Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter sphaeroides Ago (RsAgo) with guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for guide release and Ago recycling.

The conventional SEM sample preparation needs multiple steps including fixing,rinsing,and dehydrating.Because the volume of microbiology sample or unicellular sample is so small,so each step of preparation needs centrifugal collection.However,multiple centrifuging may cause loss of the sample.In this study,filter paper pack is used to collect sample.This method significantly improves the efficacy of the sample preparation and enhances the observing effect of the sample.

To capture a state of the enzyme in complex with an intact substrate, we developed and adopted a novel freezing method in crystal preparation procedure. Neither the elimination of the catalytically indispensable ligands, nor mutation or modification of the active site is required. At -20 degrees C, we soaked the crystal of 6-phosphate-beta-glucosidase (Bg1A) in the liquor containing p-nitrophenyl-beta-D-glucopyranoside-6-phosphate (pNPbetaG6P). The diffraction data at 2 A resolution was collected and an intact and unambiguous electron density map of pNPbetaG6P was obtained. These results provide an effective method for the research of cryoenzymology and the intermediate state of enzyme-substrate complex in the future.

During the step of rinsing biological samples for SEM observation,the PBS buffer is usually used and it is easily crystallized on the surface of samples so as to cover the structure.For microbiology with the characteristics of the cell wall,We use the deionized water instead of the PBS buffer to rinse the fixative so that not only the sample shape is without distortion but also the surface of the sample is clean which is better for observation.

Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms, and therefore enables these organisms to survive at extreme temperatures. Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms, the structural basis of protein thermostability is not yet fully understood. The histidine-containing phosphocarrier (HPr) protein, a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species, is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors. In this study, the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified. Crystal structure with good quality has been determined at 2.3 Å resolution, which provides a firm foundation for exploring the thermostable mechanism. However, it shows that the crystal structure is conserved and no clue can be obtained from this single structure. Furthermore, detailed comparison of sequence and structure with the homologs from meso- or thermophilic bacteria shows no obvious rule for thermostability, but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr. Thus, mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria. Using site-directed mutations and the circular dichroism method, thermostability is evaluated, and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses, indicating that mutations cause instability in the HPrs. Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability, and is critical in engineering efficient enzymes that can work at a high temperature.

Prp20p is the homolog of mammalian RCC1 (regulator of chromosome condensation 1) in Saccharomyces cerevisiae, which acts as the guanine nucleotide exchange factor (GEF) for Gsp1p (yeast Ran). Prp20p plays multiple roles in mRNA metabolism, nucleocytoplasmic transport and mitosis regulation. Prp20p also functions as a linker between chromatin and nuclear pore complex (NPC) which regulates the NPC-mediated boundary activity (BA). Prp20p contains an N-terminal nuclear localization signal (NLS) and a typical RCC1-like domain (RLD). Here we present the 1.9Å crystal structure of the RCC1-like domain of Prp20p, which exhibits a classical seven-bladed β-propeller. We also proved that the additional β-wedge in Prp20p is essential for the interaction between Prp20p and Gsp1p. Based on this structure, we built a complex model of Prp20p and Gsp1p which was optimized by molecular dynamics (MD) simulations. Our model reveals that Prp20p and RCC1 share similar Ran GTPase binding mode. In addition, we also studied the histone-binding property of Prp20p in vitro.

In archaea and eukaryotes, the nascent polypeptide-associated complex (NAC) is one of the cytosolic chaperones that contact the nascent polypeptide chains as they emerge from the ribosome and assist in post-translational processes. The eukaryotic NAC is a heterodimer, and its two subunits form a stable complex through a dimerizing domain called the NAC domain. In addition to acting as a protein translation chaperone, the NAC subunits also function individually in transcriptional regulation. Here we report the crystal structure of the human NAC domain, which reveals the manner of human NAC dimerization. On the basis of the structure, we identified a region in the NAC domain of the human NAC alpha-subunit as a new nucleic acid-binding region, which is blocked from binding nucleic acids in the heterodimeric complex by a helix region in the beta-subunit.

