Thermodynamics of metal ion binding. 1. Metal ion binding by wild-type carbonic anhydrase

Understanding the energetic consequences of molecular structure in aqueous solution is a prerequisite to the rational design of synthetic motifs with predictable properties. Such properties include ligand binding and the collapse of polymer chains into discrete three-dimensional structures. Despite advances in macromolecular structure determination, correlations of structure with high-resolution thermodynamic data remain limited. Here we compare thermodynamic parameters for the binding of Zn(II), Cu(II), and Co(II) to human carbonic anhydrase II. These calorimetrically determined values are interpreted in terms of high-resolution X-ray crystallographic data. While both zinc and cobalt are bound with a 1:1 stoichiometry, CAII binds two copper ions. Considering only the high-affinity site, there is a diminution in the enthalpy of binding through the series Co(II) --> Zn(II) --> Cu(II) that mirrors the enthalpy of hydration; this observation reinforces the notion that the thermodynamics of solute association with water is at least as important as the thermodynamics of solute-solute interaction and that these effects must be considered when interpreting association in aqueous solution. Additionally, DeltaC(p) data suggest that zinc binding to CAII proceeds with a greater contribution from desolvation than does binding of either copper or cobalt, suggesting Nature optimizes binding by optimizing desolvation.     

Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.     

Biochemical and structural characterization of Salmonella typhimurium glyoxalase II: new insights into metal ion selectivity

Glyoxalase II is a hydrolytic enzyme part of the glyoxalase system, responsible for detoxifying several cytotoxic compounds employing glutathione. Glyoxalase II belongs to the superfamily of metallo-beta-lactamases, with a conserved motif able to bind up to two metal ions in their active sites, generally zinc. Instead, several eukaryotic glyoxalases II have been characterized with different ratios of iron, zinc, and manganese ions. We have expressed a gene coding for a putative member of this enzyme superfamily from Salmonella typhimurium that we demonstrate, on the basis of its activity, to be a glyoxalase II, named GloB. Recombinant GloB expressed in Escherichia coli was purified with variable amounts of iron, zinc, and manganese. All forms display similar activities, as can be shown from protein expression in minimal medium supplemented with specific metal ions. The crystal structure of GloB solved at 1.4 A shows a protein fold and active site similar to those of its eukaryotic homologues. NMR and EPR experiments also reveal a conserved electronic structure at the metal site. GloB is therefore able to accommodate these different metal ions and to carry out the hydrolytic reaction with similar efficiencies in all cases. The metal promiscuity of this enzyme (in contrast to other members of the same superfamily) can be accounted for by the presence of a conserved Asp residue acting as a second-shell ligand that is expected to increase the hardness of the metal binding site, therefore favoring iron uptake in glyoxalases II.     

Structural parameterization of the binding enthalpy of small ligands

A major goal in ligand and drug design is the optimization of the binding affinity of selected lead molecules. However, the binding affinity is defined by the free energy of binding, which, in turn, is determined by the enthalpy and entropy changes. Because the binding enthalpy is the term that predominantly reflects the strength of the interactions of the ligand with its target relative to those with the solvent, it is desirable to develop ways of predicting enthalpy changes from structural considerations. The application of structure/enthalpy correlations derived from protein stability data has yielded inconsistent results when applied to small ligands of pharmaceutical interest (MW < 800). Here we present a first attempt at an empirical parameterization of the binding enthalpy for small ligands in terms of structural information. We find that at least three terms need to be considered: (1) the intrinsic enthalpy change that reflects the nature of the interactions between ligand, target, and solvent; (2) the enthalpy associated with any possible conformational change in the protein or ligand upon binding; and, (3) the enthalpy associated with protonation/deprotonation events, if present. As in the case of protein stability, the intrinsic binding enthalpy scales with changes in solvent accessible surface areas. However, an accurate estimation of the intrinsic binding enthalpy requires explicit consideration of long-lived water molecules at the binding interface. The best statistical structure/enthalpy correlation is obtained when buried water molecules within 5-7 A of the ligand are included in the calculations. For all seven protein systems considered (HIV-1 protease, dihydrodipicolinate reductase, Rnase T1, streptavidin, pp60c-Src SH2 domain, Hsp90 molecular chaperone, and bovine beta-trypsin) the binding enthalpy of 25 small molecular weight peptide and nonpeptide ligands can be accounted for with a standard error of +/-0.3 kcal x mol(-1).     

