Structural analysis of chorismate synthase from Plasmodium falciparum: a novel target for antimalaria drug discovery

The shikimate pathway in Plasmodium falciparum provides several targets for designing novel antiparasitic agents for the treatment of malaria. Chorismate synthase (CS) is a key enzyme in the shikimate pathway which catalyzes the seventh and final step of the pathway. P. falciparum chorismate synthase (PfCS) is unique in terms of enzymatic behavior, cellular localization and in having two additional amino acid inserts compared to any other CS. The structure of PfCS along with cofactor FMN was predicted by homology modeling using crystal structure of Helicobacter pylori chorismate synthase (HpCS). The quality of the model was validated using structure analysis servers and molecular dynamics. Dimeric form of PfCS was generated and the FMN binding mechanism involving movement of loop near active site has been proposed. Active site pocket has been identified and substrate 5-enolpyruvylshikimate 3-phosphate (EPSP) along with screened potent inhibitors has been docked. The study resulted in identification of putative inhibitors of PfCS with binding efficiency in nanomolar range. The selected putative inhibitors could lead to the development of anti-malarial drugs.     

Cloud hunting: doryphagy,a form of selective autophagy that degrades centriolar satellites

ABSTRACT

The selective clearance of cellular components by macroautophagy (hereafter autophagy) is critical for maintaining cellular homeostasis. In this punctum, we summarize and discuss our recent findings regarding a novel type of selective autophagy that targets centriolar satellites (CS) for degradation, a process we termed doryphagy from the Greek word “doryphoros”, standing for “satellite”. CS are microtubule-associated protein complexes that regulate centrosome composition. We show that CS degradation is mediated through a direct interaction between GABARAPs and an LC3-interacting region (LIR) motif in the CS protein PCM1. Autophagy-deficient systems accumulate large abnormal CS and consequently display centrosome reorganization and abnormal mitoses. Our findings provide a mechanistic link between autophagy deficiency and centrosome abnormalities and exemplify how mammalian Atg8-family proteins (mATG8s) can regulate substrate specificity.     

Cloning,characterization and expression of a cDNA encoding a granulin-like polypeptide in Ciona savignyi

Previous study in our laboratory confirmed that a novel polypeptide, CS5931 derived from Ciona savignyi possesses potent antitumor activity. In the present study, the full length cDNA of CS5931 precursor, termed Cs-pgrn-1 was cloned. The complete cDNA sequence of this gene consists of 685 bp containing an open reading frame (ORF) of 522 bp (173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin (GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares high homology with Ciona intestinalis GRN and is conserved during evolution. The polypeptide also shows high similarity with human GRN A, B, and C. Prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A. The CS5931 was expressed using a prokaryotic expression system and the purified polypeptide inhibited the growth of several tumor cell lines in vitro via apoptotic pathway. Our study revealed that CS5931 has the potential to be developed as a novel antitumor agent.     

Mast Cells Produce a Unique Chondroitin Sulfate Epitope

The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells.     

Enzyme–substrate complexes of allosteric citrate synthase: Evidence for a novel intermediate in substrate binding

The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.     

Small heat shock proteins prevent aggregation of citrate synthase and bind to the N-terminal region which is absent in thermostable forms of citrate synthase

Citrate synthase (CS) is often used in chaperone assays since this thermosensitive enzyme aggregates at moderately increased temperatures. Small heat shock proteins (sHsps) are molecular chaperones specialized in preventing the aggregation of other proteins, termed substrate proteins, under conditions of transient heat stress. To investigate the mechanism whereby sHsps bind to and stabilize a substrate protein, we here used peptide array screening covering the sequence of porcine CS (P00889). Strong binding of sHsps was detected to a peptide corresponding to the most N-terminal α-helix in CS (amino acids Leu₁₃ to Gln₂₇). The N-terminal α-helices in the CS dimer intertwine with the C-terminus in the other subunit and together form a stem-like structure which is protruding from the CS dimer. This stem-like structure is absent in thermostable forms of CS from thermophilic archaebacteria like Pyrococcus furiosus and Sulfolobus solfatacarium. These data therefore suggest that thermostabilization of thermosensitive CS by sHsps is achieved by stabilization of the C- and N-terminae in the protruding thermosensitive softspot, which is absent in thermostable forms of the CS dimer.     

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.     

