Affinity shift of ATP upon glycerol binding to a glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Glycerol kinase (GK) is a key enzyme of glycerol metabolism. It participates in glycolysis and lipid membrane biosynthesis. A hexamer of GK from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1(Tk-GK) was identified as a substrate-binding form of the enzyme. Here, the X-ray crystal structure analysis and the biochemical analysis was done and the relationships between its unique oligomer structure and substrate binding affinity were investigated. Wild type GK and mutant K271E GK, which disrupts the hexamer formation interface, were crystallized with and without their substrates and analyzed at 2.19-3.05 Å resolution. In the absence of glycerol, Tk-GK was a dimer in solution. In the presence of its glycerol substrate, however, it became a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer were in closed form as a result of domain-motion. The closed form of Tk-GK had tenfold higher ATP affinity than the open form of Tk-GK. The hexamer structure stabilized the closed conformation and enhanced ATP binding affinity when the GK was bound to glycerol. This molecular mechanism is quite simple activity regulation mechanism among known GKs.

Crystal structure of the Fab region of a neutralizing antibody against granulocyte-macrophage colony-stimulating factor.

An increased level of granulocyte-macrophage colony-stimulating factor has a potential role in the development of autoimmune diseases, and the neutralization of its activity by monoclonal antibodies is a promising therapy for some diseases. Here, the crystal structure of the Fab region of EV1007, a fully human antibody expressed in Chinese hamster ovary cells that was developed from human peripheral blood mononuclear cells, is described. The structure closely resembles that of MB007, which is the Fab region of the same antibody expressed in Escherichia coli [Blech et al. (2012), Biochem. J. 447, 205-215], except at the hinge regions between the immunoglobulin domains and the H3 loop region. This paper presents evidence for the flexibility of the hinge and H3 loop regions of the antibody based on the comparison of two independently solved crystal structures.

Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain.

Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the determination of the structure of the long α-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long α-helical domain has three α-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight into the Lip system is discussed.

Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension.

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC-Est1∼6, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome-derived enzymes, LC-Est1 is characterized by the presence of a long N-terminal extension (LNTE, residues 26-283) between a putative signal peptide (residues 1-25) and a C-terminal esterase domain (residues 284-510). A putative esterase from Candidatus Solibacter usitatus (CSu-Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC-Est1. To examine whether LC-Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC-Est1 (residues 26-510), LC-Est1C (residues 284-510), and LC-Est1C* (residues 304-510) were overproduced in E. coli, purified, and characterized. LC-Est1C* was only used for structural analysis. The crystal structure of LC-Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC-Est1C was lower than that of LC-Est1 by 60%, although its substrate specificity was similar to that of LC-Est1. LC-Est1C was less stable than LC-Est1 by 3.3°C. These results suggest that LNTE of LC-Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.

Structure, activity, and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain.

A metagenome-derived glycoside hydrolase family 9 enzyme with an N-terminal immunoglobulin-like (Ig-like) domain, leaf-branch compost (LC)-CelG, was characterized and its crystal structure was determined. LC-CelG did not hydrolyze p-nitrophenyl cellobioside but hydrolyzed CM-cellulose, indicating that it is endoglucanase. LC-CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5-9. Its denaturation temperature was 81.4°C, indicating that LC-CelG is a thermostable enzyme. The structure of LC-CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29-31%) amino acid sequence identities to LC-CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC-CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC-CelG. Removal of the Ig-like domain reduced the activity and stability of LC-CelG by 100-fold and 6.3°C, respectively. Removal of the Gln40- and Asp99-mediated interactions between the Ig-like and catalytic domains destabilized LC-CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig-like domain contributes to the stabilization of LC-CelG mainly due to the Gln40- and Asp99-mediated interactions. Because the LC-CelG derivative lacking the Ig-like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig-like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain.

Structure and stability of metagenome-derived glycoside hydrolase family 12 cellulase (LC-CelA) a homolog of Cel12A from Rhodothermus marinus.