Zn2+ regulation of ornithine transcarbamoylase. II. Metal binding site

Two types of conformational changes are mediated in Escherichia coli ornithine transcarbamoylase by the metal ion zinc. Upon binding of zinc in rapid equilibrium, the enzyme undergoes an allosteric transition. In the absence of substrates, the zinc-bound enzyme further undergoes a slow isomerization with a concomitant activity loss. Three metal ions are tightly complexed in the isomerized enzyme as determined by gel chromatography and atomic absorption spectroscopy. Since the enzyme is a trimer composed of identical subunits, one zinc ion is bound per enzyme monomer. With the application of site-directed mutagenesis, the cysteinyl residue at position 273 of the enzyme has been identified as a metal ligand. When this residue is replaced by an alanine, zinc is no longer a tight-binding inhibitor and does not promote isomerization. The alteration in the action of zinc on the mutant enzyme is attributed to a reduced metal affinity. The mutant enzyme, when saturated by the metal, displays an intrinsic allostery unchanged from that of the wild-type; an identical Hill coefficient of 1.5 is found for zinc binding to the Ala273 and wild-type enzymes. Cys273 is also a binding site of L-ornithine. At pH 8.5, the Ala273 enzyme binds the substrate analog L-norvaline ten times more weakly and exhibits a kcat/Kmorn that is 27 times less than that of the wild-type enzyme. This finding supports our earlier interpretation that the zinc-induced ornithine co-operativity of ornithine transcarbamoylase is caused by direct competition between L-ornithine and the metal for the same site. As controls, each of the remaining three cysteinyl residues of the bacterial ornithine transcarbamoylase has also been replaced with alanine. These sulfhydryl groups are found not to be related to zinc complexation, ornithine binding or enzyme allostery.     

The thermodynamics of lanthanide ion binding to adriamycin

We have examined the thermodynamics of lanthanide ion binding to adriamycin by monitoring the effects of variations in temperature on the dissociation constants of various lanthanide ion complexes of the drug. These constants were obtained by analyzing the extent of quenching of the fluorecence of adriamycin in the presence of lanthanide ions in terms of an equilibrium binding process. Our binding model included the following features, all of which are supported by evidence derived from previous published reports, vide infra. The lanthanides form 1:1 complexes with adriamycin. The binding is dependent on the pH of the solution, indicating that only the nonprotonated amine form of the drug participates in lanthanide ion binding. And finally the drug self-associates in solution to for a dimeric species. Our present results indicate that the binding process is almost completely independent of temperature, indicating that the enthalpy of complex formation is extremely small. The entropy terms are consistent with the formation of a complex in which the adriamycin acts as a bidentate ligand. Our results suggest that the lanthanide complexes are isostructural, at least as far as the adriamycin is concerned, throughout the lanthanide series.     

Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole

The mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT) binds to an occluded, nonpolar cavity in the mouse major urinary protein-I (MUP-I). The thermodynamics of this interaction have been characterized using isothermal titration calorimetry (ITC). MUP-I-SBT binding is accompanied by a large favorable enthalpy change (DeltaH = -11.2 kcal/mol at 25 degrees C), an unfavorable entropy change (-TDeltaS = 2.8 kcal/mol at 25 degrees C), and a negative heat capacity change [DeltaC(p)() = -165 cal/(mol K)]. Thermodynamic analysis of binding between MUP-I and several 2-alkyl-4,5-dihydrothiazole ligands indicated that the alkyl chain contributes more favorably to the enthalpy and less favorably to the entropy of binding than would be expected on the basis of the hydrophobic desolvation of short-chain alcohols. However, solvent transfer experiments indicated that desolvation of SBT is accompanied by a net unfavorable change in enthalpy (DeltaH = +1.0 kcal/mol) and favorable change in entropy (-TDeltaS = -1.8 kcal/mol). These results are discussed in terms of the possible physical origins of the binding thermodynamics, including (1) hydrophobic desolvation of both the protein and the ligand, (2) formation of a buried water-mediated hydrogen bond network between the protein and ligand, (3) formation of strong van der Waals interactions, and (4) changes in the structure, dynamics, and/or hydration of the protein upon binding.     

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.     

