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Binding Cavity (binding + cavity)
Selected AbstractsMapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesisFEBS JOURNAL, Issue 18 2007Petra Ml, ochová Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1, site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N -acetyl- l -aspartyl- l -glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1, pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the ,fine-tuning' of GCPII substrate specificity. [source] Binding of Warfarin Influences the Acid-Base Equilibrium of H242 in Sudlow Site I of Human Serum AlbuminPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 5 2006Jennifer L. Perry ABSTRACT Sudlow Site I of human serum albumin (HSA) is located in subdomain IIA of the protein and serves as a binding cavity for a variety of ligands. In this study, the binding of warfarin (W) is examined using computational techniques and isothermal titration calorimetry (ITC). The structure of the docked warfarin anion (W,) to Site I is similar to that revealed by X-ray crystallography, with a calculated binding constant of 5.8 × 105M,1. ITC experiments (pH 7.13 and I = 0.1) carried out in three different buffers (MOPs, phosphate and Tris) reveal binding of W, is accompanied by uptake of 0.30 ± 0.02 protons from the solvent. This measurement suggests that the binding of W, is stabilized by an ion-pair interaction between protonated H242 and the phenoxide group of W,. [source] Solution structure of the PWWP domain of the hepatoma-derived growth factor familyPROTEIN SCIENCE, Issue 3 2005Nobukazu Nameki Abstract Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded ,-barrel with a PWWP-specific long loop connecting ,2 and ,3 (PR-loop), followed by a helical region including two ,-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the ,1/,2 loop (,-, arch) and the ,3/,4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity. [source] Water and urea interactions with the native and unfolded forms of a ,-barrel proteinPROTEIN SCIENCE, Issue 12 2003Kristofer Modig CD, circular dichroism; I-FABP, intestinal fatty acid-binding protein; MRD, magnetic relaxation dispersion; NOE, nuclear Overhauser effect Abstract A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and energetics of the denatured state. Here, we use 17O and 2H magnetic relaxation dispersion (MRD) to study urea-induced denaturation of intestinal fatty acid-binding protein (I-FABP). MRD is among the few methods that can provide molecular-level information about protein solvation in native as well as denatured states, and it is used here to simultaneously monitor the interactions of urea and water with the unfolding protein. Whereas CD shows an apparently two-state transition, MRD reveals a more complex process involving at least two intermediates. At least one water molecule binds persistently (with residence time >10 nsec) to the protein even in 7.5 M urea, where the large internal binding cavity is disrupted and CD indicates a fully denatured protein. This may be the water molecule buried near the small hydrophobic folding core at the D,E turn in the native protein. The MRD data also provide insights about transient (residence time <1 nsec) interactions of urea and water with the native and denatured protein. In the denatured state, both water and urea rotation is much more retarded than for a fully solvated polypeptide. The MRD results support a picture of the denatured state where solvent penetrates relatively compact clusters of polypeptide segments. [source] Protein self-association in solution: The bovine , -lactoglobulin dimer and octamerPROTEIN SCIENCE, Issue 11 2003Michael Gottschalk Abstract We have used proton magnetic relaxation dispersion (MRD) to study the self-association of bovine , -lactoglobulin variant A (BLG-A) as a function of temperature at pH 4.7 (dimer,octamer equilibrium) and as a function of NaCl concentration at pH 2.5 (monomer,dimer equilibrium). The MRD method identifies coexisting oligomers from their rotational correlation times and determines their relative populations from the associated dispersion amplitudes. From MRD-derived correlation times and hydrodynamic model calculations, we confirm that BLG-A dimers associate to octamers below room temperature. The tendency for BLG-A dimers to assemble into octamers is found to be considerably weaker than in previous light scattering studies in the presence of buffer salt. At pH 2.5, the MRD data are consistent with an essentially complete transition from monomers in the absence of salt to dimers in 1 M NaCl. Because of an interfering relaxation dispersion from nanosecond water exchange, we cannot determine the oligomer populations at intermediate salt concentrations. This nanosecond dispersion may reflect intersite exchange of water molecules trapped inside the large binding cavity of BLG-A. [source] New insights into intracellular lipid binding proteins: The role of buried waterPROTEIN SCIENCE, Issue 10 2002Christian Lücke Abstract The crystal structures of most intracellular lipid binding proteins (LBPs) show between 5 and 20 internally bound water molecules, depending on the presence or the absence of ligand inside the protein cavity. The structural and functional significance of these waters has been discussed for several LBPs based on studies that used various biophysical techniques. The present work focuses on two very different LBPs, heart-type fatty acid binding protein (H-FABP) and ileal lipid binding protein (ILBP). Using high-resolution nuclear magnetic resonance spectroscopy, certain resonances belonging to side-chain protons that are located inside the water-filled lipid binding cavity were observed. In the case of H-FABP, the pH- and temperature-dependent behavior of selected side-chain resonances (Ser82 OgH and the imidazole ring protons of His93) indicated an unusually slow exchange with the solvent, implying that the intricate hydrogen-bonding network of amino-acid side-chains and water molecules in the protein interior is very rigid. In addition, holo H-FABP appeared to display a reversible self-aggregation at physiological pH. For ILBP, on the other hand, a more solvent-accessible protein cavity was deduced based on the pH titration behavior of its histidine residues. Comparison with data from other LBPs implies that the evolutionary specialization of LBPs for certain ligand types was not only because of mutations of residues directly involved in ligand binding but also to a refinement of the internal water scaffold. [source] N -Ethylazatribenzo-21-crown-7ACTA CRYSTALLOGRAPHICA SECTION C, Issue 3 2000Jeffrey C. Bryan The structure of the title compound, 25-ethyl-2,5,12,15,22,28-hexaoxa-25-azatetracyclo[27.4.0.06,11.016,21]tritriaconta-1(29),6(11),7,9,16(21),17,19,30,32-nonaene, C28H33NO6, does not exhibit a binding cavity for cations, but is collapsed in on itself. The conformation is unique among known tribenzo-21-crown-7 structures, and may be a result of intermolecular (C,H,,) and intramolecular (C,H,O) hydrogen bonding. [source] Expression, purification, crystallization and structure of human adipocyte lipid-binding protein (aP2)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2006Eric Marr Human adipocyte lipid-binding protein (aP2) belongs to a family of intracellular lipid-binding proteins involved in the transport and storage of lipids. Here, the crystal structure of human aP2 with a bound palmitate is described at 1.5,Å resolution. Unlike the known crystal structure of murine aP2 in complex with palmitate, this structure shows that the fatty acid is in a folded conformation and that the loop containing Phe57 acts as a lid to regulate ligand binding by excluding solvent exposure to the central binding cavity. [source] Metal Binding Properties of Fluorescent Analogues of Trichogin GA,IV: A Conformational Study by Time-Resolved Spectroscopy and Molecular Mechanics InvestigationsCHEMBIOCHEM, Issue 1 2009Mariano Venanzi Prof. Abstract The metal ion binding properties of two fluorescent analogues of trichogin GA,IV, which is a natural undecapeptide showing significant antimicrobial activity, were studied by circular dichroism, time-resolved optical spectroscopy, and molecular mechanics calculations. Binding of CaII and GdIII to the peptides investigated was shown to promote a structural transition from highly helical conformations to folded structures characterized by formation of a loop that embedded the metal ion. Time-resolved spectroscopy revealed that peptide dynamics is also remarkably affected by ion binding: peptide-backbone motions slowed down to the microsecond time scale. Finally, molecular mechanics calculations emphasized the role of the central Gly5-Gly6 motif, which allowed for the twisting of the peptide segment that gave rise to the formation of the binding cavity. [source] Site-Specific Investigation of the Steady-State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier ProteinCHEMISTRY - A EUROPEAN JOURNAL, Issue 37 2010Dr. Clelia Cogliati Abstract The investigation of multi-site ligand,protein binding and multi-step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D 1H,15N line-shape analysis, which allows a reliable investigation of ligand binding occurring on micro- to millisecond timescales, have been extended to model a two-step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule,ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi-step events could be modelled, for several residues, with a two-step binding mechanism. The protein in the ligand-bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed. [source] Combined Pharmacophore Modeling, Docking, and 3D QSAR Studies of ABCB1 and ABCC1 Transporter InhibitorsCHEMMEDCHEM, Issue 11 2009Abstract Quinazolinones, indolo- and pyrrolopyrimidines with inhibitory effects toward ABCB1 (P-gp) and ABCC1 (MRP1) transporters were studied by pharmacophore modeling, docking, and 3D QSAR to describe the binding preferences of the proteins. The pharmacophore overlays between dual and/or highly selective inhibitors point to binding sites of different topology and physiochemical properties for MRP1 and P-gp. Docking of selective inhibitors into the P-gp binding cavity by the use of a structural model based on the recently resolved P-gp structure confirms the P-gp pharmacophore features identified, and reveals the interactions of some functional groups and atoms in the structures with particular protein residues. The 3D QSAR analysis of the dual-effect inhibitors allows satisfactory prediction of the selectivity index of the compounds and outlines electrostatics as most important for selectivity. The results from the combined modeling approach complement each other and could improve our understanding of the protein,ligand interactions involved, and could aid in the development of highly selective and potent inhibitors of P-gp and MRP1. [source] | ||||||||||