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1.
Gendelman R Burton-Wurster NI MacLeod JN Lust G 《The Journal of biological chemistry》2003,278(13):11175-11181
Binding of fibronectin to the small proteoglycan decorin plays an important role in cell differentiation and cell migration. The cartilage-specific (V+C)(-) fibronectin isoform, in which nucleotides that normally encode the protein segments V, III(15), and I(10) are spliced out, is one of the major splice variants present in cartilage matrices. Full-length and truncated cDNA constructs were used to express recombinant versions of fibronectin. Results demonstrated that the (V+C)(-) isoform has a higher affinity for decorin. Dissociation constants for decorin and fibronectin interaction were calculated to be 93 nm for the V(+)C(+) isoform and 24 nm and 223 nm for (V+C)(-) fibronectin. Because heparin competed with decorin competitively, binding of decorin to fibronectin likely occurs at a heparin-binding region. We propose that alternative splicing of the V and C regions changes the global conformation of fibronectin in such a way that it opens an additional decorin-binding site. This conformational change is responsible for the higher affinity of the (V+C)(-) fibronectin isoform for decorin. 相似文献
2.
Crystallographic investigation of peptide binding sites in the N-domain of the ClpA chaperone 总被引:2,自引:0,他引:2
Escherichia coli ClpA, an Hsp100/Clp chaperone and an integral component of the ATP-dependent ClpAP protease, participates in the dissolution and degradation of regulatory proteins and protein aggregates. ClpA consists of three functional domains: an N-terminal domain and two ATPase domains, D1 and D2. The N-domain is attached to D1 by a mobile linker and is made up of two tightly bound, identically folded alpha-helical bundles related by a pseudo 2-fold symmetry. Between the halves of the pseudo-dimer is a large flexible acidic loop that becomes better ordered upon binding of the small adaptor protein, ClpS. We have identified a number of structural features in the N-domain, including a Zn(++) binding motif, several interfaces for binding to ClpS, and a prominent hydrophobic surface area that binds peptides in different configurations. These structural motifs may contribute to binding of protein or peptide substrates with weak affinity and broad specificity. Kinetic studies comparing wild-type ClpA to a mutant ClpA with its N-domain deleted show that the N-domains contribute to the binding of a non-specific protein substrate but not of a folded substrate with the specific SsrA recognition tag. A functional model is proposed in which the N-domains in ClpA function as tentacles to weakly hold on to proteins thereby enhancing local substrate concentration. 相似文献
3.
M Pak J R Hoskins S K Singh M R Maurizi S Wickner 《The Journal of biological chemistry》1999,274(27):19316-19322
ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation. 相似文献
4.
Hsp70-class molecular chaperones interact with diverse polypeptide substrates, but there is limited information on the structures of different Hsp70-peptide complexes. We have used a site-directed fluorescence labeling and quenching strategy to investigate the orientation of different peptides bound to DnaK from Escherichia coli. DnaK was selectively labeled on opposite sides of the substrate-binding domain (SBD) with the fluorescent probe bimane, and the ability of peptides containing N- or C-terminal tryptophan residues to quench bimane fluorescence was measured. Tryptophan-labeled derivatives of the model peptide NRLLLTG bound with the same forward orientation previously observed in the crystal structure of the DnaK(SBD)-NRLLLTG complex. Derivatives of this peptide containing arginine in the C-terminal rather than N-terminal region, NTLLLRG, also bound in the forward direction indicating that charged residues in the flanking regions of the peptide are not the major determinant of peptide binding orientation. We also tested peptides having proline in one (ELPLVKI) or two (ELPPVKI) central positions. Tryptophan derivatives of each of these peptides bound with a strong preference for the reverse direction relative to that observed for the NRLLLTG and NTLLLRG peptides. Computer modeling the peptides NRLLLTG and ELPPVKI in both the forward and reverse orientations into the DnaK(SBD) indicated that differential hydrogen-bonding patterns and steric constraints of the central peptide residues are likely causes for differences in their binding orientations. These findings establish that DnaK is able to bind substrates in both forward and reverse orientations and suggest that the central residues of the peptide are the major determinants of directional preference. 相似文献
5.
Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation
The cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding of substrate proteins bearing "tag" sequences, such as the 11-residue ssrA sequence appended to proteins translationally stalled at ribosomes. Unfolding is coupled to translocation through a central channel into the associating protease, ClpP. To explore the topology and mechanism of ClpA action, we carried out chemical crosslinking and functional studies. Whereas a tag from RepA protein crosslinked proximally to the flexible N domains, the ssrA sequence in GFP-ssrA crosslinked distally in the channel to a segment of the distal ATPase domain (D2). Single substitutions placed in this D2 loop, and also in two apparently cooperating proximal (D1) loops, abolished binding of ssrA substrates and unfolded proteins lacking tags and blocked unfolding of GFP-RepA. Additionally, a substitution adjoining the D2 loop allowed binding of ssrA proteins but impaired their translocation. This loop, as in homologous nucleic-acid translocases, may bind substrates proximally and, coupled with ATP hydrolysis, translocate them distally, exerting mechanical force that mediates unfolding. 相似文献
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The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group. 相似文献
9.
