deviated by 23 Šfrom that of the wild type. This result indicates that 1510-N can greatly change the form of its dimer. Because of crystallographic symmetry in space group P65, a sixfold helical structure is constructed using the 1510-N dimer as a basic unit. This helical structure may be common to STOPP structures.

Unusual thermal disassembly of the SPFH domain oligomer from Pyrococcus horikoshii.

Stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins are membrane proteins that are widely conserved from bacteria to mammals. The molecular functions of these proteins have not been established. In mammals, the domain is often found in raft-associated proteins such as flotillin and podocin. We determined the structure of the SPFH domain of PH0470 derived from Pyrococcus horikoshii using NMR. The structure closely resembles that of the SPFH domain of the paralog PH1511, except for two C-terminal helices. The results show that the SPFH domain forms stable dimers, trimers, tetramers, and multimers, although it lacks the coiled-coil region for oligomerization, which is a highly conserved region in this protein family. The oligomers exhibited unusual thermodynamic behavior, as determined by circular dichroism, NMR, gel filtration, chemical cross-linking, and analytical ultracentrifugation. The oligomers were converted into monomers when they were heated once and then cooled. This transition was one-way and irreversible. We propose a mechanism of domain swapping for forming dimers as well as successive oligomers. The results of this study provide what to our knowledge are new insights into the common molecular function of the SPFH domain, which may act as a membrane skeleton through oligomerization by domain swapping.

Implication for buried polar contacts and ion pairs in hyperthermostable enzymes.

Understanding the structural basis of thermostability and thermoactivity, and their interdependence, is central to the successful future exploitation of extremophilic enzymes in biotechnology. However, the structural basis of thermostability is still not fully characterized. Ionizable residues play essential roles in proteins, modulating protein stability, folding and function. The dominant roles of the buried polar contacts and ion pairs have been reviewed by distinguishing between the inside polar contacts and the total intramolecular polar contacts, and by evaluating their contribution as molecular determinants for protein stability using various protein structures from hyperthermophiles, thermophiles and mesophilic organisms. The analysis revealed that the remarkably increased number of internal polar contacts in a monomeric structure probably play a central role in enhancing the melting temperature value up to 120 degrees C for hyperthermophilic enzymes from the genus Pyrococcus. These results provide a promising contribution for improving the thermostability of enzymes by modulating buried polar contacts and ion pairs.

Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii.

Archaeal/eukaryotic primases form a heterodimer consisting of a small catalytic subunit (PriS) and a large subunit (PriL). The heterodimer complex synthesizes primer oligoribonucleotides that are required for chromosomal replication. Here, we describe crystallographic and biochemical studies of the N-terminal domain (NTD) of PriL (PriL(NTD); residues 1-222) that bind to PriS from a hyperthermophilic archaeon, Pyrococcus horikoshii, at 2.9 A resolution. The PriL(NTD) structure consists of two subdomains, the helix-bundle and twisted-strand domains. The latter is structurally flexible, and is expected to contain a PriS interaction site. Pull-down and surface plasmon resonance analyses of structure-based deletion and alanine scanning mutants showed that the conserved hydrophobic Tyr155-Tyr156-Ile157 region near the flexible region is the PriS-binding site, as the Y155A/Y156A/I157A mutation markedly reduces PriS binding, by 1000-fold. These findings and a structural comparison with a previously reported PriL(NTD)-PriS complex suggest that the presented alternative conformations of the twisted-strand domain facilitate the heterodimer assembly.

Molecular structure of a novel membrane protease specific for a stomatin homolog from the hyperthermophilic archaeon Pyrococcus horikoshii.

Membrane-bound proteases are involved in various regulatory functions. Our previous study indicated that the N-terminal region of an open reading frame, PH1510 (residues 16-236, designated as 1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii, is a serine protease with a catalytic Ser-Lys dyad that specifically cleaves the C-terminal hydrophobic residues of a membrane protein, the stomatin-homolog PH1511. In humans, an absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis, but the function of stomatin is not fully understood. Here, we report the crystal structure of 1510-N in dimeric form. Each active site of 1510-N is rich in hydrophobic residues, which accounts for the substrate-specificity. The monomer of 1510-N shows structural similarity to one monomer of Escherichia coli ClpP, an ATP-dependent tetradecameric protease. But, their oligomeric forms are different. Major contributors to dimeric interaction in 1510-N are the alpha7 helix and beta9 strand, both of which are missing from ClpP. While the long handle region of ClpP contributes to the stacking of two heptameric rings, the corresponding L2 loop of 1510-N is disordered because the region has little interaction with other residues of the same molecule. The catalytic Ser97 of 1510-N is in almost the same location as the catalytic Ser97 of E.coli ClpP, whereas another residue, Lys138, presumably forming the catalytic dyad, is located in the disordered L2 region of 1510-N. These findings suggest that the binding of the substrate to the catalytic site of 1510-N induces conformational changes in a region that includes loop L2 so that Lys138 approaches the catalytic Ser97.