Zinc coordination geometry and ligand binding affinity: the structural and kinetic analysis of the second-shell serine 228 residue and the methionine 180 residue of the aminopeptidase from Vibrio proteolyticus

The chemical properties of zinc make it an ideal metal to study the role of coordination strain in enzymatic rate enhancement. The zinc ion and the protein residues that are bound directly to the zinc ion represent a functional charge/dipole complex, and polarization of this complex, which translates to coordination distortion, may tune electrophilicity, and hence, reactivity. Conserved protein residues outside of the charge/dipole complex, such as second-shell residues, may play a role in supporting the electronic strain produced as a consequence of functional polarization. To test the correlation between charge/dipole polarity and ligand binding affinity, structure-function studies were carried out on the dizinc aminopeptidase from Vibrio proteolyticus. Alanine substitutions of S228 and M180 resulted in catalytically diminished enzymes whose crystal structures show very little change in the positions of the metal ions and the protein residues. However, more detailed inspections of the crystal structures show small positional changes that account for differences in the zinc ion coordination geometry. Measurements of the binding affinity of leucine phosphonic acid, a transition state analogue, and leucine, a product, show a correlation between coordination geometry and ligand binding affinity. These results suggest that the coordination number and polarity may tune the electrophilicity of zinc. This may have provided the evolving enzyme with the ability to discriminate between reaction coordinate species.     

Factorising ligand affinity: a combined thermodynamic and crystallographic study of trypsin and thrombin inhibition.

The binding of a series of low molecular weight ligands towards trypsin and thrombin has been studied by isothermal titration calorimetry and protein crystallography. In a series of congeneric ligands, surprising changes of protonation states occur and are overlaid on the binding process. They result from induced pK(a) shifts depending on the local environment experienced by the ligand and protein functional groups in the complex (induced dielectric fit). They involve additional heat effects that must be corrected before any conclusion on the binding enthalpy (DeltaH) and entropy (DeltaS) can be drawn. After correction, trends in both contributions can be interpreted in structural terms with respect to the hydrogen bond inventory or residual ligand motions. For all inhibitors studied, a strong negative heat capacity change (DeltaC(p)) is detected, thus binding becomes more exothermic and entropically less favourable with increasing temperature. Due to a mutual compensation, Gibbs free energy remains virtually unchanged. The strong negative DeltaC(p) value cannot solely be explained by the removal of hydrophobic surface portions of the protein or ligand from water exposure. Additional contributions must be considered, presumably arising from modulations of the local water structure, changes in vibrational modes or other ordering parameters. For thrombin, smaller negative DeltaC(p) values are observed for ligand binding in the presence of sodium ions compared to the other alkali ions, probably due to stabilising effects on the protein or changes in the bound water structure.     

Equilibrium binding studies of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus

A tryptophan-shifted mutant of phosphofructokinase (PFK) from Bacillus stearothermophilus has been constructed. This mutant, which is functionally similar to wild-type, provides the opportunity to examine the allosteric properties of PFK under equilibrium conditions. The unique fluorescence properties of the tryptophan-shifted mutant enzyme, W179F/F230W, have been utilized to deduce the thermodynamics of ligand binding and the allosteric perturbations in the absence of catalytic turnover. Specifically, phospho(enol)pyruvate (PEP) and MgADP binding to the mutant PFK can be directly observed using tryptophan fluorescence, and dissociation constants for these ligands have been measured to be equal to 2.71 +/- 0.04 and 90.4 +/- 3.5 microM, respectively. In addition, the homotropic couplings for the allosteric ligands have been assessed for the first time. PEP binds cooperatively with a Hill number of 2.9 +/- 0.3, while MgADP binding is not cooperative. The equilibrium couplings between these ligands and the substrate fructose 6-phosphate (Fru-6-P) have also been determined and follow the same trends with temperature observed under steady-state kinetic assay conditions using wild-type PFK, indicating that the presence of bound MgATP has little influence on the allosteric interactions. Like wild-type PFK, the coupling free energies for the mutant result from largely compensating enthalpy and entropy components at 25 degrees C. Furthermore, the sign of each coupling free energy, which signifies the nature of the allosteric effect, is opposite that of the enthalpy contribution and is therefore due to the larger absolute value of the associated entropy change. This characteristic stands in direct contrast to the thermodynamic basis of the allosteric response in the homologous PFK from E. coli in which the sign of the coupling free energy is established by the sign of the coupling enthalpy.     

Order changes within receptor systems upon ligand binding: receptor tightening/oligomerisation and the interpretation of binding parameters

Recent hydrogen-deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (DeltaH) and order (DeltaS) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e. it shows positive or negative cooperativity between ligand binding and the receptor structure. In the case of membrane-bound receptors, such as G protein-coupled receptors (GPCRs) and ligand gated ion channel receptors (LGICRs), most binding studies have focussed on association/dissociation constants. Where these have been broken down into enthalpic and entropic contributions, the phenomenon of "thermodynamic discrimination" between antagonists and agonists has often been noted; e.g. for a receptor where agonist binding is predominantly enthalpy driven, antagonist binding is predominantly entropy driven and vice versa. These data have not previously been considered in terms of the tightening, or loosening, of receptor structures that respectively occurs upon positively, or negatively, cooperative binding of ligand. Nor have they been considered in light of the homo- and hetero-oligomerisation of GPCRs and the possibility of ligand-induced changes in oligomerisation. Here, we argue that analysis of the DeltaH and DeltaS of ligand binding may give useful information on ligand-induced changes in membrane-bound receptor oligomers, relevant to the differing effects of agonists and antagonists.     