Bovine mitochondrial ribosomes possess a high affinity binding site for guanine nucleotides 总被引:1,自引:0,他引:1
Mammalian mitochondrial ribosomes possess a binding site for guanine nucleotides. GTP binds in unit stoichiometry and with high affinity (Kd = 15.3 +/- 2.8 nM) to the small subunit of bovine mitochondrial ribosomes. This binding activity survives high salt washes, indicating that the nucleotide binds to an integral site within this subunit. GDP also binds to the small subunit with high affinity (Kd = 17 +/- 5.8 nm) and in unit stoichiometry. The GTP binding activity can be competed with GDP but not appreciably by other nucleotides, indicating that both GTP and GDP bind specifically and to the same site. The non-hydrolyzable analogs of GTP, guanylyl-5'-imidophosphate, and guanylyl-(beta,gamma-methylene)- diphosphonate also bind to the small subunit, but with reduced affinity. These results indicate that mammalian mitochondrial ribosomes, unlike other ribosomes, are able to interact directly with guanosine triphosphate, suggesting that the bound GTP may be involved in a novel regulatory mechanism in mitochondrial protein synthesis. 相似文献
10.
《Molecular membrane biology》2013,30(3):246-260
Abstract Background: The involvement of aquaporin (AQP) water and small solute channels in the etiology of several diseases, including cancer, neuromyelitis optica and body fluid imbalance disorders, has been suggested previously. Furthermore, results obtained in a mouse model suggested that AQP9 function contributes to hyperglycemia in type-2 diabetes. In addition, the physiological role of several AQP family members remains poorly understood. Small molecule inhibitors of AQPs are therefore desirable to further study AQP physiological and pathophysiological functions. Methods: The binding of recently established AQP9 inhibitors to a homology model of AQP9 was investigated by molecular dynamics simulations and molecular docking. Putative inhibitor binding sites identified with this procedure were modified by site-directed mutagenesis. Active compounds were measured in a mammalian cell water permeability assay of mutated AQP9 isoforms and tested for changes in inhibitory effects. Controls: Three independent cell lines were established for each mutated AQP9 isoform and functionality of mutant isoforms was established. Principal findings: We have identified putative binding sites of recently established AQP9 inhibitors. This information facilitated successful identification of novel AQP9 inhibitors with low micromolar IC50 values in a cell based assay by in silico screening of a compound library targeting specifically this binding site. Significance: We have established a successful strategy for AQP small molecule inhibitor identification. AQP inhibitors may be relevant as experimental tools, to enhance our understanding of AQP function, and in the treatment of various diseases. 相似文献
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Qiu LY Swarts HG Tonk EC Willems PH Koenderink JB De Pont JJ 《The Journal of biological chemistry》2006,281(19):13533-13539
P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349-32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase beta1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the NH4+-stimulated ATPase activity in membranes of transfected Sf9 cells. 相似文献
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Preparations of photosystem II complex from spinach chloroplasts with Triton X-100 were treated with 1 M KCl to release 17 KDa and 23 KDa polypeptides. The inhibited oxygen evolution activity could be reactivated by adding high concentration (mM) of Ca++ or by reconstituting 17 KDa and 23 KDa polypeptides which were found to promote high affinity binding of Ca++ to the reconstituted membranes (Ghanotakis et al. FEBS (1984) 170, 169-173). Inclusion of 50 mM Ca++ during KCl treatment did not prevent the release of 17 KDa and 23 KDa polypeptides but protected oxygen evolution from being inactivated. It is explained by preservation of the high affinity binding site for Ca++ if, Ca++ is present during the salt treatment even though depletion of 17 KDa and 23 KDa polypeptides usually results in replacement by a low affinity (mM) binding site for Ca++. It also implies that the high affinity binding site is not located on 17 KDa and 23 KDa polypeptides. 相似文献
14.
Jüse U Arntzen M Højrup P Fleckenstein B Sollid LM 《Bioorganic & medicinal chemistry》2011,19(7):2470-2477
Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount of HLA molecules, giving a selective force in the binding. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format. 相似文献
15.