Differences in substrate specificity of C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides by DNA polymerases from thermophilic bacteria, archaea, and phages.

The pyrimidine bases of RNA are uracil (U) and cytosine (C), while thymine (T) and C are used for DNA. The C(5) position of C and U is unsubstituted, whereas the C(5) of T is substituted with a Me group. Miller et al. hypothesized that various C(5)-substituted uracil derivatives were formed during chemical evolution, and that C(5)-substituted U derivatives may have played important roles in the transition from an 'RNA world' to a 'DNA-RNA-protein world'. Hyperthermophilic bacteria and archaea are considered to be primitive organisms that are evolutionarily close to the universal ancestor of all life on earth. Thus, we examined the substrate specificity of several C(5)-substituted or C(5)-unsubstituted dUTP and dCTP analogs for several DNA polymerases from hyperthermophilic bacteria, hyperthermophilic archaea, and viruses during PCR or primer extension reaction. The substrate specificity of the C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides varied greatly depending on the type of DNA polymerase. The significance of this difference in substrate specificity in terms of the origin and evolution of the DNA replication system is discussed briefly.

A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.

Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain. We show that D363, H365 and H454 are the essential residues, while D407, N453, H500, H563 and H565 are critical residues for the activity. We provide experimental evidence demonstrating that manganese, rather than magnesium, is the preferable metal ion for the nuclease activity of PolD. We also show that DP1 alone is insufficient to perform full catalysis, which additionally requires the formation of the PolD complex and manganese ion. We found that a 21 amino acid, subunit-interacting peptide of the sequence from cysteine cluster II of the large subunit (DP2) stimulates the exonuclease activity of DP1 and the internal deletion mutants of PolD lacking the 21-aa sequence. This indicates that the putative zinc finger motif of the cysteine cluster II is deeply involved in the nucleolytic catalysis.

A hyperthermostable D-ribose-5-phosphate isomerase from Pyrococcus horikoshii characterization and three-dimensional structure.

A gene homologous to D-ribose-5-phosphate isomerase (EC 5.3.1.6) was found in the genome of Pyrococcus horikoshii. D-ribose-5-phosphate isomerase (PRI) is of particular metabolic importance since it catalyzes the interconversion between the ribose and ribulose forms involved in the pentose phosphate cycle and in the process of photosynthesis. The gene consisting of 687 bp was overexpressed in Escherichia coli, and the resulting enzyme showed activity at high temperatures with an optimum over 90 degrees C. The crystal structures of the enzyme, free and in complex with D-4-phosphoerythronic acid inhibitor, were determined. PRI is a tetramer in the crystal and in solution, and each monomer has a new fold consisting of two alpha/beta domains. The 3D structures and the characterization of different mutants indicate a direct or indirect catalytic role for the residues E107, D85, and K98.

X-ray structure of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii.

The beta-glycosidase of the hyperthermophilic Archaeon Pyrococcus horikoshii is a membrane-bound enzyme with the preferred substrate of alkyl-beta-glycosides. In this study, the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme were investigated by X-ray crystallography and docking simulation. The enzyme was crystallized in the presence of a neutral surfactant, and the crystal structure was solved by the molecular replacement method and refined at 2.5 A. The main-chain fold of the enzyme belongs to the (betaalpha)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. We propose that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate.

Crystal structure of the stomatin operon partner protein from Pyrococcus horikoshii indicates the formation of a multimeric assembly.

Stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins are found in the lipid raft microdomains of various cellular membranes. Stomatin/STOPP (stomatin operon partner protein) gene pairs are present in both archaeal and bacterial species, and their protein products may be involved in the quality control of membrane proteins. In the present study, the crystal structure of the C-terminal soluble domain of STOPP PH1510 (1510-C) from the hyperthermophilic archaeon Pyrococcus horikoshii was determined at 2.4 Å resolution. The structure of 1510-C had a compact five-stranded ß-barrel fold known as an oligosaccharide/oligonucleotide-binding fold (OB-fold). According to crystal packing, 1510-C could assemble into multimers based on a dimer as a basic unit. 1510-C also formed a large cylinder-like structure composed of 24 subunits or a large triangular prism-like structure composed of 12 subunits. These results indicate that 1510-C functions as a scaffold protein to form the multimeric assembly of STOPP and stomatin.

Domain structures and inter-domain interactions defining the holoenzyme architecture of archaeal d-family DNA polymerase.

Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information.

Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales.

The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein-protein complexes, which suggested the possibility of self-assembling of STOPP. Protein-protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin.