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.     

Inhibitor coordination interactions in the binuclear manganese cluster of arginase

Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. The structure and stability of the binuclear manganese cluster are critical for catalytic activity as it activates the catalytic nucleophile, metal-bridging hydroxide ion, and stabilizes the tetrahedral intermediate and its flanking states. Here, we report X-ray structures of a series of inhibitors bound to the active site of arginase, and each inhibitor exploits a different mode of coordination with the Mn(2+)(2) cluster. Specifically, we have studied the binding of fluoride ion (F(-); an uncompetitive inhibitor) and L-arginine, L-valine, dinor-N(omega)-hydroxy-L-arginine, descarboxy-nor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid. Some inhibitors, such as fluoride ion, dinor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid, cause the net addition of one ligand to the Mn(2+)(2) cluster. Other inhibitors, such as descarboxy-nor-N(omega)-hydroxy-L-arginine, simply displace the metal-bridging hydroxide ion of the native enzyme and do not cause any net change in the metal coordination polyhedra. The highest affinity inhibitors displace the metal-bridging hydroxide ion (and sometimes occupy a Mn(2+)(A) site found vacant in the native enzyme) and maintain a conserved array of hydrogen bonds with their alpha-amino and -carboxylate groups.     

Het-PDB Navi.: a database for protein-small molecule interactions

The genomes of more than 100 species have been sequenced, and the biological functions of encoded proteins are now actively being researched. Protein function is based on interactions between proteins and other molecules. One approach to assuming protein function based on genomic sequence is to predict interactions between an encoded protein and other molecules. As a data source for such predictions, knowledge regarding known protein-small molecule interactions needs to be compiled. We have, therefore, surveyed interactions between proteins and other molecules in Protein Data Bank (PDB), the protein three-dimensional (3D) structure database. Among 20,685 entries in PDB (April, 2003), 4,189 types of small molecules were found to interact with proteins. Biologically relevant small molecules most often found in PDB were metal ions, such as calcium, zinc, and magnesium. Sugars and nucleotides were the next most common. These molecules are known to act as cofactors for enzymes and/or stabilizers of proteins. In each case of interactions between a protein and small molecule, we found preferred amino acid residues at the interaction sites. These preferences can be the basis for predicting protein function from genomic sequence and protein 3D structures. The data pertaining to these small molecules were collected in a database named Het-PDB Navi., which is freely available at http://daisy.nagahama-i-bio.ac.jp/golab/hetpdbnavi.html and linked to the official PDB home page.     

C-H. . .pi interactions in the metal-porphyrin complexes with chelate ring as the H acceptor

Specific C-H. . .pi interactions with the pi-system of porphyrinato chelate ring were found in crystal structures of transition metal complexes from the Cambridge Structural Database and statistical analysis of geometrical parameters for intramolecular and intermolecular interactions was done. By density functional theory calculations on a model system it was evaluated that an interaction energy is above 1.5 kcal/mol and that the strongest interaction occurs when the distance between hydrogen atom and the center of the chelate ring is 2.6 A. This prediction is in good agreement with the distances for intermolecular interactions found in the crystal structures. In many cases the intramolecular interaction distances are much shorter than 2.6 A, and these short distances are caused by geometrical constrains. The C-H. . .pi interactions with chelate ring of porphyrinato ligand can influence the structure, contribute to its stability, and play some role in the function of biomolecules with metalo porphyrins.     

Biological recognition of phosphate and sulfate

The biological recognition of trigonal pyramids has been studied in a statistical analysis of the Cambridge Structural Data Base. The preferential stereochemistry of pyramidal anion-Lewis acid interactions is easily described for the phosphonyl dianion (R-PO3(2-), as found in the phosphate group) and the sulfonyl monoanion (R-SO3-, as found in the sulfate group) by trans/gauche conformational terminology. Interactions between these pyramidal anions and metals generally prefer gauche geometry (as defined by the R-X = O-Mn+ torsion angle, X = P or S). Interactions between phosphonyls and hydrogen bond donors likewise display a preference for gauche orientation. Interactions between sulfonyls and hydrogen bond donors exhibit a preference for gauche stereochemistry and also cluster in eclipsed regions. These hydrogen bond motifs provide a greater understanding of the function of pyramidal anions in biological structure and function. For example, interactions of the sulfate monoanion are important for the binding of heparin, a sulfated glycosaminoglycan, to antithrombin III.     

