共查询到20条相似文献，搜索用时 0 毫秒
1.
The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25-residue peptide taken from the CaMBD, joined by a 5-glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25-residue CaMBD peptide without the glycine linker at a resolution of 2.1 A. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N-lobe from one molecule and C-lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully-designed CaM-sepharose pull-down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg-408, Val-409, and Phe-410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N- and C-lobes, ultimately keeping these lobes apart and forcing \"head-to-tail\" dimerization to attain the requisite N- and C-lobe pairing for CaMBD binding. 相似文献
2.
We have developed a nonredundant protein-RNA docking benchmark dataset, which is derived from the available bound and unbound structures in the Protein Data Bank involving polypeptide and nucleic acid chains. It consists of nine unbound-unbound cases where both the protein and the RNA are available in the free form. The other 36 cases are of unbound-bound type where only the protein is available in the free form. The conformational change upon complex formation is calculated by a distance matrix alignment method, and based on that, complexes are classified into rigid, semi-flexible, and full flexible. Although in the rigid body category, no significant conformational change accompanies complex formation, the fully flexible test cases show large domain movements, RNA base flips, etc. The benchmark covers four major groups of RNA, namely, t-RNA, ribosomal RNA, duplex RNA, and single-stranded RNA. We find that RNA is generally more flexible than the protein in the complexes, and the interface region is as flexible as the molecule as a whole. The structural diversity of the complexes in the benchmark set should provide a common ground for the development and comparison of the protein-RNA docking methods. The benchmark can be freely downloaded from the internet. 相似文献
3.
S. Krebs D. Rognan J. A. Lpez de Castro 《Protein science : a publication of the Protein Society》1999,8(7):1393-1399
B*2701 differs from all other HLA-B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA-B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2-containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H-bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA-B27-bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long-range effect of the polymorphic residue 74 of HLA-B27, by inducing a conformational switch of the conserved Lys70. 相似文献
4.
Cloninger MJ 《Current opinion in chemical biology》2002,6(6):742-748
In the past year, significant advances have been made in the synthesis and study of glycodendrimers and peptide dendrimers. Application of these dendrimers to the study of carbohydrate-protein and protein-protein interactions has facilitated the understanding of these processes. In addition, dendrimers show great promise as DNA- and drug-delivery systems. 相似文献
5.
The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions. 相似文献
6.
The trypsin-like serine proteases comprise a structurally similar family of proteins with a wide diversity of biological functions. Members of this family play roles in digestion, hemostasis, immune responses, and cancer metastasis. Bovine trypsin is an archetypical member of this family that has been extensively characterized both functionally and structurally, and that preferentially hydrolyzes Arg/Lys-Xaa peptide bonds. We have used molecular dynamics (MD) simulations to study bovine trypsin complexed with the two noncovalent small-molecule ligands, benzamidine and tranylcypromine, that have the same hydrogen-bond donating moieties as Arg and Lys side-chains, respectively. Multiple (10) simulations ranging from 1 ns to 2.2 ns, with explicit water molecules and periodic boundary conditions, were performed. The simulations reveal that the trypsin binding pocket residues are relatively rigid regardless of whether there is no ligand, a high-affinity ligand (benzamidine), or a low-affinity ligand (tranylcypromine). The thermal average of the conformations sampled by benzamidine bound to trypsin is planar and consistent with the planar internal geometry of the benzamidine crystallographic model coordinates. However, the most probable bound benzamidine conformations are +/-25 degrees out of plane, implying that the observed X-ray electron density represents an average of densities from two mirror symmetric, nonplanar conformations. Solvated benzamidine has free energy minima at +/-45 degrees , and the induction of a more planar geometry upon binding is associated with approximately 1 kcal/mol of intramolecular strain. Tranylcypromine's hydrogen-bonding pattern in the MD differs substantially from that inferred from the X-ray electron density. Early in simulations of this system, tranylcypromine adopts an alternative binding conformation, changing from the crystallographic conformation, with a direct hydrogen bond between its amino moiety and the backbone oxygen of Gly219, to one having a bridging water molecule. This result is consistently seen with the CHARMM22, Amber, or OPLS-AA force fields. The trypsin-tranylcypromine hydrogen-bonding pattern observed in the simulations also occurs as the crystallographic binding mode of the Lys15 side-chain of bovine pancreatic trypsin inhibitor bound to trypsin. In this latter cocrystal, a bridging crystallographic water does reside between the side-chain's amino group and the trypsin Gly219 backbone oxygen. Furthermore, the trypsin-tranylcypromine simulations sample two different stable noncrystallographic binding poses. These data suggest that some of the electron density ascribed to tranylcypromine in the X-ray model is rather due to a bound water molecule, and that multiple tranylcypromine binding conformations (crystallographic disorder) may be the cause of ambiguous electron density. The combined trypsin-benzamidine and trypsin- tranylcypromine results highlight the ability of simulations to augment protein-ligand complex structural data by deconvoluting the effects of thermal and structural averaging, and by finding energetically optimal ligand and bound water positions for weakly bound ligands. 相似文献
7.
