Periplasmic chitooligosaccharide-binding protein requires a three-domain organization for substrate translocation.

Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc)n (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and the negative control of chitin signal transduction in Vibrios. Most translocation processes by SBPs across the inner membrane have been explained thus far by two-domain open/closed mechanism. Here we propose three-domain mechanism of the (GlcNAc)n translocation based on experiments using a recombinant VcCBP, SBP specific for (GlcNAc)n from Vibrio cholerae. X-ray crystal structures of unliganded or (GlcNAc)3-liganded VcCBP solved at 1.2-1.6 Å revealed three distinct domains, the Upper1, Upper2 and Lower domains for this protein. Molecular dynamics simulation indicated that the motions of the three domains are independent and that in the (GlcNAc)3-liganded state the Upper2/Lower interface fluctuated more intensively, compared to the Upper1/Lower interface. The Upper1/Lower interface bound two GlcNAc residues tightly, while the Upper2/Lower interface appeared to loosen and release the bound sugar molecule. The three-domain mechanism proposed here was fully supported by binding data obtained by thermal unfolding experiments and ITC, and may be applicable to other translocation systems involving SBPs belonging to the same cluster.

Protein expression and purification, molecular interaction, and X-ray crystallographic analysis of baculovirus protein PK2.

Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by eIF2α kinases is a common mechanism to regulate the initiation of translation under stress conditions. The PK2 protein from baculovirus Autographica californica multiple nucleopolyhedrovirus (AcMNPV) binds and inhibits eIF2α kinases to ensure efficient virus propagation. The C-terminal region of PK2 shares a homology with the C-lobe of eIF2α kinases, but the N-terminal region of PK2 is unique to the orthologous proteins. In order to understand the detailed structure and function of PK2, both the full-length PK2 and its N-terminal truncated protein (PK2Δ22) were expressed as a His-tag fusion protein in Escherichia coli and purified by three steps of chromatography. Notably, the cysteine mutant, PK2 C181S/C211S, promotes the solubility and stability of the PK2 protein. The results of the size-exclusion chromatography showed that the full-length PK2 exists in both multimeric and monomeric forms, and the molecular interaction of PK2 and the eIF2α kinase domain. The purified proteins were used further to screen various conditions to obtain these crystals. Crystals of the full-length PK2 and PK2Δ22 were obtained by a sitting-drop vapour-diffusion method using lithium sulfate and PEG3350 as the precipitant, respectively. The crystal of PK2 belonged to space group P41212, and diffracted X-rays to 2.7 Å resolution. The asymmetric unit contained four molecules of the protein, and the solvent content was 67.4%. Whereas, the crystal of the PK2Δ22 belonged to space group P212121, diffracted X-rays to 2.8 Å resolution. The asymmetric unit contained three molecules of the protein, and the solvent content was 48.1%. The crystallographic study of the PK2 protein will provide mechanistic insights into the inhibition of eIF2α kinase by the PK2 protein, and also pave the way for the improvement of the baculovirus-based protein expression system.

Crystal Structures of Csm2 and Csm3 in the Type III-A CRISPR-Cas Effector Complex.

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (Cas) genes encode CRISPR RNAs (crRNA) and Cas proteins, respectively, which play important roles in the adaptive immunity system (CRISPR-Cas system) in prokaryotes. The crRNA and Cas proteins form ribonucleoprotein effector complexes to capture and degrade invading genetic materials with base complementarity to the crRNA guide sequences. The Csm complex, a type III-A effector complex, comprises five Cas proteins (Csm1-Csm5) and a crRNA, which co-transcriptionally degrades invading DNA and RNA. Here we report the crystal structures of the Staphylococcus epidermidis Csm2 (SeCsm2) and Thermoplasma volcanium Csm3 (TvCsm3) at 2.4- and 2.7-Å resolutions, respectively. SeCsm2 adopts a monomeric globular fold by itself, in striking contrast to the previously reported Thermotoga maritima Csm2, which adopted an extended conformation and formed a dimeric structure. We propose that the globular monomeric form is the bona fide structure of Csm2. TvCsm3 forms a filamentous structure in the crystals. The molecular arrangement of TvCsm3 is similar to that of the stacked Cmr4 proteins in the Cmr complex, suggesting the functionally relevant architecture of the present Csm3 structure. We constructed model structures of the Csm complex, which revealed that Csm3 binds the crRNA and periodically deforms the crRNA-target duplex by a similar mechanism to that of Cmr4 in the Cmr complex. The model and mutational analysis suggest that the conserved lysine residue of Csm2 is important for target RNA binding, and Csm2 stabilizes the active structure of the Csm complex to facilitate the reaction.