Preferential interactions of trehalose,L-arginine.HCl and sodium chloride with therapeutically relevant IgG1 monoclonal antibodies

Preferential interactions of weakly interacting formulation excipients govern their effect on the equilibrium and kinetics of several reactions of protein molecules in solution. Using vapor pressure osmometry, we characterized the preferential interactions of commonly used excipients trehalose, L-arginine.HCl and NaCl with three therapeutically-relevant, IgG1 monoclonal antibodies that have similar size and shape, but differ in their surface hydrophobicity and net charge. We further characterized the effect of these excipients on the reversible self-association, aggregation and viscosity behavior of these antibody molecules. We report that trehalose, L-arginine.HCl and NaCl are all excluded from the surface of the three IgG1 monoclonal antibodies, and that the exclusion behavior is linearly related to the excipient molality in the case of trehalose and NaCl, whereas a non-linear behavior is observed for L-arginine.HCl. Interestingly, we find that the magnitude of trehalose exclusion depends upon the nature of the protein surface. Such behavior is not observed in case of NaCl and L-arginine.HCl as they are excluded to the same extent from the surface of all three antibody molecules tested in this study. Analysis of data presented in this study provides further insight into the mechanisms governing excipient-mediated stabilization of mAb formulations.     

iPfam: visualization of protein-protein interactions in PDB at domain and amino acid resolutions

SUMMARY: There are many resources that contain information about binary interactions between proteins. However, protein interactions are defined by only a subset of residues in any protein. We have implemented a web resource that allows the investigation of protein interactions in the Protein Data Bank structures at the level of Pfam domains and amino acid residues. This detailed knowledge relies on the fact that there are a large number of multidomain proteins and protein complexes being deposited in the structure databases. The resource called iPfam is hosted within the Pfam UK website. Most resources focus on the interactions between proteins; iPfam includes these as well as interactions between domains in a single protein. AVAILABILITY: iPfam is available on the Web for browsing at http://www.sanger.ac.uk/Software/Pfam/iPfam/; the source-data for iPfam is freely available in relational tables via the ftp site ftp://ftp.sanger.ac.uk/pub/databases/Pfam/database_files/.     

Ion–dipole interactions and their functions in proteins

Ion–dipole interactions in biological macromolecules are formed between atomic or molecular ions and neutral protein dipolar groups through either hydrogen bond or coordination. Since their discovery 30 years ago, these interactions have proven to be a frequent occurrence in protein structures, appearing in everything from transporters and ion channels to enzyme active sites to protein–protein interfaces. However, their significance and roles in protein functions are largely underappreciated. We performed PDB data mining to identify a sampling of proteins that possess these interactions. In this review, we will define the ion–dipole interaction and discuss several prominent examples of their functional roles in nature.     

Non-hydrogen bond interactions involving the methionine sulfur atom

Of all the nonbonded interactions, hydrogen bond, because of its geometry involving polar atoms, is the most easily recognizable. Here we characterize two interactions involving the divalent sulfur of methionine (Met) residues that do not need any participation of proton. In one an oxygen atom of the main-chain carbonyl group or a carboxylate side chain is used. In another an aromatic atom interacting along the face of the ring is utilized. In these, the divalent sulfur behaves as an electrophile and the other electron-rich atom, a nucleophile. The stereochemistry of the interaction is such that the nucleophile tends to approach approximately along the extension of one of the covalent bonds to S. The nitrogen atom of histidine side chain is extensively used in these nonbonded contacts. There is no particular geometric pattern in the interaction of S with the edge of an aromatic ring, except when an N-H group in involved, which is found within 40 degrees from the perpendicular to the sulfide plane, thus defining the geometry of hydrogen bond interaction involving the sulfur atom. As most of the Met residues which partake in such stereospecific interactions are buried, these would be important for the stability of the protein core, and their incorporation in the binding site would be useful for molecular recognition and optimization of the site's affinity for partners (especially containing aromatic and heteroaromatic groups). Mutational studies aimed at replacing Met by other residues would benefit from the delineation of these interactions.     