On the molecular basis of the high affinity binding of basic amino acids to LAOBP,a periplasmic binding protein from Salmonella typhimurium

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high‐affinity binding of ligands to proteins is still limited; such is the case for l ‐lysine–l ‐arginine–l ‐ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l ‐arginine, l ‐lysine, and l ‐ornithine with nanomolar affinity and to l ‐histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l ‐histidine and l ‐arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~?300 cal mol^?1 K^?1, most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000‐fold higher affinity of LAOBP for l ‐arginine as compared with l ‐histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy‐driven micromolar affinity toward l ‐arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley & Sons, Ltd.     

Conformational dynamics and thermodynamics of protein-ligand binding studied by NMR relaxation

Protein conformational dynamics can be critical for ligand binding in two ways that relate to kinetics and thermodynamics respectively. First, conformational transitions between different substates can control access to the binding site (kinetics). Secondly, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics). In the present paper, I focus on the second topic, summarizing our recent results on the role of conformational entropy in ligand binding to Gal3C (the carbohydrate-recognition domain of galectin-3). NMR relaxation experiments provide a unique probe of conformational entropy by characterizing bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side chain order parameters, we have estimated the contributions from conformational entropy to the free energy of binding. Overall, the conformational entropy of Gal3C increases upon ligand binding, thereby contributing favourably to the binding affinity. Comparisons with the results from isothermal titration calorimetry indicate that the conformational entropy is comparable in magnitude to the enthalpy of binding. Furthermore, there are significant differences in the dynamic response to binding of different ligands, despite the fact that the protein structure is virtually identical in the different protein-ligand complexes. Thus both affinity and specificity of ligand binding to Gal3C appear to depend in part on subtle differences in the conformational fluctuations that reflect the complex interplay between structure, dynamics and ligand interactions.     

Computational protocol for predicting the binding affinities of zinc containing metalloprotein-ligand complexes

Zinc is one of the most important metal ions found in proteins performing specific functions associated with life processes. Coordination geometry of the zinc ion in the active site of the metalloprotein-ligand complexes poses a challenge in determining ligand binding affinities accurately in structure-based drug design. We report here an all atom force field based computational protocol for estimating rapidly the binding affinities of zinc containing metalloprotein-ligand complexes, considering electrostatics, van der Waals, hydrophobicity, and loss in conformational entropy of protein side chains upon ligand binding along with a nonbonded approach to model the interactions of the zinc ion with all the other atoms of the complex. We examined the sensitivity of the binding affinity predictions to the choice of Lennard-Jones parameters, partial atomic charges, and dielectric treatments adopted for system preparation and scoring. The highest correlation obtained was R2 = 0.77 (r = 0.88) for the predicted binding affinity against the experiment on a heterogenous dataset of 90 zinc containing metalloprotein-ligand complexes consisting of five unique protein targets. Model validation and parameter analysis studies underscore the robustness and predictive ability of the scoring function. The high correlation obtained suggests the potential applicability of the methodology in designing novel ligands for zinc-metalloproteins. The scoring function has been web enabled for free access at www.scfbio-iitd.res.in/software/drugdesign/bapplz.jsp as BAPPL-Z server (Binding Affinity Prediction of Protein-Ligand complexes containing Zinc metal ions).     

High resolution structure of an alternate form of the ferric ion binding protein from Haemophilus influenzae

The periplasmic iron binding protein of pathogenic Gram-negative bacteria performs an essential role in iron acquisition from transferrin and other iron sources. Structural analysis of this protein from Haemophilus influenzae identified four amino acids that ligand the bound iron: His(9), Glu(57), Tyr(195), and Tyr(196). A phosphate provides an additional ligand, and the presence of a water molecule is required to complete the octahedral geometry for stable iron binding. We report the 1.14-A resolution crystal structure of the iron-loaded form of the H. influenzae periplasmic ferric ion binding protein (FbpA) mutant H9Q. This protein was produced in the periplasm of Escherichia coli and, after purification and conversion to the apo form, was iron-loaded. H9Q is able to bind ferric iron in an open conformation. A surprising finding in the present high resolution structure is the presence of EDTA located at the previously determined anion ternary binding site, where phosphate is located in the wild type holo and apo structures. EDTA contributes four of the six coordinating ligands for iron, with two Tyr residues, 195 and 196, completing the coordination. This is the first example of a metal binding protein with a bound metal.EDTA complex. The results suggest that FbpA may have the ability to bind and transport iron bound to biological chelators, in addition to bare ferric iron.     

