Rigid Binding Research Articles

Force measurements on the molecular interactions between ligand (RGD) and human platelet α IIbβ 3 receptor system

The peptide sequence arginine-glycine-aspartate (RGD) found in fibrinogen, von Willebrand factor, fibronectin, and vitronectin, plays a critical role in platelet adhesion and thrombus formation, when bound to the platelet α IIbβ 3 integrin receptor. Using atomic force microscopy (AFM), we have measured the debonding interaction between an RGD peptide-modified AFM probe tip and a human platelet surface from pN to nN levels of force. The peptide sequence, GSSSGRGDSPA, which contains the biologically active RGDSP sequence with a hydrophilic spacer sequence (GSSSG), was covalently coupled to AFM probe tips. Direct measurements on the debonding force for the RGD ligand – α IIbβ 3 platelet receptor system were carried out in Tyrode buffer at room temperature. Our results show three distinct distributions of debonding forces at a loading rate of 12 nN/s, from which we estimate the debonding force for the single ligand–receptor to be ∼93 pN. The results also show evidence for considerable extension in the flexible sample surface during the debonding process, and a linear correlation between the debonding force and the logarithm of the rate of loading. From our analysis, the zero kinetic off-rate K off(0), the single molecular binding energy E b, and the transition state x B, assuming rigid binding, were extracted from the data, and estimated to be 22.6 s −1, −2.64×10 −20 J and 0.1 nm, respectively.

Zirconium Complexes of Amine−Bis(phenolate) Ligands as Catalysts for 1-Hexene Polymerization: Peripheral Structural Parameters Strongly Affect Reactivity

Novel amine bis(phenolate) zirconium dibenzyl complexes were synthesized in quantitative yields from a versatile family of chelating amine−bis((2-hydroxyaryl)methyl) ligand precursors, their X-ray structures solved, and their reactivity in the polymerization of 1-hexene in the presence of B(C6F5)3 studied. Several minor peripheral structural modifications were studied and found to have a major influence on the catalyst performance. Thus, a variety of reactivities, ranging from extremely high to negligible, were obtained, demonstrating a unique structure−reactivity relationship. This relationship is partially revealed from the crystal structures of the precatalysts, indicating similar [ONO] ligand cores in all structures solved. A correlation between the solid and the solution structures is obtained from 1H NMR spectra, which reveal a rigid binding of the ligand to the metal. The solid structures are therefore proposed to serve as reliable references when studying structure−reactivity relationships. The mo...

Grafting of protein-protein binding sites

A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Cα and Cβ atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

HTTP `Next Generation'

We report on the results of the Protocol Design Group of the W3C's HTTP `Next Generation' Activity. The group produced and measured a prototype that shows that it is possible, largely using familiar engineering principles, to make simultaneous improvements in the following problem areas of HTTP/1.1: (1) the layering of other application protocols over HTTP; (2) modularity and extensibility; (3) networking performance and fairness; (4) the rigid binding between identifiers and protocol stacks; and (5) the opacity of layered traffic to firewalls. The prototype also suggests that these can be done in a way that may lead to unifying the Web with related middleware systems such as COM, CORBA, and Java RMI.

Compulsory Order of Substrate Binding to Herpes Simplex Virus Type 1 Thymidine Kinase: A CALORIMETRIC STUDY

Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK.dT complex. At pH 7.5 and 25 degrees C, the binding constants are 1.9 x 10(5) m(-1) for dT and 3.9 x 10(6) m(-1) for ATP binding to the binary HSV1 TK.dT complex. Binding of both substrates is enthalpy-driven and opposed by a large negative entropy change. The heat capacity change (DeltaCp) obtained from DeltaH in the range of 10-25 degrees C is -360 cal K(-1) mol(-1) for dT binding and -140 cal K(-1) mol(-1) for ATP binding. These large DeltaCp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of DeltaCp and TDeltaS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK.dT.ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.

Suitability of immobilized metal affinity chromatography for protein purification from canola.