Gluconate 5‐dehydrogenase (Ga5DH) is an NADP(H)‐dependent enzyme that catalyzes a reversible oxidoreduction reaction between D ‐gluconate and 5‐keto‐ D ‐gluconate, thereby regulating the flux of this important carbon and energy source in bacteria. Despite the considerable amount of physiological and biochemical knowledge of Ga5DH, there is little physical or structural information available for this enzyme. To this end, we herein report the crystal structures of Ga5DH from pathogenic Streptococcus suis serotype 2 in both substrate‐free and liganded (NADP + / D ‐gluconate/metal ion) quaternary complex forms at 2.0 Å resolution. Structural analysis reveals that Ga5DH adopts a protein fold similar to that found in members of the short chain dehydrogenase/reductase (SDR) family, while the enzyme itself represents a previously uncharacterized member of this family. In solution, Ga5DH exists as a tetramer that comprised four identical ∼29 kDa subunits. The catalytic site of Ga5DH shows considerable architectural similarity to that found in other enzymes of the SDR family, but the S. suis protein contains an additional residue (Arg104) that plays an important role in the binding and orientation of substrate. The quaternary complex structure provides the first clear crystallographic evidence for the role of a catalytically important serine residue and also reveals an amino acid tetrad RSYK that differs from the SYK triad found in the majority of SDR enzymes. Detailed analysis of the crystal structures reveals important contributions of Ca 2+ ions to active site formation and of specific residues at the C‐termini of subunits to tetramer assembly. Because Ga5DH is a potential target for therapy, our findings provide insight not only of catalytic mechanism, but also suggest a target of structure‐based drug design.

The leukocyte Ig-like receptor (LILR/ILT/LIR) family comprises 13 members that are either activating or inhibitory receptors, regulating a broad range of cells in the immune responses. LILRB1 (ILT2), LILRB2 (ILT4) and LILRA1 (LIR6) can recognize MHC (major histocompatibility complex) class I or class I-like molecules, and LILRB1/HLA-A2, LILRB1/UL18 and LILRB2/HLA-G complex (extracellular domains D1D2) structures have been solved recently. The details of binding to MHC have been described. Despite high levels of sequence similarity among LILRA1, LILRA2 (ILT1), LILRA3 (ILT6) and LILRB1/B2, all earlier experiments showed that LILRA2 does not bind to MHC, but the reason is unknown. Here, we report the LILRA2 extracellular D1D2 domain crystal structure at 2.6 A resolution, which reveals structural shifts of the corresponding MHC-binding amino acid residues in comparison with LILR B1/B2, explaining its non-binding to MHC molecules. We identify some key residues with great influence on the local structure, which exist only in the MHC-binding receptors. Moreover, we show that LILRA2 forms a domain-swapped dimer. Further work with these key swapping residues yields a monomeric form, confirming that the domain-swapping is primarily amino acid sequence-specific. The structure described here supports the dimer conformation in solution observed earlier, and implies a stress-induced regulation by dimerization, consistent with its function as a heat shock promoter.

Nisin is a cationic antimicrobial peptide produced by some lactic acid bacteria. However, expression of nisin resistance protein (NSR) could confer nisin resistance on some non-nisin-producing Lactococcus lactis. To deeply elucidate molecular mechanism underlying NSR-mediated nisin resistance, an NSR mutant with N-terminal 38 amino acid residues deleted (NSRΔ38) was overexpressed in Escherichia coli by fusion with GST. Purified NSRΔ38 was obtained through glutathione (GSH) affinity chromatography followed by cleavage of GST tag. Putative proteolytic activity of NSRΔ38 was determined in vitro against nisin. Antimicrobial activity analysis revealed that nisin lost its bactericidal activity after incubation with NSRΔ38. Further reversed-phase high performance liquid chromatography (RP-HPLC) analysis indicated that NSRΔ38 displayed proteolytic activity against nisin, thus inactivating the antimicrobial peptide. The current study paves the way for in-depth functional studies on NSR.

ABSTRACT Mannonate dehydratase (ManD) is found only in certain bacterial species, where it participates in the dissimilation of glucuronate. ManD catalyzes the dehydration of d -mannonate to yield 2-keto-3-deoxygluconate (2-KDG), the carbon and energy source for growth. Selective inactivation of ManD by drug targeting is of therapeutic interest in the treatment of human Streptococcus suis infections. Here, we report the overexpression, purification, functional characterization, and crystallographic structure of ManD from S. suis . Importantly, by Fourier transform mass spectrometry, we show that 2-KDG is formed when the chemically synthesized substrate ( d -mannonate) is incubated with ManD. Inductively coupled plasma-mass spectrometry revealed the presence of Mn 2+ in the purified protein, and in the solution state catalytically active ManD exists as a homodimer of two 41-kDa subunits. The crystal structures of S. suis ManD in native form and in complex with its substrate and Mn 2+ ion have been solved at a resolution of 2.9 Å. The core structure of S. suis ManD is a TIM barrel similar to that of other members of the xylose isomerase-like superfamily. Structural analyses and comparative amino acid sequence alignments provide evidence for the importance of His311 and Tyr325 in ManD activity. The results of site-directed mutagenesis confirmed the functional role(s) of these residues in the dehydration reaction and a plausible mechanism for the ManD-catalyzed reaction is proposed.