Molecular insights into replication initiation by Qß replicase using ribosomal protein S1.

Ribosomal protein S1, consisting of six contiguous OB-folds, is the largest ribosomal protein and is essential for translation initiation in Escherichia coli. S1 is also one of the three essential host-derived subunits of Qß replicase, together with EF-Tu and EF-Ts, for Qß RNA replication in E. coli. We analyzed the crystal structure of Qß replicase, consisting of the virus-encoded RNA-dependent RNA polymerase (ß-subunit), EF-Tu, EF-Ts and the N-terminal half of S1, which is capable of initiating Qß RNA replication. Structural and biochemical studies revealed that the two N-terminal OB-folds of S1 anchor S1 onto the ß-subunit, and the third OB-fold is mobile and protrudes beyond the surface of the ß-subunit. The third OB-fold mainly interacts with a specific RNA fragment derived from the internal region of Qß RNA, and its RNA-binding ability is required for replication initiation of Qß RNA. Thus, the third mobile OB-fold of S1, which is spatially anchored near the surface of the ß-subunit, primarily recruits the Qß RNA toward the ß-subunit, leading to the specific and efficient replication initiation of Qß RNA, and S1 functions as a replication initiation factor, beyond its established function in protein synthesis.

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR.

(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

Structural basis of stereospecific reduction by quinuclidinone reductase.

Chiral molecule (R)-3-quinuclidinol, a valuable compound for the production of various pharmaceuticals, is efficiently synthesized from 3-quinuclidinone by using NADPH-dependent 3-quinuclidinone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of RrQR and the structure-based mutational analysis. The enzyme forms a tetramer, in which the core of each protomer exhibits the α/ß Rossmann fold and contains one molecule of NADPH, whereas the characteristic substructures of a small lobe and a variable loop are localized around the substrate-binding site. Modeling and mutation analyses of the catalytic site indicated that the hydrophobicity of two residues, I167 and F212, determines the substrate-binding orientation as well as the substrate-binding affinity. Our results revealed that the characteristic substrate-binding pocket composed of hydrophobic amino acid residues ensures substrate docking for the stereospecific reaction of RrQR in spite of its loose interaction with the substrate.

Translocation and rotation of tRNA during template-independent RNA polymerization by tRNA nucleotidyltransferase.

The 3'-terminal CCA (CCA-3' at positions 74-76) of tRNA is synthesized by CCA-adding enzyme using CTP and ATP as substrates, without a nucleic acid template. In Aquifex aeolicus, CC-adding and A-adding enzymes collaboratively synthesize the CCA-3'. The mechanism of CCA-3' synthesis by these two enzymes remained obscure. We now present crystal structures representing CC addition onto tRNA by A. aeolicus CC-adding enzyme. After C74 addition in an enclosed active pocket and pyrophosphate release, the tRNA translocates and rotates relative to the enzyme, and C75 addition occurs in the same active pocket as C74 addition. At both the C74-adding and C75-adding stages, CTP is selected by Watson-Crick-like hydrogen bonds between the cytosine of CTP and conserved Asp and Arg residues in the pocket. After C74C75 addition and pyrophosphate release, the tRNA translocates further and drops off the enzyme, and the CC-adding enzyme terminates RNA polymerization.

Structure of the Ca(2+)-saturated C-terminal domain of scallop troponin C in complex with a troponin I fragment.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin that triggers the contraction of striated muscles. In scallops, the striated muscles consume little ATP energy in sustaining strong contractile forces. The N-terminal domain of TnC works as the Ca(2+) sensor in vertebrates, whereas scallop TnC uses the C-terminal domain as the Ca(2+) sensor, suggesting that there are differences in the mechanism of the Ca(2+)-dependent regulation of muscles between invertebrates and vertebrates. Here, we report the crystal structure of Akazara scallop (Chlamys nipponensis akazara) adductor muscle TnC C-terminal domain (asTnCC) complexed with a short troponin I fragment (asTnIS) and Ca(2+). The electron density of a Ca(2+) ion is observed in only one of the two EF-hands. The EF-hands of asTnCC can only be in the fully open conformation with the assistance of asTnIS. The number of hydrogen bonds between asTnCC and asTnIS is markedly lower than the number in the vertebrate counterparts. The Ca(2+) modulation on the binding between asTnCC and asTnIS is weaker, but structural change of the complex depending on Ca(2+) concentration was observed. Together, these findings provide a detailed description of the distinct molecular mechanism of contractile regulation in the scallop adductor muscle from that of vertebrates.

Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens.

(R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Šresolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2(1), with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, ß = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V(M) = 2.08 Å(3) Da(-1)).

Mechanism for template-independent terminal adenylation activity of Qß replicase.

The genomic RNA of Qß virus is replicated by Qß replicase, a template-dependent RNA polymerase complex. Qß replicase has an intrinsic template-independent RNA 3'-adenylation activity, which is required for efficient viral RNA amplification in the host cells. However, the mechanism of the template-independent 3'-adenylation of RNAs by Qß replicase has remained elusive. We determined the structure of a complex that includes Qß replicase, a template RNA, a growing RNA complementary to the template RNA, and ATP. The structure represents the terminal stage of RNA polymerization and reveals that the shape and size of the nucleotide-binding pocket becomes available for ATP accommodation after the 3'-penultimate template-dependent C-addition. The stacking interaction between the ATP and the neighboring Watson-Crick base pair, between the 5'-G in the template and the 3'-C in the growing RNA, contributes to the nucleotide specificity. Thus, the template for the template-independent 3'-adenylation by Qß replicase is the RNA and protein ribonucleoprotein complex.

Molecular basis for RNA polymerization by Qß replicase.

Core Qß replicase comprises the Qß virus-encoded RNA-dependent RNA polymerase (ß-subunit) and the host Escherichia coli translational elongation factors EF-Tu and EF-Ts. The functions of the host proteins in the viral replicase are not clear. Structural analyses of RNA polymerization by core Qß replicase reveal that at the initiation stage, the 3'-adenine of the template RNA provides a stable platform for de novo initiation. EF-Tu in Qß replicase forms a template exit channel with the ß-subunit. At the elongation stages, the C-terminal region of the ß-subunit, assisted by EF-Tu, splits the temporarily double-stranded RNA between the template and nascent RNAs before translocation of the single-stranded template RNA into the exit channel. Therefore, EF-Tu in Qß replicase modulates RNA elongation processes in a distinct manner from its established function in protein synthesis.

Mechanism for the alteration of the substrate specificities of template-independent RNA polymerases.

PolyA polymerase (PAP) adds a polyA tail onto the 3'-end of RNAs without a nucleic acid template, using adenosine-5'-triphosphate (ATP) as a substrate. The mechanism for the substrate selection by eubacterial PAP remains obscure. Structural and biochemical studies of Escherichia coli PAP (EcPAP) revealed that the shape and size of the nucleobase-interacting pocket of EcPAP are maintained by an intra-molecular hydrogen-network, making it suitable for the accommodation of only ATP, using a single amino acid, Arg(197). The pocket structure is sustained by interactions between the catalytic domain and the RNA-binding domain. EcPAP has a flexible basic C-terminal region that contributes to optimal RNA translocation for processive adenosine 5'-monophosphate (AMP) incorporations onto the 3'-end of RNAs. A comparison of the EcPAP structure with those of other template-independent RNA polymerases suggests that structural changes of domain(s) outside the conserved catalytic core domain altered the substrate specificities of the template-independent RNA polymerases.

Assembly of Q{beta} viral RNA polymerase with host translational elongation factors EF-Tu and -Ts.

Replication and transcription of viral RNA genomes rely on host-donated proteins. Qbeta virus infects Escherichia coli and replicates and transcribes its own genomic RNA by Qbeta replicase. Qbeta replicase requires the virus-encoded RNA-dependent RNA polymerase (beta-subunit), and the host-donated translational elongation factors EF-Tu and -Ts, as active core subunits for its RNA polymerization activity. Here, we present the crystal structure of the core Qbeta replicase, comprising the beta-subunit, EF-Tu and -Ts. The beta-subunit has a right-handed structure, and the EF-Tu:Ts binary complex maintains the structure of the catalytic core crevasse of the beta-subunit through hydrophobic interactions, between the finger and thumb domains of the beta-subunit and domain-2 of EF-Tu and the coiled-coil motif of EF-Ts, respectively. These hydrophobic interactions are required for the expression and assembly of the Qbeta replicase complex. Thus, EF-Tu and -Ts have chaperone-like functions in the maintenance of the structure of the active Qbeta replicase. Modeling of the template RNA and the growing RNA in the catalytic site of the Qbeta replicase structure also suggests that structural changes of the RNAs and EF-Tu:Ts should accompany processive RNA polymerization and that EF-Tu:Ts in the Qbeta replicase could function to modulate the RNA folding and structure.

Mechanism for the definition of elongation and termination by the class II CCA-adding enzyme.