This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his(6)-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co(2+) with iminodiacetate (IDA) as the chelating ligand, beta-glucuronidase-his(6) (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu(2+) < Ni(2+) < Zn(2+) < Co(2+) in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his(6) in forming a tridentate with Me(2+)-IDA than in forming a bidentate with Me(2+)-NTA. The more flexible binding allows for multisite interactions over the protein.

Suitability of immobilized metal affinity chromatography for protein purification from canola

This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his6-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co2+ with iminodiacetate (IDA) as the chelating ligand, β-glucuronidase-his6 (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu2+ < Ni2+ < Zn2+ < Co2+ in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his6 in forming a tridentate with Me2+–IDA than in forming a bidentate with Me2+–NTA. The more flexible binding allows for multisite interactions over the protein. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 52–58, 2000.

Grafting of Protein-Protein Interaction Epitope

Transferring the biological function of one protein to another is a key issue in understanding the structure and function relationship of proteins. We have developed a strategy for grafting protein-protein interaction epitopes. As a first step, residues at the interface of the ligand protein which strongly interact with the receptor protein were identified. Then protein scaffolds were docked onto receptor protein based on geometric complementarity. Only high docking score matches were saved. For each saved match, the scaffold protein was accepted if it had suitable positions for grafting key interaction residues of the ligand protein. These candidate residues were mutated to corresponding residues in the ligand protein at each relevant position and the mutated scaffold protein was co-minimized with receptor protein. Finally, the minimized complexes were evaluated by a scoring function deduced from statistical analysis of rigid binding data sets. As a test case, the binding epitope of barstar, the inhibitor of barnase, was grafted onto smaller proteins. Pheromone Er-1(PDB entry 1erc) has been found to be a good scaffold. The calculated binding free energy for mutated Pheromone Er-1 is equivalent to that of barstar.

Efficient electrostatic solvation model for protein-fragment docking

A method is presented for the fast evaluation of the binding energy of a protein-small molecule complex with electrostatic solvation. It makes use of a fast preprocessing step based on the assumption that the main contribution to electrostatic desolvation upon ligand binding originates from the displacement of the first shell of water molecules. For a rigid protein, the precomputation of the energy contributions on a set of grids allows the estimation of the energy in solution of about 300 protein-fragment binding modes per second on a personal computer. The docking procedure is applied to five rigid binding sites whose size ranges from 17 residues to a whole protein of 107 amino acids. Using a library of 70 mainly rigid molecules, known micromolar inhibitors or close analogs are docked and prioritized correctly. The docking based rank-ordering of the library requires about 5 h and is proposed as a complementary approach to structure-activity relationships by nuclear magnetic resonance. Proteins 2001;42:256-268.

The X-ray three-dimensional structure of avidin

Avidin is a basic, highly stable, homotetrameric protein, isolated from bird egg-white, binding up to four molecules of d-biotin with extremely high affinity ( K d∼10 −15 M). The protein has been the object of different crystallographic investigations. In all the crystal structures, the four avidin subunits display almost exact 222 symmetry. Each avidin chain (128 amino acids) is arranged in a eight-stranded antiparallel β-barrel, whose inner region defines the d-biotin binding site. The molecular bases of d-biotin affinity can be recognised in a fairly rigid binding site, which is sterically complementary to the shape and polarity of the incoming vitamin, and is readily accessible in the apoprotein structure. Avidin displays remarkable structural and functional relationships to the acidic protein sretpavidin, isolated from Streptomyces avidinii.

Ribozyme cleavage of a 2,5-phosphodiester linkage: mechanism and a restricted divalent metal-ion requirement.

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3',5'-phosphodiester linkage at the cleavage site; however, a 2',5'-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2',3'-cyclic phosphate and 5'-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3',5'-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2',5'-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2',5'-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5' to the cleavage site.