Heat shock response (HSR) is a ubiquitous cellular mechanism that copes with a variety of stresses. This response is mediated by a family of transcriptional activators, heat shock factors (HSFs), which are under tight regulation. HSF binding protein 1 (HSBP1) is a negative regulator of HSR and is reported to bind specifically with the active trimeric form of HSF1, thus inhibiting its activity. HSBP1 contains heptad‐repeats in the primary sequence and was believed to stay in a trimer form in solution. We report the crystal structure of the trimerization domain of the M30I/L55P mutant of human HSBP1 at 1.8 Å resolution. In this crystal form, the HSBP1 fragment of residues 6–53 forms a continuous, 11‐turn long helix. The helix self‐associates to form a parallel, symmetrical, triple coiled‐coil helix bundle, which further assembles into a dimer of trimers in a head‐to‐head fashion. Solution study confirmed that the wild‐type HSBP1 shares similar biophysical properties with the crystallized variant. Furthermore, we identified Ser31, which buried its polar side chain in the hydrophobic interior of the helix bundle, as a stability weak‐spot. Substitution of this residue with Ile increases the melting temperature by 24°C, implicating that this conserved serine residue is maintained at position 31 for functional purposes. Proteins 2009. © 2008 Wiley‐Liss, Inc.

In this paper,the free fatty acids composition of horse oil was analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry (GC-MS) after methylesterification treatment.The results showed that large amounts of saturated,monounsaturated and polyunsaturated fatty acids (PUFAs) were contained in the horse oil,among which,the contents of palmitic acid,oleic acid and linoleic acid are the most,the sum of relative contents of oleic acid and linoleic acid is more than 50%.Oleic acid’s ability to quench singlet-oxygen in vitro was detected by Bioluminescence Detection Technology.The results demonstrated that it has good antioxidant function in a certain concentration range,the concentration of oleic acid in horse oil is 47.5 mg/g,it can be speculated that horse oil will also have antioxidant function in a certain concentration range.

6-Phospho-β-glucosidase (PbgL,EC 3.2.1.86) catalyzes the hydrolysis of some kinds of phospho-disaccharides imported by phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS). An open reading frame (ORF TTE0337) from thermophilic bacterium Thermoanaerobacter tengcongensis, encoding a novel 6-phospho-β-glucosidase (436 amino acid residues) was cloned and actively expressed in E. coli system. Sequence analysis indicates that it belongs to the glycoside hydrolase family 4(GH4), with 62% identity to LicH from Bacillus subtilis and 40% identity to BglT from Thermotoga Maritima. The recombinant form of PbgL (rPbgL) was purified as a single band with molecular weight of about 50 kD on SDS-PAGE, in agreement with the molecular weight deduced from the amino acid sequences. The recombinant enzyme requires Mn 2+ ,NAD+ and reducing agents for catalysis, and exhibits substrate specificity for pNPβG6P with optimal catalytic activity at 85 ℃. Western immunoblots using highly specific polyclonal antibody against rPbgL revealed the expression of the enzyme in the modified MB medium grown cells of T. tengcongensis. Crystals of rPbgL were obtained and the diffraction data were collected to 2.4  resolution. Structure determination by the molecular replacement method is in progress.

The infection of rhesus macaques (Macaca mulatta) by the SIV is the best animal model for studying HIV infection and for AIDS vaccine development. A prevalent MHC class I allele, Mamu-A*01, is known to correlate with containment of SIV, which has been extensively explored in studies of CTL-based vaccination concepts. We determined the crystal structures of Mamu-A*01 complexed with two immunodominant SIV epitopes: the nonamer CM9 of group-specific Ag (Gag, 181-189; CTPYDINQM) and the octamer TL8 of transcription activator (Tat, 28-35; TTPESANL). The overall structures of the two Mamu-A*01 complexes are similar to other MHC class I molecules. Both structures confirm the presence of an absolutely conserved proline anchor residue in the P3 position of the Ag, bound to a D pocket of the Mamu-A*01 H chain with optimal surface complementarity. Like other MHC/peptide complex structures, the P2 and C-terminal residues of the epitopes are also important for anchoring to the MHC molecule, whereas the middle residues form an arch and their side chains are directed into solvent. These two structures reveal details of how Mamu-A*01 interacts with two well-studied epitopes at the atomic level. We discuss the structural basis of CTL escape, based on molecular models made possible by these two structures. The results we present in this study are most relevant for the rational design of Mamu-A*01-restricted CTL epitopes with improved binding, as a step toward development of AIDS vaccines.