Ten genes encoding novel cellulases with putative signal peptides at the N-terminus, termed pre-LC-CelA-J, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre-LC-CelG have a 14-29 residue long flexible linker (FL) between the signal peptide and the catalytic domain. LC-CelA without a signal peptide (residues 20-261), which shows 76% amino acid sequence identity to Cel12A from Rhodothermus marinus (RmCel12A), was overproduced in Escherichia coli, purified and characterized. LC-CelA exhibited its highest activity across a broad pH range (pH 5-9) and at 90 °C, indicating that LC-CelA is a highly thermostable cellulase, like RmCel12A. The crystal structure of LC-CelA was determined at 1.85 Å resolution and is nearly identical to that of RmCel12A determined in a form without the FL. Both proteins contain two disulfide bonds. LC-CelA has a 16-residue FL (residues 20-35), most of which is not visible in the electron density map, probably due to structural disorder. However, Glu34 and Pro35 form hydrogen bonds with the central region of the protein. ΔFL-LC-CelA (residues 36-261) and E34A-LC-CelA with a single Glu34 â†’ Ala mutation were therefore constructed and characterized. ΔFL-LC-CelA and E34A-LC-CelA had lower melting temperatures (T m) than LC-CelA by 14.7 and 12.0 °C respectively. The T m of LC-CelA was also decreased by 28.0 °C in the presence of dithiothreitol. These results suggest that Glu34-mediated hydrogen bonds and the two disulfide bonds contribute to the stabilization of LC-CelA.

Rational design of a glycosynthase by the crystal structure of ß-galactosidase from Bacillus circulans (BgaC) and its use for the synthesis of N-acetyllactosamine type 1 glycan structures.

The crystal structure of ß-galactosidase from Bacillus circulans (BgaC) was determined at 1.8Å resolution. The overall structure of BgaC consists of three distinct domains, which are the catalytic domain with a TIM-barrel structure and two all-ß domains (ABDs). The main-chain fold and steric configurations of the acidic and aromatic residues at the active site were very similar to those of Streptococcus pneumoniae ß(1,3)-galactosidase BgaC in complex with galactose. The structure of BgaC was used for the rational design of a glycosynthase. BgaC belongs to the glycoside hydrolase family 35. The essential nucleophilic amino acid residue has been identified as glutamic acid at position 233 by site-directed mutagenesis. Construction of the active site mutant BgaC-Glu233Gly gave rise to a galactosynthase transferring the sugar moiety from α-d-galactopyranosyl fluoride (αGalF) to different ß-linked N-acetylglucosamine acceptor substrates in good yield (40-90%) with a remarkably stable product formation. Enzymatic syntheses with BgaC-Glu233Gly afforded the stereo- and regioselective synthesis of ß1-3-linked key galactosides like galacto-N-biose or lacto-N-biose.

Structural basis for salt-dependent folding of ribonuclease H1 from halophilic archaeon Halobacterium sp. NRC-1.

RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNase H1) requires ⩾2M NaCl, ⩾10mM MnCl2, or ⩾300mM MgCl2 for folding. To understand the structural basis for this salt-dependent folding of Halo-RNase H1, the crystal structure of Halo-RNase H1 was determined in the presence of 10mM MnCl2. The structure of Halo-RNase H1 highly resembles those of metagenome-derived LC11-RNase H1 and Sulfolobus tokodaii RNase H1 (Sto-RNase H1), except that it contains two Mn(2+) ions at the active site and has three bi-aspartate sites on its surface. To examine whether negative charge repulsion at these sites are responsible for low-salt denaturation of Halo-RNase H1, a series of the mutant proteins of Halo-RNase H1 at these sites were constructed. The far-UV CD spectra of these mutant proteins measured in the presence of various concentrations of NaCl suggest that these mutant proteins exist in an equilibrium between a partially folded state and a folded state. However, the fraction of the protein in a folded state is nearly 0% for the active site mutant, 40% for the bi-aspartate site mutant, and 70% for the mutant at both sites in the absence of salt. The active site mutant requires relatively low concentration (∼0.5M) of salt for folding. These results suggest that suppression of negative charge repulsion at both active and bi-aspartate sites by salt is necessary to yield a folded protein.

Structural and mechanistic insights into the kynurenine aminotransferase-mediated excretion of kynurenic acid.

Kynurenine aminotransferase (KAT) is a homodimeric pyridoxal protein that mediates the catalytic conversion of kynurenine (KYN) to kynurenic acid (KYA), an endogenous N-methyl-d-aspartate (NMDA) receptor antagonist. KAT is involved in the biosynthesis of glutamic and aspartic acid, functions as a neurotransmitter for the NMDA receptor in mammals, and is regulated by allosteric mechanisms. Its importance in various diseases such as schizophrenia makes KAT a highly attractive drug target. Here, we present the crystal structure of the Pyrococcus horikoshii KAT (PhKAT) in complex with pyridoxamine phosphates (PMP), KYN, and KYA. Surprisingly, the PMP was bound to the LYS-269 of phKAT by forming a covalent hydrazine bond. This crystal structure clearly shows that an amino group of KYN was transaminated to PLP, which forms a Schiff's base with the LYS-269 of the KYN. Thus, our structure confirms that the PMPs represent an intermediate state during the KAT reaction. Thus, PhKAT catalyzes the sequential conversion of KYN to KYA via the formation of an intermediate 4-(2-aminophenyl)-2,4-dioxobutanoate (4AD), which is spontaneously converted to KYA in the absence of an amino group acceptor. Furthermore, we identified the two entry and exit sites of the PhKAT homodimer for KYN and KYA, respectively. The structural data on PhKAT presented in this manuscript contributes to further the understanding of transaminase enzyme reaction mechanisms.