Chiral Molecular Recognition in a Tripeptide Benzylviologen Cyclophane Host

A cationic chiral cyclophane was synthesized and studied as a host for chiral and racemic π-donor molecules. The cyclophane host has a rigid binding cavity flanked by (S)-(valine-leucine-alanine) and N,N‘-dibenzyl-4,4‘-bipyridinium subunits, which allow for hydrogen-bonding and π-stacking interactions with included aromatic guest molecules. 1H NMR binding titrations were performed with several different pharmaceutically interesting guest molecules including β-blockers, NSAIDs, and amino acids and amino acid derivatives. The host−guest complexation constants were generally small for neutral and cationic guests (0−39 M-1 at 20 °C in water/acetone mixtures). However, a (R)/(S) enantioselectivity ratio of 13 ± 5 was found for DOPA, a strongly π-donating cationic guest. Two-dimensional NOESY 1H NMR spectra confirm that (R)-DOPA binds inside the cavity of the host and that there is no measurable interaction of the cavity with (S)-DOPA under the same conditions.

X-ray structures of apo and tungstate derivatives of vanadium chloroperoxidase from the fungus Curvularia inaequalis

The X-ray structures of the apo and tungstate forms of vanadium chloroperoxidase (CPO) from the fungus Curvularia inaequalis have been solved by difference Fourier techniques using the atomic model of native chloroperoxidase. In the 2.50 Å crystal structure ( R = 17.1%) of apo CPO, a water solvent molecule is found in place of the vanadate cofactor. The 2.30 Å crystal structure of tungstate CPO ( R = 16.2%) reveals the binding of the tungstate to the metal-cofactor binding site. It binds in a similar fashion to the hydrogen vanadate(V) group in the native form. However, the tungsten atom is not bound or is only weakly bound to the NE2 nitrogen atom of histidine 496 in contrast to native CPO where the vanadium atom forms a bond to the NE2 nitrogen atom of histidine 496. The overall protein structure and the metal-cofactor binding site of apo and tungstate CPO remain virtually unchanged. This means that the CPO protein matrix provides a rigid preformed metal-cofactor binding site. The binding mode of vanadate or tungstate can be described as rack-induced bonding.

Dibenzotropylidene phosphanes (TROPPs): synthesis and coinage metal complexes

Dibenzocycloheptatrienyl phosphanes (dibenzotropylidene phosphanes=TROPPR) 11a–c may be easily prepared from dibenzocycloheptatrienyl chloride 8 and the secondary phosphanes R2PH [9a: R=Ph; 9b: R=4-Me-C6H4; 9c: R=cyclohexyl (Cyc)] in good yields. Alternatively, the di(tert-butyl)phosphanyl substituted TROPPBut derivative 4 was obtained along with the phosphane 5 by a mechanistically still unknown rearrangement of a strained phosphorus ylide I. The conformations of these new phosphanes were established by X-ray analyses performed for the compounds TROPPBut4 and TROPPPh11a. The R2P moiety is bonded to an axial position of the central seven-membered ring, which adopts a boat conformation. Thereby a rigid concave binding site containing a phosphane and an olefin function is formed, which should allow the synthesis of a wide range of transition metal complexes. In order to test how far the particular shape of the TROPP-type ligands enforces metal–olefin interactions, the coinage metal complexes [(TROPPPh)Cu(µ2-Cl)]2, 13, [(TROPPPh)Ag(µ2-O2SOCF3)]2, 16, [(TROPPPh)2Ag][O3SCF3], 17 and [(TROPPPh)AuCl], 19 were prepared. These were completely characterized by multinuclear NMR experiments in solution and the solid state, as well as by X-ray analyses. The structural and NMR data show that the interaction between the metal center M and the olefin moiety of the TROPP ligand is weak and decreases in the order Cu>Ag>Au. Indeed, for 19 (M=Au) no interaction could be observed. In the silver complex 17, coupling between an Ag nucleus and a proton of a bonded olefin was determined for the first time [J(109/107AgH)=0.4 Hz]. In solution the complexes derived from TROPP-type ligands seem to have an enhanced (kinetic) stability.