In this paper, the free fatty acids composition of the mixed snake oil of Agkistrodon and Zaocys was analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry(GC-MS). The results showed that large amounts of saturated、monounsaturated and polyunsaturated fatty acids(PUFAs)were contained in the snake oil, among which, the content of oleic acid is the most. And the ratio between omega- 6 series of PUFAs and omega-3 series of PUFAs is 8, in an ideal ratio range(between5-10).Snake oil's ability to quench singlet-oxygen in vitro was detected by Bioluminescence Detection Technology. The results demonstrated that some natural active ingredients with antioxidant function were contained in snake oil, which made it have good an- tioxidant function in a certain concentration range.

The Slit-Robo (sr) GTPase-activating protein (GAPs) are important components in the intracellular pathway mediating Slit-Robo signaling in axon guidance and cell migration. We report the first crystal structure of the srGAP1 SH3 domain at 1.8-Å resolution. The unusual side chain conformation of the conserved Phe-13 in the P1 pocket renders the ligand binding pocket shallow and narrow, which contributes toward the low binding affinity. Moreover, the opposing electrostatic charge and the hydrophobic properties of the P3 specificity pocket are consistent with the observed binding characteristics of the srGAP1 SH3 domain to its ligand. Surface plasmon resonance experiments indicate that the srGAP1 SH3 domain interacts with its natural ligand inaCtoN orientation. The srGAP1 SH3 domain can bind to both the CC2 and CC3 motifs in vitro. The N-terminal two acidic residues in the CC3 motif recognition site are necessary for srGAP1 SH3 domain binding. A longer CC3 peptide (CC3-FL) binds with greater affinity than its shorter counterpart, suggesting that the residues surrounding the proline-rich core are important for protein-peptide interactions. Our study reveals previously unknown properties of the srGAP-Robo interaction. Our data provide a structural basis for the srGAP-Robo interaction, consistent with the role of the Robo intracellular domain in interacting with other downstream signaling molecules and mediating versatile and dynamic responses to axon guidance and cell migration cues.

FKBP family proteins are immunophilins which process peptidylprolyl isomerase (PPIase) domain and they can all bind FK506, a macrolide immunosuppressant.FKBP52 is a FKBP protein, which can be separated into four domains.The first (FK1) and the second (FK2) domains are similar with FKBP12.The third domain includes three tetratricopeptide repeat (TPR) motives and the forth domain contains calmodulin binding-site.We have overexpressed and purified FKBP52 full-length and three segments of FKBP52, including FKBP52-FK1, N(1-260) and C(145-459).The crystals of FKBP52-FK1, N(1-260), C(145-459) have been obtained, as well as the complex of C(145-459) and a Cterminal pentapeptide MEEVD from Hsp90.The three dimensional structure of FKBP52 has been defined based on the crystal structures of N(1-260) and C(145-459).The structures have indicated the pattern of natural substrates binding to the active site of PPIase and the reason why the FKBP52-FK506 complex is not able to inhibit calcineurin activity, and has interpreted why FK2 has no PPIase activity.The functional differences between FKBP52 and FKBP51 have been clarified by comparing their structures.A hormone-signaling model based on FKBPs / Hsp90 / hormone receptor complex assembly has been proposed and FKBP51 is regarded as a negative feedback factor of FKBP52 in hormone signaling.

FKBP family proteins are immunophilins which process peptidylprolyl isomerase (PPIase) domain and they can all bind FK506, a macrolide immunosuppressant. FKBP52 is a FKBP protein, which can be separated into four domains. The first (FK1) and the second (FK2) domains are similar with FKBP12. The third domain includes three tetratricopeptide repeat (TPR) motives and the forth domain contains calmodulin binding-site. We have overexpressed and purified FKBP52 full-length and three segments of FKBP52, including FKBP52-FK1, N(1-260) and C(145-459). The crystals of FKBP52-FK1, N(1-260), C(145-459) have been obtained, as well as the complex of C(145-459) and a Cterminal pentapeptide MEEVD from Hsp90. The three dimensional structure of FKBP52 has been defined based on the crystal structures of N(1-260) and C(145-459). The structures have indicated the pattern of natural substrates binding to the active site of PPIase and the reason why the FKBP52-FK506 complex is not able to inhibit calcineurin activity, and has interpreted why FK2 has no PPIase activity. The functional differences between FKBP52 and FKBP51 have been clarified by comparing their structures. A hormone-signaling model based on FKBPs / Hsp90 / hormone receptor complex assembly has been proposed and FKBP51 is regarded as a negative feedback factor of FKBP52 in hormone signaling.