Formation of the high-affinity calcium binding site in pro-subtilisin E with the insertion sequence IS1 of pro-Tk-subtilisin.

Subtilisin E is activated from its inactive precursor Pro-subtilisin E by autoprocessing and degradation of the propeptide. Subtilisin E has two calcium binding sites, the high-affinity Ca1 site and the low-affinity Ca2 site. The Ca1 site is conserved in various subtilisin-like proteases and is important for stability. This site is not formed in Pro-subtilisin E, because the structural rearrangement of the N-terminal region of the subtilisin domain upon autoprocessing is necessary for the formation of this site. As a result, Pro-subtilisin E is not fully folded. In contrast, Pro-Tk-subtilisin from Thermococcus kodakarensis is fully folded, because it does not require the structural rearrangement upon autoprocessing for the formation of the Ca1 site due to the presence of the insertion sequence IS1 between the propeptide and subtilisin domains. To examine whether the Ca1 site is formed in Pro-subtilisin E by inserting IS1 between the propeptide and subtilisin domains, the Pro-subtilisin E mutant with this insertion, IS1-Pro-subtilisin E, and its active site mutants, IS1-Pro-S221A and IS1-Pro-S221C, were constructed and characterized. The crystal structure of IS1-Pro-S221A revealed that this protein is fully folded and the Ca1 site is formed. In this structure, IS1 serves as a linker that brings the N-terminus of the subtilisin domain near the Ca1 site. IS1-Pro-S221A in a calcium-bound form was more stable than that in a calcium-free form by 13.1 °C. IS1-Pro-S221C was more rapidly autoprocessed than Pro-S221C. These results suggest that IS1 facilitates the formation of the Ca1 site and the complete folding of Pro-subtilisin E and thereby accelerates its autoprocessing.

Role of N-terminal extension of Bacillus stearothermophilus RNase H2 and C-terminal extension of Thermotoga maritima RNase H2.

Bacillus stearothermophilus RNase H2 (BstRNH2) and Thermotoga maritima RNase H2 (TmaRNH2) have N-terminal and C-terminal extensions, respectively, as compared with Aquifex aeolicus RNase H2 (AaeRNH2). To analyze the role of these extensions, BstRNH2 and TmaRNH2 without these extensions were constructed, and their biochemical properties were compared with those of their intact partners and AaeRNH2. The far-UV CD spectra of all proteins were similar, suggesting that the protein structure is not significantly altered by removal of these extensions. However, both the junction ribonuclease and RNase H activities of BstRNH2 and TmaRNH2, as well as their substrate-binding affinities, were considerably decreased by removal of these extensions. The stability of BstRNH2 and TmaRNH2 was also decreased by removal of these extensions. The activity, substrate binding affinity and stability of TmaRNH2 without the C-terminal 46 residues were partly restored by the attachment of the N-terminal extension of BstRNH2. These results suggest that the N-terminal extension of BstRNH2 functions as a substrate-binding domain and stabilizes the RNase H domain. Because the C-terminal extension of TmaRNH2 assumes a helix hairpin structure and does not make direct contact with the substrate, this extension is probably required to make the conformation of the substrate-binding site functional. AaeRNH2 showed comparable junction ribonuclease activity to those of BstRNH2 and TmaRNH2, and was more stable than these proteins, indicating that bacterial RNases H2 do not always require an N-terminal or C-terminal extension to increase activity, substrate-binding affinity, and/or stability.

Crystallization and X-ray structure determination of a thermoalkalophilic lipase from Geobacillus SBS-4S.

A thermoalkalophilic lipase (LIPSBS) from the newly isolated Geobacillus strain SBS-4S which hydrolyzes a wide range of fatty acids has been characterized. In the present study, the crystallization of purified LIPSBS using the sitting-drop vapour-diffusion method and its X-ray diffraction studies are described. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 55.13, b = 71.75, c = 126.26 Å. The structure was determined at 1.6 Å resolution by the molecular-replacement method using the lipase from G. stearothermophilus L1 as a model.

Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures.