目的：在大肠杆菌中构建、表达和纯化抗血管内皮生长因子（VEGF）的Fab片段（兰尼单抗,ranibizumab）,通过发酵条件的控制实现其在大肠杆菌周质和胞外的高效分泌表达,并检测其抗VEGF的活性。方法：以pET30a为质粒载体,构建了Fd链和L链前都含有OmpA信号肽、SD序列和T7 promoter的克隆载体pET30a（+）-LC-HC,转化BL21（DE3）表达菌株,并进行了培养基、温度和IPTG诱导浓度的条件优化。结果：确定Fab片段在大肠杆菌分泌表达摇瓶发酵最佳条件为：在含有1.5% Tryptone,1% Yeast Extract,0.5% Glucose,0.15% NaCl,0.1% NH4Cl,0.08% MgCl2·6H2O的1L培养基的摇瓶中,按照10%的接种量,37℃摇床培养至对数生长后期（OD600为2左右）,添加0.1mmol/L IPTG诱导剂,于16℃条件下诱导表达过夜（16h左右）。用周质破菌提取分泌至大肠杆菌周质腔的Fab片段,同时用中空纤维柱浓缩发酵培养基,最后用ProteinG亲和层析柱一步纯化洗脱,经SDS-PAGE检测分析和Brandford法测蛋白浓度得出纯化的Fab抗体片段纯度在90%以上,分泌表达纯化量为0.4mg/L。以VEGF165作为结合抗原,间接ELISA分析纯化后的Fab抗体EC50=30ng/ml。继续用该培养基在3.7L体积发酵罐中进行2L体积的发酵,获得最终的菌体产率为30g/L,可亲和纯化Fab抗体量为1.94mg/L。结论：成功实现了Fab抗体片段在大肠杆菌中的高效分泌表达,且具有很高的活性,为规模化制备Fab抗体片段提供了研究依据。 相似文献
8.
Bert A. Van Der Reijden Claudia A.J. Erpelinck-Verschueren BOB L
WENBERG Joop H. Jansen 《Protein science : a publication of the Protein Society》1999,8(7):1557-1561
Triad1 was recently identified as a nuclear RING finger protein, which is up-regulated during retinoic acid induced granulocytic differentiation of acute leukemia cells. Here we show that a cysteine-rich domain (C6HC), present in Triad1, is conserved in at least 24 proteins encoded by various eukaryotes. The C6HC consensus pattern C-x(4)-C-x(14-30)-C-x(1-4)-C-x(4)-C-x(2)-C-x(4)-H-x(4)-C defines this structure as the fourth family member of the zinc-binding RING, LIM, and LAP/PHD fingers. Strikingly, in 22 of 24 proteins the C6HC domain is flanked by two RING finger structures. We have termed the novel C6HC motif DRIL (double RING finger linked). The strong conservation of the larger tripartite TRIAD (two RING fingers and DRIL) structure indicates that the three subdomains are functionally linked and identifies a novel class of proteins. 相似文献
9.