Crystal structure of enterotoxigenic Escherichia coli colonization factor CS6 reveals a novel type of functional assembly

Coli surface antigen 6 (CS6) is a widely expressed enterotoxigenic Escherichia coli (ETEC) colonization factor that mediates bacterial attachment to the small intestinal epithelium. CS6 is a polymer of two protein subunits CssA and CssB, which are secreted and assembled on the cell surface via the CssC/CssD chaperone usher (CU) pathway. Here, we present an atomic resolution model for the structure of CS6 based on the results of X‐ray crystallographic, spectroscopic and biochemical studies, and suggest a mechanism for CS6‐mediated adhesion. We show that the CssA and CssB subunits are assembled alternately in linear fibres by the principle of donor strand complementation. This type of fibre assembly is novel for CU assembled adhesins. We also show that both subunits in the fibre bind to receptors on epithelial cells, and that CssB, but not CssA, specifically recognizes the extracellular matrix protein fibronectin. Taken together, structural and functional results suggest that CS6 is an adhesive organelle of a novel type, a hetero‐polyadhesin that is capable of polyvalent attachment to different receptors.     

Chondroitin sulfate-D promotes neurite outgrowth by acting as an extracellular ligand for neuronal integrin αVβ3

BackgroundChondroitin sulfate (CS) chains are prominent extra/pericellular matrix components in the central nervous system (CNS) and can exert positive or negative regulatory effects on neurite outgrowth, depending on the CS structure and the amount. Despite the remarkable abilities of highly sulfated forms of CS chains to enhance neurite outgrowth, the neuronal recognition systems for such promotional CS chains, including CS-D polysaccharide, remain to be fully elucidated.MethodsWe explored the molecular basis of the CS-D-mediated neurite extension using primary hippocampal neurons cultured on substrate precoated with CS-D polysaccharides, and evaluated functional involvement of a distinct integrin heterodimer as a novel neuronal CS receptor for CS-D.ResultsWe identified an extracellular matrix receptor, integrin αVβ3, as a functional receptor for CS-D. CS-D, but not CS-C (a precursor form of CS-D) showed significant binding affinity toward recombinant integrin αVβ3 heterodimer and activated intracellular signaling(s) involving focal adhesion kinase (FAK) and Src/Fyn kinase. Functional blockade of the respective players for integrin signaling abrogated the promotional effects of CS-D. We also found the existence of CS-D-induced integrin activation system in neuronal stem/progenitor cell population.ConclusionsThe neuronal cell surface integrin αVβ3 can function as a CS receptor for a highly sulfated CS subtype, CS-D.General significanceOur findings are the first to demonstrate that CS-dependent neurite outgrowth promotion is exerted via direct activation of specific integrin heterodimers on neuronal cell surfaces, providing new insights into understanding the CS-sensing machineries that regulate CNS development and regeneration.     

Structures of mesophilic and extremophilic citrate synthases reveal rigidity and flexibility for function

Citrate synthase (CS) catalyses the entry of carbon into the citric acid cycle and is highly‐conserved structurally across the tree of life. Crystal structures of dimeric CSs are known in both “open” and “closed” forms, which differ by a substantial domain motion that closes the substrate‐binding clefts. We explore both the static rigidity and the dynamic flexibility of CS structures from mesophilic and extremophilic organisms from all three evolutionary domains. The computational expense of this wide‐ranging exploration is kept to a minimum by the use of rigidity analysis and rapid all‐atom simulations of flexible motion, combining geometric simulation and elastic network modeling. CS structures from thermophiles display increased structural rigidity compared with the mesophilic enzyme. A CS structure from a psychrophile, stabilized by strong ionic interactions, appears to display likewise increased rigidity in conventional rigidity analysis; however, a novel modified analysis, taking into account the weakening of the hydrophobic effect at low temperatures, shows a more appropriate decreased rigidity. These rigidity variations do not, however, affect the character of the flexible dynamics, which are well conserved across all the structures studied. Simulation trajectories not only duplicate the crystallographically observed symmetric open‐to‐closed transitions, but also identify motions describing a previously unidentified antisymmetric functional motion. This antisymmetric motion would not be directly observed in crystallography but is revealed as an intrinsic property of the CS structure by modeling of flexible motion. This suggests that the functional motion closing the binding clefts in CS may be independent rather than symmetric and cooperative. Proteins 2014; 82:2657–2670. © 2014 Wiley Periodicals, Inc.     