A family I.3 lipase from Pseudomonas sp. MIS38 (PML) is characterized by the presence of two lids (lid1 and lid2) that greatly change conformation upon substrate binding. While lid1 represents the commonly known lid in lipases, lid2 is unique to PML and other family I.3 lipases. To clarify the role of lid2 in PML, a lid2 deletion mutant (ΔL2-PML) was constructed by deleting residues 35-64 of PML. ΔL2-PML requires calcium ions for both lipase and esterase activities as does PML, suggesting that it exhibits activity only when lid1 is fully open and anchored by the catalytically essential calcium ion, as does PML. However, when the enzymatic activity was determined using triacetin, the activity of PML exponentially increased as the substrate concentration reached and increased beyond the critical micellar concentration, while that of ΔL2-PML did not. These results indicate that PML undergoes interfacial activation, while ΔL2-PML does not. The activities of ΔL2-PML for long-chain triglycerides significantly decreased while its activity for fatty acid ethyl esters increased, compared with those of PML. Comparison of the tertiary models of ΔL2-PML in a closed and open conformation, which are optimized by molecular dynamics simulation, with the crystal structures of PML suggests that the hydrophobic surface area provided by lid1 and lid2 in an open conformation is considerably decreased by the deletion of lid2. We propose that the hydrophobic surface area provided by these lids is necessary to hold the micellar substrates firmly to the active site and therefore lid2 is required for interfacial activation of PML.

Structure and stability of a thermostable carboxylesterase from the thermoacidophilic archaeon Sulfolobus tokodaii.

The hormone-sensitive lipase (HSL) family is comprised of carboxylesterases and lipases with similarity to mammalian HSL. Thermophilic enzymes of this family have a high potential for use in biocatalysis. We prepared and crystallized a carboxylesterase of the HSL family from Sulfolobus tokodaii (Sto-Est), and determined its structures in the presence and absence of an inhibitor. Sto-Est forms a dimer in solution and the crystal structure suggests the presence of a stable biological dimer. We identified a residue close to the dimer interface, R267, which is conserved in archaeal enzymes of HSL family and is in close proximity to the same residue from the other monomer. Mutations of R267 to Glu, Gly and Lys were conducted and the resultant R267 mutants were characterized and crystallized. The structures of R267E, R267G and R267K are highly similar to that of Sto-Est with only slight differences in atomic coordinates. The dimerized states of R267E and R267G are unstable under denaturing conditions or at high temperature, as shown by a urea-induced dimer dissociation experiment and molecular dynamics simulation. R267E is the most unstable mutant protein, followed by R267G and R267K, as shown by the thermal denaturation curve and optimum temperature for activity. From the data, we discuss the importance of R267 in maintaining the dimer integrity of Sto-Est.

Characteristic features of kynurenine aminotransferase allosterically regulated by (alpha)-ketoglutarate in cooperation with kynurenine.

Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5'-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.

Structure and characterization of RNase H3 from Aquifex aeolicus.

The crystal structure of ribonuclease H3 from Aquifex aeolicus (Aae-RNase H3) was determined at 2.0 Å resolution. Aae-RNase H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase H domain (C-domain). The structure of the C-domain highly resembles that of Bacillus stearothermophilus RNase H3 (Bst-RNase H3), except that it contains three disulfide bonds, and the fourth conserved glutamate residue of the Asp-Glu-Asp-Glu active site motif (Glu198) is located far from the active site. These disulfide bonds were shown to contribute to hyper-stabilization of the protein. Non-conserved Glu194 was identified as the fourth active site residue. The structure of the N-domain without the C-domain also highly resembles that of Bst-RNase H3. However, the arrangement of the N-domain relative to the C-domain greatly varies for these proteins because of the difference in the linker size between the domains. The linker of Bst-RNase H3 is relatively long and flexible, while that of Aae-RNase H3 is short and assumes a helix formation. Biochemical characterizations of Aae-RNase H3 and its derivatives without the N- or C-domain or with a mutation in the N-domain indicate that the N-domain of Aae-RNase H3 is important for substrate binding, and uses the flat surface of the ß-sheet for substrate binding. However, this surface is located far from the active site and on the opposite side to the active site. We propose that the N-domain of Aae-RNase H3 is required for initial contact with the substrate. The resulting complex may be rearranged such that only the C-domain forms a complex with the substrate.

Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1.

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca(2+). The Ca(2+)-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca(2+) binding loop region that modulate the Ca(2+)-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca(2+)-binding loop region of C2B domain. The results indicate that Ca(2+)-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca(2+)-bound Synaptotagmin-1 associated synaptic vesicles.

Activity, stability, and structure of metagenome-derived LC11-RNase H1, a homolog of Sulfolobus tokodaii RNase H1.