A.H.M. Nurun Nabi Kazal Boron Biswas Tsutomu Nakagawa Atsuhiro Ichihara Tadashi Inagami Fumiaki Suzuki 《Biochimica et Biophysica Acta - Proteins and Proteomics》2009,1794(12):1838-1847
An important role of the decoy peptide sequence has recently been suggested in vitro for the binding of prorenin to the (pro)renin receptor [(P)RR]. In this study, other prospective crucial regions in renin and prorenin responsible for their interaction with (P)RR were investigated using various kinds of peptides, e.g., the “hinge” S149QGVLKEDVF158 designed from the structure of renin also common to prorenin, L1PPTDTTTF8P, L1PPTDTTTFKRIFLKR15P and the decoy (R10PIFLKRMPSI19P) designed from the predicted structure of prorenin. For the kinetic analysis, the recombinant h(P)RR was immobilized on the biosensor surface through a specific anti-(P)RR antibody. In case of the equilibrium state analysis, the (P)RR was directly adsorbed on plastic wells for observing the bindings of renin/prorenin. The dissociation constants (KD) for the bindings of renin and prorenin to the pre-adsorbed receptors were 4.5 and 1.0 nM, respectively, similar to those stated in the kinetic study by BIAcore assay. The “hinge” region peptide bound to (P)RR in a dose-dependent manner with a KD estimated 17.0 nM which was five times higher than that of the decoy. The KD values for L1PPTDTTTF8P and L1PPTDTTTFKRIFLKR15P were 52 and 7.6 nM, respectively. The “hinge” peptide, as the decoy, inhibited the bindings of renin and prorenin to (P)RR. The inhibition constant (Ki) for the binding of renin and prorenin by the decoy and “hinge” were 16.7 and 15.1, and 37.1 and 30.7 nM, respectively. These in vitro studies suggest that renin has a single and prorenin has at least two high affinity binding sites for the (P)RR. 相似文献
16.
The nucleocapsid protein (NC) of HIV-1 is a small zinc finger protein that contributes to multiple steps of the viral life cycle, including the proper encapsidation of HIV RNA. This is accomplished through an interaction between NC and a region at the 5'-end of the RNA, defined as the Psi element. However, the specificity of NC for Psi or for RNA in general is not well understood. To study this problem, we used SELEX to identify high affinity RNA ligands that bind to NC. A 'winner' molecule (SelPsi), as well as a subregion of Psi RNA, were further characterized to understand the interaction between NC and SelPsi and its relationship to the interaction between NC and Psi. The comparison makes predictions about the sequence and structure of a high affinity binding site within the HIV-1 Psi element. 相似文献
17.
A novel binding assay identifies high affinity ligands to the rosiglitazone binding site of mitoNEET
Geldenhuys WJ Funk MO Awale PS Lin L Carroll RT 《Bioorganic & medicinal chemistry letters》2011,21(18):5498-5501
A novel outer mitochondrial membrane protein containing [2Fe-2S] clusters, mitoNEET was first identified through its binding to the anti-diabetic drug pioglitazone. Pioglitazone belongs to a family of drugs that are peroxisome proliferator-activated receptor (PPAR) gamma agonists, collectively known as glitazones. With the lack of pharmacological tools available to fully elucidate mitoNEET's function, we developed a binding assay to probe the glitazone binding site with the aim of developing selective and high affinity compounds. We used multiple thiazolidine-2,4-dione (TZD), 2-thioxothiazolidin-4-one (TTD), and 2-iminothiazolidin-4-one (ITD) compounds to establish several trends to enhance ligand development for the purpose of elucidating mitoNEET function. 相似文献
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Specific binding of human alpha interferon to a high affinity cell surface binding site on bovine kidney cells 总被引:11,自引:0,他引:11
Virus-induced human alpha interferon (HuIFN-alpha) derived from Namalwa cells and purified to a specific activity of 2 X 10(8) units/mg of protein was radiolabeled with 125I-labeled Bolton and Hunter reagent to a specific activity of 4-12 microCi/micrograms of protein. The binding of this 125I-IFN to bovine kidney cells was examined at 4 degrees C. Scatchard analysis of the binding data indicate the presence of 650 binding sites/cell and binding of the ligand with an apparent Kd of 6 X 10(-11) M. Trypsin or acid treatment of cells to which 125I-IFN was bound resulted in the release of greater than or equal to 77% of the radioactivity, indicating a majority of radiolabeled material was bound to the cell surface. Antibodies against human leukocyte IFN but not antibodies against human fibroblast IFN inhibited the binding of radiolabeled IFN to the cells. The binding of 125I-IFN was not inhibited by a 75-fold molar excess of mouse IFN but was inhibited 30% by a 200-fold molar excess of human beta (fibroblast) IFN. These data are compatible with the Lower biological activities of these IFNs on bovine kidney cells. Several Escherichia coli derived HuIFN-alpha s inhibited the binding of the radiolabeled IFN to the same extent as native HuIFN-alpha s, but four fragments of HuIFN-alpha 1, an E. coli-derived 86 amino acid NH2-terminal fragment as well as 3 different synthetic carboxy-terminal fragments of 140, 56, or 46 amino acids did not inhibit binding. 相似文献
20.
The high affinity binding site on polyoma virus DNA for the viral large-T protein. 总被引:36,自引:2,他引:34
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In order to map the high affinity binding site for the viral large-T protein on polyoma virus DNA, we have developed an assay which does not require purified protein. It is based on the specific elution of the large-T ATPase activity from calf thymus DNA cellulose by recombinant DNA molecules including known sequences of the viral DNA. Using this assay, a high affinity binding site has been mapped on the early region side of the ori region. Binding requires the integrity of a sequence /AGAGGC/TTCC/AGAGGC/ (nucleotides 49 to 64 in the DNA sequence of the A2 strain). Similar repeats of a PuGPuGGC sequence within less than 20 bases are not found within the viral coding regions, but are strikingly common in the control regions of papovaviruses and other eukaryotic DNAs. 相似文献