The CCA-adding enzyme synthesizes the CCA sequence at the 3' end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA-adding enzyme and its complexes with CTP or ATP were determined. The structure-based replacement of both the catalytic heads and nucleobase-interacting neck domains of the phylogenetically closely related Aquifex aeolicus A-adding enzyme by the corresponding domains of the T. maritima CCA-adding enzyme allowed the A-adding enzyme to add CCA in vivo and in vitro. However, the replacement of only the catalytic head domain did not allow the A-adding enzyme to add CCA, and the enzyme exhibited (A, C)-adding activity. We identified the region in the neck domain that prevents (A, C)-adding activity and defines the number of nucleotide incorporations and the specificity for correct CCA addition. We also identified the region in the head domain that defines the terminal A addition after CC addition. The results collectively suggest that, in the class II CCA-adding enzyme, the head and neck domains collaboratively and dynamically define the number of nucleotide additions and the specificity of nucleotide selection.

Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra.

(R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 91.3, c = 265.4 A, and diffracted X-rays to 2.2 A resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%.

Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme.

CCA-adding enzyme builds the 3'-end CCA of tRNA without a nucleic acid template. The mechanism for the maintenance of fidelity during the CCA-adding reaction remains elusive. Here, we present almost a dozen complex structures of the class I CCA-adding enzyme and tRNA mini-helices (mini-D(73)N(74), mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75); D(73) is a discriminator nucleotide and N is either A, G, or U). The mini-D(73)N(74) complexes adopt catalytically inactive open forms, and CTP shifts the enzymes to the active closed forms and allows N(74) to flip for CMP incorporation. In contrast, unlike the catalytically active closed form of the mini-D(73)C(74)C(75) complex, the mini-D(73)N(74)C(75) and mini-D(73)C(74)N(75) complexes adopt inactive open forms. Only the mini-D(73)C(74)U(75) accepts AMP to a similar extent as mini-D(73)C(74)C(75), and ATP shifts the enzyme to a closed, active form and allows U(75) to flip for AMP incorporation. These findings suggest that the 3'-region of RNA is proofread, after two nucleotide additions, in the closed, active form of the complex at the AMP incorporation stage. This proofreading is a prerequisite for the maintenance of fidelity for complete CCA synthesis.

Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer.

Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.0 A resolution. The structure revealed that the RNase IIIb domain can form a tightly associated homodimer, which is similar to the dimers of the bacterial RNase III domains and the two RNase III domains of Giardia Dicer. Biochemical analysis showed that the RNase IIIb homodimer can cleave double-stranded RNAs (dsRNAs), and generate short dsRNAs with 2 nt 3' overhang, which is characteristic of RNase III products. The RNase IIIb domain contained two magnesium ions per monomer around the active site. The distance between two Mg-1 ions is approximately 20.6 A, almost identical with those observed in bacterial RNase III enzymes and Giardia Dicer, while the locations of two Mg-2 ions were not conserved at all. We presume that Mg-1 ions act as catalysts for dsRNA cleavage, while Mg-2 ions are involved in RNA binding.

Crystal structure of the PIN domain of human telomerase-associated protein EST1A.

Saccharomyces cerevisiae Est1p is a telomerase-associated protein essential for telomere length homeostasis. hEST1A is one of the three human Est1p homologues and is considered to be involved not only in regulation of telomere elongation or capping but also in nonsense-mediated degradation of RNA. hEST1A is composed of two conserved regions, Est1p homology and PIN (PilT N-terminus) domains. The present study shows the crystal structure of the PIN domain at 1.8 A resolution. The overall structure is composed of an alpha/beta fold or a core structure similar to the counterpart of 5' nucleases and an extended structure absent from archaeal PIN-domain proteins and 5' nucleases. The structural properties of the PIN domain indicate its putative active center consisting of invariant acidic amino acid residues, which is geometrically similar to the active center of 5' nucleases and an archaeal PAE2754 PIN-domain protein associated with exonuclease activity.

Crystallization and preliminary X-ray analysis of the PIN domain of human EST1A.

Human EST1A (ever shorter telomeres 1A) is associated with most or all active telomerase in cell extracts and is involved either directly or indirectly in telomere elongation and telomere capping. The C-terminal region of EST1A contains the PIN (PilT N-terminus) domain, a putative nuclease domain. The PIN domain of human EST1A was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 107.3, b = 51.6, c = 100.5 angstroms, beta = 119.3 degrees, and diffracted X-rays to 1.8 angstroms resolution. The asymmetric unit contained two molecules of the PIN domain and the solvent content was 57%.