Ypt and Rab GTPases: insight into functions through novel interactions.

Ypt/Rab GTPases are key regulators of vesicular transport in eukaryotic cells. During the past two years, a number of new Ypt/Rab-interacting proteins have been identified and shown to serve as either upstream regulators or downstream effectors. Proteins that interact with these regulators and effectors of Ypt/Rabs have also been identified, and together they provide new insights into Ypt/Rab mechanisms of action. The picture that emerges from these studies suggests that Ypt/Rabs function in multiple and diverse aspects of vesicular transport. In addition, not only are Ypt/Rabs highly conserved, but their functions and interactions are as well. Interestingly, crosstalk among Ypt/Rabs and between Ypt/Rabs and other signaling factors, suggest the possibility of coordination of the individual vesicular transport steps and of the protein transport machinery with other cellular processes.     

Structures and interactions of the core histone tail domains

The core histone tail domains are "master control switches" that help define the structural and functional characteristics of chromatin at many levels. The tails modulate DNA accessibility within the nucleosome, are essential for stable folding of oligonucleosome arrays into condensed chromatin fibers, and are important for fiber-fiber interactions involved in higher order structures. Many nuclear signaling pathways impinge upon the tail domains, resulting in posttranslational modifications that are likely to alter the charge, structure, and/or interactions of the core histone tails or to serve as targets for the binding of ancillary proteins or other enzymatic functions. However, currently we have only a marginal understanding of the molecular details of core histone tail conformations and contacts. Here we review data related to the structures and interactions of the core histone tail domains and how these domains and posttranslational modifications therein may define the structure and function of chromatin.     

Crystal structures representing the Michaelis complex and the thiouronium reaction intermediate of Pseudomonas aeruginosa arginine deiminase

L-arginine deiminase (ADI) catalyzes the irreversible hydrolysis of L-arginine to citrulline and ammonia. In a previous report of the structure of apoADI from Pseudomonas aeruginosa, the four residues that form the catalytic motif were identified as Cys406, His278, Asp280, and Asp166. The function of Cys406 in nucleophilic catalysis has been demonstrated by transient kinetic studies. In this study, the structure of the C406A mutant in complex with L-arginine is reported to provide a snapshot of the enzyme.substrate complex. Through the comparison of the structures of apoenzyme and substrate-bound enzyme, a substrate-induced conformational transition, which might play an important role in activity regulation, was discovered. Furthermore, the position of the guanidinium group of the bound substrate relative to the side chains of His278, Asp280, and Asp166 indicated that these residues mediate multiple proton transfers. His278 and Asp280, which are positioned to activate the water nucleophile in the hydrolysis of the S-alkylthiouronium intermediate, were replaced with alanine to stabilize the intermediate for structure determination. The structures determined for the H278A and D280A mutants co-crystallized with L-arginine provide a snapshot of the S-alkylthiouronium adduct formed by attack of Cys406 on the guanidinium carbon of L-arginine followed by the elimination of ammonia. Asp280 and Asp166 engage in ionic interactions with the guanidinium group in the C406A ADI. L-arginine structure and might orient the reaction center and participate in proton transfer. Structure determination of D166A revealed the apoD166A ADI. The collection of structures is interpreted in the context of recent biochemical data to propose a model for ADI substrate recognition and catalysis.     