Deciphering metal ion preference and primary coordination sphere robustness of a designed zinc finger with high‐resolution mass spectrometry

Small zinc finger (ZnF) motifs are promising molecular scaffolds for protein design owing to their structural robustness and versatility. Moreover, their characterization provides important insights into protein folding in general. ZnF motifs usually possess an exceptional specificity and high affinity towards Zn(II) ion to drive folding. While the Zn(II) ion is canonically coordinated by two cysteine and two histidine residues, many other coordination spheres also exist in small ZnFs, all having four amino acid ligands. Here we used high‐resolution mass spectrometry to study metal ion binding specificity and primary coordination sphere robustness of a designed zinc finger, named MM1. Based on the results, MM1 possesses high specificity for zinc with sub‐micromolar binding affinity. Surprisingly, MM1 retains metal ion binding affinity even in the presence of selective alanine mutations of the primary zinc coordinating amino acid residues.     

Functional and computational studies of the ligand-associated metal binding site of beta3 integrins

A combination of experimental and computational approaches was used to provide a structural context for the role of the beta3 integrin subunit ligand-associated metal binding site (LIMBS) in the binding of physiological ligands to beta3 integrins. Specifically, we have carried out (1) adhesion assays on cells expressing normal alphaIIbeta3, normal alphaVbeta3, or the corresponding beta3 D217A LIMBS mutants; and (2) equilibrium and nonequilibrium (steered) molecular dynamics (MD) simulations of eptifibatide in complex with either a fully hydrated normal alphaIIbeta3 integrin fragment (alphaIIb beta-propeller and the beta3 betaA (I-like), hybrid, and PSI domains) or the equivalent beta3 D217A mutant. Normal alphaIIbeta3 expressing cells adhered to immobilized fibrinogen and echistatin, whereas cells expressing the alphaIIbeta3 D217A LIMBS mutant failed to adhere to either ligand. Similarly, the equivalent alphaVbeta3 mutant was unable to support adhesion to vitronectin or fibrinogen. The alphaIIbeta3 D217A mutation increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the beta3 PSI domain, indicating that this mutation induced allosteric changes in the protein. Steered MD simulating the unbinding of eptifibatide from either normal alphaIIbeta3 or the equivalent beta3 D217A mutant suggested that the reduction in ligand binding caused by the LIMBS mutant required the loss of both the LIMBS and the metal ion-dependent adhesion site (MIDAS) metal ions. Our computational results indicate that the LIMBS plays a crucial role in ligand binding to alphaIIbeta3 by virtue of its effects on the coordination of the MIDAS.     

Merging the binding sites of aldose and aldehyde reductase for detection of inhibitor selectivity-determining features

Inhibition of human aldose reductase (ALR2) evolved as a promising therapeutic concept to prevent late complications of diabetes. As well as appropriate affinity and bioavailability, putative inhibitors should possess a high level of selectivity for ALR2 over the related aldehyde reductase (ALR1). We investigated the selectivity-determining features by gradually mapping the residues deviating between the binding pockets of ALR1 and ALR2 into the ALR2 binding pocket. The resulting mutational constructs of ALR2 (eight point mutations and one double mutant) were probed for their influence towards ligand selectivity by X-ray structure analysis of the corresponding complexes and isothermal titration calorimetry (ITC). The binding properties of these mutants were evaluated using a ligand set of zopolrestat, a related uracil derivative, IDD388, IDD393, sorbinil, fidarestat and tolrestat. Our study revealed induced-fit adaptations within the mutated binding site as an essential prerequisite for ligand accommodation related to the selectivity discrimination of the ligands. However, our study also highlights the limits of the present understanding of protein-ligand interactions. Interestingly, binding site mutations not involved in any direct interaction to the ligands in various cases show significant effects towards their binding thermodynamics. Furthermore, our results suggest the binding site residues deviating between ALR1 and ALR2 influence ligand affinity in a complex interplay, presumably involving changes of dynamic properties and differences of the solvation/desolvation balance upon ligand binding.     

Analysis of zinc binding sites in protein crystal structures

The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.