Hydrogen bonds and salt bridges across protein-protein interfaces.

To understand further, and to utilize, the interactions across protein-protein interfaces, we carried out an analysis of the hydrogen bonds and of the salt bridges in a collection of 319 non-redundant protein-protein interfaces derived from high-quality X-ray structures. We found that the geometry of the hydrogen bonds across protein interfaces is generally less optimal and has a wider distribution than typically observed within the chains. This difference originates from the more hydrophilic side chains buried in the binding interface than in the folded monomer interior. Protein folding differs from protein binding. Whereas in folding practically all degrees of freedom are available to the chain to attain its optimal configuration, this is not the case for rigid binding, where the protein molecules are already folded, with only six degrees of translational and rotational freedom available to the chains to achieve their most favorable bound configuration. These constraints enforce many polar/charged residues buried in the interface to form weak hydrogen bonds with protein atoms, rather than strongly hydrogen bonding to the solvent. Since interfacial hydrogen bonds are weaker than the intra-chain ones to compete with the binding of water, more water molecules are involved in bridging hydrogen bond networks across the protein interface than in the protein interior. Interfacial water molecules both mediate non-complementary donor-donor or acceptor-acceptor pairs, and connect non-optimally oriented donor-acceptor pairs. These differences between the interfacial hydrogen bonding patterns and the intra-chain ones further substantiate the notion that protein complexes formed by rigid binding may be far away from the global minimum conformations. Moreover, we summarize the pattern of charge complementarity and of the conservation of hydrogen bond network across binding interfaces. We further illustrate the utility of this study in understanding the specificity of protein-protein associations, and hence in docking prediction and molecular (inhibitor) design.

Role of Arg-277 in the binding of pyridoxal 5'-phosphate to Trypanosoma brucei ornithine decarboxylase.

The pyridoxal 5'-phosphate (PLP) binding site in Trypanosoma brucei ornithine decarboxylase (ODC) has been studied by site-directed mutagenesis and spectroscopy. The beta/alpha barrel model proposed for the eukaryotic ODC structure predicts that the phosphate group of PLP is stabilized by interactions with a Gly-rich loop (residues 235-237) and by a salt bridge to Arg-277 [Grishin, N. V., Phillips, M. A., & Goldsmith, E. J. (1995) Protein Sci. 4, 1291-1304]. Mutation of Arg-277 to Ala increases the K(m) for PLP by 270-fold compared to that of wild-type ODC while reducing k(cat) by only 2-fold at pH 8. PLP binding affinity was measured directly by ultrafiltration; the K(d) for PLP is at least 20-fold higher in the mutant enzyme at pH 8. In addition, R277A ODC also has weaker binding affinities for a series of cofactor analogs than the wild-type enzyme. These results demonstrate that Arg-277 is necessary for high-affinity PLP binding by ODC. The 31P NMR spectra of ODC suggest that the phosphate is bound in a strained conformation as a dianion to both wild-type and R277A ODC. However, the 31P chemical shift for R277A ODC (6.7 ppm) is 0.5 ppm downfield from that observed for the wild-type enzyme, indicating that the environment of the enzyme-bound phosphate is altered in the mutant enzyme. The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, corresponding to the titration of a protonated species with a pK(a) of approximately 8.5. Concomitant with these changes are a decreased k(cat) and an altered absorption spectra which arises from bound PLP. PLP bound to wild-type ODC has a 31P chemical shift and a CD signal observable over the entire tested pH range (7-9). In contrast, for R277A ODC between pH 8 and 9, the 31P chemical shift becomes solution-like and the CD signal is abolished. The data suggest that for R277A ODC the rigid PLP binding mode which characterizes the wild-type enzyme is lost at high pH. Thus, multiple interactions between the wild-type active site and PLP maintain the cofactor in a constrained conformation that is essential for efficient catalysis, tempering the consequence of the removal of any single interaction.