Chaperone activity of tobacco HSP18, a small heat-shock protein, is inhibited by ATP

NtHSP18P (HSP18), a cytosolic class I small heat-shock protein from tobacco pollen grains, was expressed in Escherichia coli. The viability of these cells was improved by 50% at 50 degrees C, demonstrating its functionality in vivo. Purified recombinant protein formed 240 kDa HSP18 oligomers, irrespective of temperature. These oligomers interacted with the model substrate citrate synthase (CS) to form large complexes in a temperature-dependent manner. Furthermore, HSP18 prevented thermally induced aggregation of CS at 45 degrees C. The fluorescence probe bis-ANS revealed the exposure of HSP18 hydrophobic surfaces at this temperature. Reactivation of chemically denatured CS was also significantly enhanced by HSP18. Surprisingly, HSP18 function was inhibited (in contrast to the related chaperone alphabeta-crystallin and plant sHSPs studied so far) by the presence of ATP in a concentration-dependent manner. The conformational changes of HSP18 imposed by ATP binding were indicated by the difference in the quenching of intrinsic tryptophan fluorescence, and implied more compact structure with ATP. Fluorescence measurements with bis-ANS showed that the conformational shift of HSP18 is suppressed in the presence of ATP. Decreased chaperone activity of HSP18 in the presence of ATP is caused by the lower affinity of conformationally blocked HSP18 for the substrate, as demonstrated by a higher susceptibility of model substrate, malate dehydrogenase, to proteolytic cleavage. Our results suggest that the chaperone activity of some plant sHSPs could be regulated by the availability of ATP in the cytoplasm, which would provide a mechanism to monitor the cell environment, control biological activity of sHSPs, and coordinate it with other ATP-dependent chaperones such as HSP70.     

Cholesterol Sulfate Alters Substrate Preference of Matrix Metalloproteinase-7 and Promotes Degradations of Pericellular Laminin-332 and Fibronectin

Localization of secreted matrix metalloproteinases (MMPs) on the cell surface is required not only for processing of cell surface proteins, but also for controlled degradation of the extracellular matrix (ECM). Our previous study demonstrated that binding of MMP-7 (matrilysin) to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of this MMP. In this study, we investigated the role of CS in the MMP-7-catalyzed degradation of protein components of ECM. We found that the degradation of laminin-332 (laminin-5) catalyzed by MMP-7 was accelerated dramatically in the presence of CS, whereas the sulfated lipid inhibited the degradation of casein catalyzed by the protease. The MMP-7-catalyzed degradation of fibronectin was partially inhibited in the presence of low concentrations of CS, whereas it was accelerated significantly at high concentrations of the lipid. Therefore, it is likely that CS alters the substrate preference of MMP-7. We also found that the proteins of which MMP-7-catalyzed degradation were accelerated by CS also had affinities for CS, suggesting that CS facilitates the proteolyses by cross-linking MMP-7 to its substrates. Moreover, MMP-7 tethered to cancer cell surface via CS degraded fibronectin and laminin-332 coated on a culture plate. The degradations of the adhesive proteins led to significant detachment of the cells from the plate. Taken together, our findings provide a novel mechanism in which cell surface CS promotes the proteolytic activities of MMP-7 toward selective substrates in the pericellular ECM, thereby contributing to cancer cell migration and metastasis.     

Cloning and Characterization of a Novel Chondroitin Sulfate/Dermatan Sulfate 4-O-Endosulfatase from a Marine Bacterium

Sulfatases are potentially useful tools for structure-function studies of glycosaminoglycans (GAGs). To date, various GAG exosulfatases have been identified in eukaryotes and prokaryotes. However, endosulfatases that act on GAGs have rarely been reported. Recently, a novel HA and CS lyase (HCLase) was identified for the first time from a marine bacterium (Han, W., Wang, W., Zhao, M., Sugahara, K., and Li, F. (2014) J. Biol. Chem. 289, 27886–27898). In this study, a putative sulfatase gene, closely linked to the hclase gene in the genome, was recombinantly expressed and characterized in detail. The recombinant protein showed a specific N-acetylgalactosamine-4-O-sulfatase activity that removes 4-O-sulfate from both disaccharides and polysaccharides of chondroitin sulfate (CS)/dermatan sulfate (DS), suggesting that this sulfatase represents a novel endosulfatase. The novel endosulfatase exhibited maximal reaction rate in a phosphate buffer (pH 8.0) at 30 °C and effectively removed 17–65% of 4-O-sulfates from various CS and DS and thus significantly inhibited the interactions of CS and DS with a positively supercharged fluorescent protein. Moreover, this endosulfatase significantly promoted the digestion of CS by HCLase, suggesting that it enhances the digestion of CS/DS by the bacterium. Therefore, this endosulfatase is a potential tool for use in CS/DS-related studies and applications.     