Divya Chandra Nitesh Sankalia Imee Arcibal Scott Banta Donald Cropek Pankaj Karande 《Peptide Science》2014,102(1):97-106
We describe a general strategy for the design and discovery of affinity peptides for a protein from its natural ligands. Our approach is guided by protein–protein interactions in natural systems and focuses on the hetero‐trimeric complex of cardiac troponin I (cTnI), C (cTnC) and T (cTnT). A key premise of this work is that cTnC and cTnT, owing to their innate ability to bind cTnI, are potential templates for the design and discovery of cTnI‐binding peptides. Relying only on the knowledge of primary sequences of cTnC and cTnT, we designed a library of short overlapping peptides that span the entirety of cTnC and cTnT and tested them for binding to cTnI. We were successful in identifying several peptides that display high affinity (1–100 nM) for cTnI. The specific implication of this work is that mimicking natural protein‐protein interactions is an excellent starting point for the discovery and rational design of peptide ligands. The knowledge of secondary or tertiary structures of the proteins involved is not a necessary precondition for this approach. Nevertheless, we show that structural information can be used to validate the results of a fragment‐based peptide design, and can be potentially beneficial for refining the lead candidates. Our approach is broadly applicable to any protein with at least one natural binding ligand with known primary sequence. For protein targets with multiple natural ligands, this approach can potentially yield several distinct affinity peptides capable of simultaneously binding the target protein via orthogonal modes or at complementary interfaces. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 97–106, 2014. 相似文献
10.
Schatz SM Kerschbaumer RJ Gerstenbauer G Kral M Dorner F Scheiflinger F 《Biotechnology and bioengineering》2003,84(4):433-438
A chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two-stage production process. Cells were first grown to 90% confluence at 37 degrees C in a proliferation phase, followed by a production phase at either 37 degrees C or 28 degrees C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28 degrees C resulted in a 14-fold increase in product yield when cells were cultivated in serum-containing medium, and in a 38-fold increase in product yield when serum-free medium was applied. 相似文献
11.
Bovine seminal ribonuclease, a homodimeric enzyme joined covalently by two interchain disulphide bonds, is an equilibrium mixture of two conformational isomers, MxM and M=M. The major form, MxM, whose crystal structure has been previously determined at 1.9 A resolution, presents the swapping of the N-terminal segments (residues 1-15) and composite active sites formed by residues of different chains. The three-dimensional domain swapping does not occur in the M=M form. The different fold of each N-terminal tail is directed by the hinge loop (residue 16-22) connecting the swapping domain to the body of the protein. Reduction and alkylation of interchain disulphide bridges produce a monomeric derivative and a noncovalent swapped dimer, which are both active. The free and nucleotide-bound forms of the monomer have been crystallized at an alkaline pH and refined at 1.45 and 1.65 A resolution, respectively. In both cases, the N-terminal fragment is folded on the main body of the protein to produce an intact active site and a chain architecture very similar to that of bovine pancreatic ribonuclease. In this new fold of the seminal chain, the hinge loop is disordered. Despite the difference between the tertiary structure of the monomer and that of the chains in the MxM form, the active sites of the two enzymes are virtually indistinguishable. Furthermore, the structure of the liganded enzyme represents the first example of a ribonuclease complex studied at an alkaline pH and provides new information on the binding of a nucleotide when the catalytic histidines are deprotonated. 相似文献
12.
Eberini I Guerini Rocco A Ientile AR Baptista AM Gianazza E Tomaselli S Molinari H Ragona L 《Proteins》2008,71(4):1889-1898
The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family. 相似文献
13.
M. V. Sidorova A. S. Molokoedov E. V. Kudryavtseva M. I. Baldin D. A. Frid M. V. Ovchinnikov Zh. D. Bespalova V. N. Bushuev 《Russian Journal of Bioorganic Chemistry》2006,32(2):136-145
Two variants of the synthesis of tridecapeptide alloferon, the active principle of antiviral preparation allokine-alpha, were developed on the basis of fragment condensation in solution or on the Merrifield resin. The solid phase variant of the synthesis was shown to be more technological; it allows the preparation of the product at a higher total yield (40% vs. 17% for conventional synthesis in solution from the starting derivatives of the C-terminal dipeptide). The by-products formed during the synthesis of alloferon were identified. 相似文献
14.