Synthesis of a tripalladium cluster containing an η1:η2:η2-bridging butadienyl ligand stabilized by a zwitterionic structure

Abstract An η1-butadienyl complex [trans-η1-CH2=C(Me)C=CH2Pd(PPh3)2Cl] (1) reacted with [(μ-η2:η2-1,3-butadiene)Pd2(PPh3)(μCl)Cl] (2) to result in displacement of the diene ligand of 2 accompanied by exchange of PPh3 of 1 with Cl anion of 2 producing a butadienyl tripalladium cluster [(μ-CH2=C(Me)C=CH2)Pd(PPh3)Cl2 · Pd2(PPh3)2(μ-Cl)] (3) stabilized by the zwitterionic structure in moderate yield. The X-ray structure analysis of 3 revealed rigid binding of [Pd2(PPh3)2(μ-Cl)]+ and [CH2 =C(Me)C=CH2Pd(PPh3)Cl2]− through the π-bond coordination of the butadienyl group to the dipalladium cation.

Room temperature (nπ∗) phosphorescence of indanetrione (anhydrous ninhydrine) in phthalic anhydride matrix

Indanetrione, a cis vicinal cyclic triketone, is found to exhibit room temperature (nπ∗) phosphorescence (RTP) in a phthalic anhydride matrix in addition to (nπ∗) fluorescence. The compound does not show RTP in benzophenone mixed crystals or in any other solvent studied. A rigid binding of the cyclic triketone in the phthalic anhydride matrix, lowering the T 1 → S 0 nonradiative rate, has been proposed as the explanation for RTP.

Multiple Wavelength Anomalous Diffraction (MAD) Crystal Structure of Rusticyanin: a Highly Oxidizing Cupredoxin with Extreme Acid Stability

The X-ray crystal structure of the oxidized form of the extremely stable and highly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidanshas been determined by the method of multiwavelength anomalous diffraction (MAD) and refined to 1.9 Å resolution. Like other cupredoxins, rusticyanin is a copper-containing metalloprotein, which is composed of a core β-sandwich fold. In rusticyanin the β-sandwich is composed of a six- and a seven-stranded β-sheet. Also like other cupredoxins, the copper ion is coordinated by a cluster of four conserved residues (His85, Cys138, His143, Met148) arranged in a distorted tetrahedron. Rusticyanin has a redox potential of 680 mV, roughly twice that of any other cupredoxin, and it is optimally active at pH values ≤2. By comparison with other cupredoxins, the three-dimensional structure of rusticyanin reveals several possible sources of the chemical differences, including more ordered secondary structure and more intersheet connectivity than other cupredoxins. The acid stability and redox potential of rusticyanin may also be enhanced over other cupredoxins by a more extensive internal hydrogen bonding network and by more extensive hydrophobic interactions surrounding the copper binding site. Finally, reduction in the number of charged residues surrounding the active site may also make a major contribution to acid stability. We propose that the resulting rigid copper binding site, which is constrained by the surrounding hydrophobic environment, structurally and electronically favours Cu(I). We propose that the two extreme chemical properties of rusticyanin are interrelated; the same unique structural features that enhance acid stability also lead to elevated redox potential.

Synthesis and solution complexation behaviour of dimeric zinc-selective bis(benzimidazole) derivatives

The synthesis of 1,1′-bis(4-tert-butylbenzyl)-2,2′-bis(benzimidazole)-4,4′-dicarboxylic acid and its N-substituted analogues is reported. Complexation of the ligand by divalent Zn, Cu, Ni, Pb and Cd ions has been studied in methanolic solution by 1H NMR, FT-IR and electrospray mass spectrometry. The ligand forms a dimeric neutral M2L2 complex selectively with zinc, favoured by the two rigid tetrahedral binding sites created by the cooperative ligation of two orthogonally disposed ligands. Overall binding affinities follow the sequence Zn > Cd ≈ Ni >Pb > Cu and solution speciation has been defined by ESMS showing non-selective formation of, for example, ML, MLH, M2L2X species with all ions but zinc, where the M2L2 species was predominant.