葡萄糖流加策略对基因工程FR-008／杀念菌素衍生物CS103补料分批发酵过程的影响

以组合生物合成技术得到的链霉菌FR-008突变株CS103为研究对象,研究了3．7L发酵罐上维持一定葡萄糖浓度对其次级代谢产物脱羧FR-008／candicidin衍生聚酮抗生素CS103生物合成的影响。当初始葡萄糖浓度20g／L,发酵过程还原糖浓度维持在10g/L时,抗生素CS103最高产量较分批发酵最高产量相比提高30％。研究了3．7L罐上补料分批发酵生产CS103的工艺,主要考察了脉冲补料、间歇流加补料和连续流加补料三种补料分批发酵工艺,并与分批发酵进行了比较。连续流加补料维持糖浓度的效果明显,最高产量达到126．9μg/mL,与分批发酵相比提高了44％左右。     

Replacement of the Catalytic Nucleophile Aspartyl Residue of Dextran Glucosidase by Cysteine Sulfinate Enhances Transglycosylation Activity

Dextran glucosidase from Streptococcus mutans (SmDG) catalyzes the hydrolysis of an α-1,6-glucosidic linkage at the nonreducing end of isomaltooligosaccharides and dextran. This enzyme has an Asp-194 catalytic nucleophile and two catalytically unrelated Cys residues, Cys-129 and Cys-532. Cys-free SmDG was constructed by replacement with Ser (C129S/C532S (2CS), the activity of which was the same as that of the wild type, SmDG). The nucleophile mutant of 2CS was generated by substitution of Asp-194 with Cys (D194C-2CS). The hydrolytic activity of D194C-2CS was 8.1 × 10⁻⁴ % of 2CS. KI-associated oxidation of D194C-2CS increased the activity up to 0.27% of 2CS, which was 330 times higher than D194C-2CS. Peptide-mapping mass analysis of the oxidized D194C-2CS (Ox-D194C-2CS) revealed that Cys-194 was converted into cysteine sulfinate. Ox-D194C-2CS and 2CS shared the same properties (optimum pH, pI, and substrate specificity), whereas Ox-D194C-2CS had much higher transglucosylation activity than 2CS. This is the first study indicating that a more acidic nucleophile (-SOO⁻) enhances transglycosylation. The introduction of cysteine sulfinate as a catalytic nucleophile could be a novel approach to enhance transglycosylation.     

Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum

Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs. Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum. In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time. Utilizing an N-terminal 6x histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step. The products of exhaustive digestion of chondroitin-4SO(4) and chondroitin-6SO(4) with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique. In addition, the Michaelis-Menten parameters were determined for these enzymes. With chondroitin-4SO(4) as the substrate, the recombinantly expressed chondroitinase AC has a K(m) of 0.8 microM and a k(cat) of 234 s(-1). This is the first report of kinetic parameters for chondroitinase AC with this substrate. With chondroitin-6SO(4) as the substrate, the enzyme has a K(m) of 0.6 microM and a k(cat) of 480 s(-1). Recombinantly expressed chondroitinase B has a K(m) of 4.6 microM and a k(cat) of 190 s(-1) for dermatan sulfate as its substrate. Efficient recombinant expression of the chondroitinases will facilitate the structure-function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS.     

Structure-based discovery of a novel non-peptidic small molecular inhibitor of caspase-3

Ac-DNLD-CHO is a novel caspase-3 specific peptide inhibitor that was rationally designed by our computational strategy. The specificity was shown to be due to the specific interaction of NLD moiety with the active site of caspase-3 on the basis of docking mode and site-directed mutagenesis analyses. Here, we computationally screened non-peptidic small molecular inhibitors of caspase-3 from our chemical library using a reliable pharmacophore derived from the specific binding mode of NLD. Through in vitro enzyme assay of the screened candidate compounds, we discovered a novel caspase-3 specific small molecular inhibitor, CS4566, which has a unique scaffold structure. The binding mode of CS4566 to caspase-3 mimics that of NLD, especially LD moiety. This represents a promising lead compound for creating non-peptidic pharmaceuticals for caspase-mediated diseases, such as neurodegenerative disorders.     