Metagenome-derived LC11-RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto-RNase H1). It lacks a C-terminal tail, which is responsible for hyperstabilization of Sto-RNase H1. Sto-RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double-stranded RNA (dsRNA). To examine whether LC11-RNase H1 also exhibits both RNase H and dsRNase activities, LC11-RNase H1 was overproduced in Escherichia coli, purified, and characterized. LC11-RNase H1 exhibited RNase H activity with similar metal ion preference, optimum pH, and cleavage mode of substrate with those of Sto-RNase H1. However, LC11-RNase H1 did not exhibit dsRNase activity at any condition examined. LC11-RNase H1 was less stable than Sto-RNases H1 and its derivative lacking the C-terminal tail (Sto-RNase H1ΔC6) by 37 and 13 °C in T(m) , respectively. To understand the structural bases for these differences, the crystal structure of LC11-RNase H1 was determined at 1.4 Å resolution. The LC11-RNase H1 structure is highly similar to the Sto-RNase H1 structure. However, LC11-RNase H1 has two grooves on protein surface, one containing the active site and the other containing DNA-phosphate binding pocket, while Sto-RNase H1 has one groove containing the active site. In addition, LC11-RNase H1 contains more cavities and buried charged residues than Sto-RNase H1. We propose that LC11-RNase H1 does not exhibit dsRNase activity because dsRNA cannot fit to the two grooves on protein surface and that LC11-RNase H1 is less stable than Sto-RNase H1ΔC6 because of the increase in cavity volume and number of buried charged residues.

Role of polar and nonpolar residues at the active site for PPIase activity of FKBP22 from Shewanella sp. SIB1.

FKBP22 from the psychotropic bacterium Shewanella sp. SIB1 is a homodimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. According to a tertiary model, several nonpolar residues including Trp157 and Phe197 form a substrate-binding cavity, and Asp137 and Arg142, which form a salt bridge, are located at the edge of this cavity. To analyze the role of these residues, nine single (D137A, R142A, W157A/F/Y, F197A/L/Y/W) and one double (D137A/R142A) mutant protein of SIB1 FKBP22 were constructed. The far- and near-UV CD spectra of these mutant proteins suggest that the mutations at Asp137 and Arg142 do not seriously affect the protein structure, while those at Trp157 and Phe197 cause a local conformational change around the mutation site. Each mutation decreased the PPIase activities of SIB1 FKBP22 for peptide and protein substrates similarly without seriously affecting chaperone function. This result indicates that SIB1 FKBP22 does not require PPIase activity for chaperone function. The PPIase activities of R142A, D137A and D137A/R142A decreased in this order, suggesting that Asp137 and Arg142 play a principal and auxiliary role in catalytic function, respectively, but Arg142 can function as a substitute of Asp137. Because the PPIase activity of SIB1 FKBP22 was not fully lost by the removal of all polar residues around the active site, the desolvation effect may also contribute to the enzymatic activity. However, the mutations of Trp157 to Phe or Phe197 to Leu greatly decrease the enzymatic activity, suggesting that the shape of the substrate-binding cavity is also important for enzymatic activity.

Crystal structure of N-domain of FKBP22 from Shewanella sp. SIB1: dimer dissociation by disruption of Val-Leu knot.

FK506-binding protein 22 (FKBP22) from the psychrotophic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) is a homodimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. Each monomer consists of the N-terminal domain responsible for dimerization and C-terminal catalytic domain. To reveal interactions at the dimer interface of SIB1 FKBP22, the crystal structure of the N-domain of SIB1 FKBP22 (SN-FKBP22, residues 1-68) was determined at 1.9 Å resolution. SN-FKBP22 forms a dimer, in which each monomer consists of three helices (α1, α2, and α3N). In the dimer, two monomers have head-to-head interactions, in which residues 8-64 of one monomer form tight interface with the corresponding residues of the other. The interface is featured by the presence of a Val-Leu knot, in which Val37 and Leu41 of one monomer interact with Val41 and Leu37 of the other, respectively. To examine whether SIB1 FKBP22 is dissociated into the monomers by disruption of this knot, the mutant protein V37R/L41R-FKBP22, in which Val37 and Leu41 of SIB1 FKBP22 are simultaneously replaced by Arg, was constructed and biochemically characterized. This mutant protein was indistinguishable from the SIB1 FKBP22 derivative lacking the N-domain in oligomeric state, far-UV CD spectrum, thermal denaturation curve, PPIase activity, and binding ability to a folding intermediate of protein, suggesting that the N-domain of V37R/L41R-FKBP22 is disordered. We propose that a Val-Leu knot at the dimer interface of SIB1 FKBP22 is important for dimerization and dimerization is required for folding of the N-domain.