Interactions and inhibition of blood coagulation factor Va involving residues 311-325 of activated protein C. 
下载免费PDF全文
下载免费PDF全文 R. M. Mesters M. J. Heeb J. H. Griffin 《Protein science : a publication of the Protein Society》1993,2(9):1482-1489
Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide-(311-325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa-1-stage coagulation assays in normal and in protein S-depleted plasma with 50% inhibition at 13 microM peptide. In a system using purified clotting factors, peptide-(311-325) inhibited APC-catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 6 microM peptide. However, peptide-(311-325) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]alpha 1-antitrypsin. Peptide-(311-325) surprisingly inhibited factor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the absence of factor Va with 50% inhibition at 8 microM peptide. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting it does not directly inhibit these enzymes. Factor Va bound in a dose-dependent manner to immobilized peptide-(311-325). Peptide-(311-315) inhibited the binding of factor Va to immobilized APC or factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
S-adenosylhomocysteine hydrolase (SAHH) is a key regulator of S-adenosylmethionine-dependent methylation reactions and an interesting pharmacologic target. We cloned the SAHH gene from Plasmodium falciparum (PfSAHH), with an amino acid sequence agreeing with that of the PlasmoDB genomic database. Even though the expressed recombinant enzyme, PfSAHH, could use 3-deaza-adenosine (DZA) as an alternative substrate in contrast to the human SAHH, it has a unique inability to substitute 3-deaza-(+/-)aristeromycin (DZAri) for adenosine. Among the analogs of DZA, including neplanocin A, DZAri was the most potent inhibitor of the PfSAHH enzyme activity, with a K(i) of about 150 nM, whether Ado or DZA was used as a substrate. When the same DZA analogs were tested for their antimalarial activity, they also inhibited the in vitro growth of P. falciparum parasites potently. Homology-modeling analysis revealed that a single substitution (Thr60-Cys59) between the human and malarial PfSAHH, in an otherwise similar SAH-binding pocket, might account for the differential interactions with the nucleoside analogs. This subtle difference in the active site may be exploited in the development of novel drugs that selectively inhibit PfSAHH. We performed a comprehensive phylogenetic analysis of the SAHH superfamily and inferred that SAHH evolved in the common ancestor of Archaea and Eukaryota, and was subsequently horizontally transferred to Bacteria. Additionally, an analysis of the unusual and uncharacterized AHCYL1 family of the SAHH paralogs extant only in animals reveals striking divergence of its SAH-binding pocket and the loss of key conserved residues, thus suggesting an evolution of novel function(s). 相似文献
16.
17.
Dealing with receptor flexibility in docking methodology is still a problem. The main reason behind this difficulty is the large number of degrees of freedom that have to be considered in this kind of calculations. In this paper, we present an automated procedure, called MADAMM, that allows flexibilization of both the receptor and the ligand during a multistaged docking with an automated molecular modeling protocol. We show that the orientation of particular residues at the interface between the protein and the ligand have a crucial influence on the way they interact during the docking process, and the standard docking methodologies failed to predict their correct mode of binding. We present some examples that demonstrate the capabilities of this approach when compared with traditional docking methodologies. 相似文献
18.
Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding. 相似文献
19.
An equimolar mixture of avian pancreatic polypeptide (aPP) fragments aPP(1-11)-NH2 and Ac-aPP(12-36) had an electronic circular dichroism (ECD) spectrum that was similar to that of whole aPP in H2O and even more so in 30% (v/v) trifluoroethanol (TFE) in 15 mM Na2HPO4, but was different from the sum of the spectra of the individual fragments. The vibrational circular dichroism (VCD) spectrum of the combined fragments in 30% (v/v) TFE in 15 mM Na2HPO4 in D2O was also similar to that of the intact aPP and unlike the sum of the VCD spectra of the fragments. The interaction of these fragments is thus sufficient to support the conformation of whole aPP. This study demonstrates that VCD, in combination with ECD, is useful for the study of protein-protein interactions. 相似文献
20.