Atomistic simulations of competition between substrates binding to an enzyme

Although the idea that electrostatic potentials generated by enzymes can guide substrates to active sites is well established, it is not always appreciated that the same potentials can also promote the binding of molecules other than the intended substrate, with the result that such enzymes might be sensitive to the presence of competing molecules. To provide a novel means of studying such "electrostatic competition" effects, computer simulation methodology has been developed to allow the diffusion and association of many solute molecules around a single enzyme to be simulated. To demonstrate the power of the methodology, simulations have been conducted on an artificial fusion protein of citrate synthase (CS) and malate dehydrogenase (MDH) to assess the chances of oxaloacetate being channeled between the MDH and CS active sites. The simulations demonstrate that the probability of channeling is strongly dependent on the concentration of the initial substrate (malate) in the solution. In fact, the high concentrations of malate used in experiments appear high enough to abolish any channeling of oxaloacetate. The simulations provide a resolution of a serious discrepancy between previous simulations and experiments and raise important questions relating to the observability of electrostatically mediated substrate channeling in vitro and in vivo.     

Preparation and biological properties of a novel composite scaffold of nano-hydroxyapatite/chitosan/carboxymethyl cellulose for bone tissue engineering

In this study, we report the physico-chemical and biological properties of a novel biodegradable composite scaffold made of nano-hydroxyapatite and natural derived polymers of chitosan and carboxymethyl cellulose, namely, n-HA/CS/CMC, which was prepared by freeze-drying method. The physico-chemical properties of n-HA/CS/CMC scaffold were tested by infrared absorption spectra (IR), transmission electron microscope(TEM), scanning electron microscope(SEM), universal material testing machine and phosphate buffer solution (PBS) soaking experiment. Besides, the biological properties were evaluated by MG63 cells and Mesenchymal stem cells (MSCs) culture experiment in vitro and a short period implantation study in vivo. The results show that the composite scaffold is mainly formed through the ionic crossing-linking of the two polyions between CS and CMC, and n-HA is incorporated into the polyelectrolyte matrix of CS-CMC without agglomeration, which endows the scaffold with good physico-chemical properties such as highly interconnected porous structure, high compressive strength and good structural stability and degradation. More important, the results of cells attached, proliferated on the scaffold indicate that the scaffold is non-toxic and has good cell biocompatibility, and the results of implantation experiment in vivo further confirm that the scaffold has good tissue biocompatibility. All the above results suggest that the novel degradable n-HA/CS/CMC composite scaffold has a great potential to be used as bone tissue engineering material.     

Conformer selection and induced fit in flexible backbone protein-protein docking using computational and NMR ensembles

Accommodating backbone flexibility continues to be the most difficult challenge in computational docking of protein-protein complexes. Towards that end, we simulate four distinct biophysical models of protein binding in RosettaDock, a multiscale Monte-Carlo-based algorithm that uses a quasi-kinetic search process to emulate the diffusional encounter of two proteins and to identify low-energy complexes. The four binding models are as follows: (1) key-lock (KL) model, using rigid-backbone docking; (2) conformer selection (CS) model, using a novel ensemble docking algorithm; (3) induced fit (IF) model, using energy-gradient-based backbone minimization; and (4) combined conformer selection/induced fit (CS/IF) model. Backbone flexibility was limited to the smaller partner of the complex, structural ensembles were generated using Rosetta refinement methods, and docking consisted of local perturbations around the complexed conformation using unbound component crystal structures for a set of 21 target complexes. The lowest-energy structure contained > 30% of the native residue-residue contacts for 9, 13, 13, and 14 targets for KL, CS, IF, and CS/IF docking, respectively. When applied to 15 targets using nuclear magnetic resonance ensembles of the smaller protein, the lowest-energy structure recovered at least 30% native residue contacts in 3, 8, 4, and 8 targets for KL, CS, IF, and CS/IF docking, respectively. CS/IF docking of the nuclear magnetic resonance ensemble performed equally well or better than KL docking with the unbound crystal structure in 10 of 15 cases. The marked success of CS and CS/IF docking shows that ensemble docking can be a versatile and effective method for accommodating conformational plasticity in docking and serves as a demonstration for the CS theory—that binding-competent conformers exist in the unbound ensemble and can be selected based on their favorable binding energies.