Inter-residue interactions in protein structures exhibit power-law behavior

Inter-residue interactions play an essential role in driving protein folding, and analysis of these interactions increases our understanding of protein folding and stability and facilitates the development of tools for protein structure and function prediction. In this work, we systematically characterized the change of inter-residue interactions at various sequence separation cutoffs using two protein datasets. The first set included 100 diverse, nonredundant and high-resolution soluble protein structures, covering all four major structural classes, all-alpha, alpha/beta, alpha+beta, and all-beta; and the second set included 20 diverse, nonredundant and high-resolution membrane protein structures, representing 19 unique superfamilies. It was shown that the average number of inter-residue interactions in structures of both datasets displays the power-law behavior. Fitting parameters of the power-law function are directly related to the structural classes analyzed. These findings provided further insight into the distribution of short-, medium-, and long-range inter-residue interactions in both soluble and membrane proteins and could be used for protein structure prediction.     

Helix-helix packing and interfacial pairwise interactions of residues in membrane proteins

Helix-helix packing plays a critical role in maintaining the tertiary structures of helical membrane proteins. By examining the overall distribution of voids and pockets in the transmembrane (TM) regions of helical membrane proteins, we found that bacteriorhodopsin and halorhodopsin are the most tightly packed, whereas mechanosensitive channel is the least tightly packed. Large residues F, W, and H have the highest propensity to be in a TM void or a pocket, whereas small residues such as S, G, A, and T are least likely to be found in a void or a pocket. The coordination number for non-bonded interactions for each of the residue types is found to correlate with the size of the residue. To assess specific interhelical interactions between residues, we have developed a new computational method to characterize nearest neighboring atoms that are in physical contact. Using an atom-based probabilistic model, we estimate the membrane helical interfacial pairwise (MHIP) propensity. We found that there are many residue pairs that have high propensity for interhelical interactions, but disulfide bonds are rarely found in the TM regions. The high propensity pairs include residue pairs between an aromatic residue and a basic residue (W-R, W-H, and Y-K). In addition, many residue pairs have high propensity to form interhelical polar-polar atomic contacts, for example, residue pairs between two ionizable residues, between one ionizable residue and one N or Q. Soluble proteins do not share this pattern of diverse polar-polar interhelical interaction. Exploratory analysis by clustering of the MHIP values suggests that residues similar in side-chain branchness, cyclic structures, and size tend to have correlated behavior in participating interhelical interactions. A chi-square test rejects the null hypothesis that membrane protein and soluble protein have the same distribution of interhelical pairwise propensity. This observation may help us to understand the folding mechanism of membrane proteins.     

Stereoselective and isozyme-selective drug interactions

A surprisingly large number of marketed drugs are racemic mixtures. The pharmacokinetic literature on racemic drugs contains a vast amount of information on drug–drug interactions derived from the measurement of total drug concentrations in plasma and urine. The appreciation of the role of stereochemistry in drug interactions with racemic warfarin resulted in a long-overdue scientific rigor being applied to the study of drug interactions. It also compelled us to recognize that much of the literature was uninterpretable. A better understanding of oxidative metabolism, particularly the complexity of the cytochrome P-450 family of enzymes, has also strengthened the scientific basis of drug interactions. We now recognize that investigators and clinicians must consider both stereoselectivity and isozyme selectivity in the study of drug interactions to understand the nature of the interaction so as to more effectively use new and potent drugs. © 1993 Wiley-Liss, Inc.     

Mobility and cytoskeletal interactions of cell adhesion receptors.

Clustering of cell adhesion receptors and their interactions with the cytoskeleton are key events in the formation and function of cell adhesion structures. On the free cell surface, cadherin molecules interact with the cytoskeleton/membrane skeleton by being bound or corralled, and such interactions are greatly enhanced by the formation of cadherin oligomers. Corralled cadherin molecules undergo hop diffusion from one compartment to an adjacent one (membrane skeleton fence model), which prompts the initial formation of small adhesion clusters at cell-cell contact sites, but larger-scale assemblies of cadherin and actin filaments might require a further co-ordinated recruitment of these molecules.     

The geometry and efficacy of cation-pi interactions in a diagonal position of a designed beta-hairpin

Cation-pi interactions are common in proteins, but their contribution to the stability and specificity of protein structure has not been well established. In this study, we examined the impact of cation-pi interactions in a diagonal position of a beta-hairpin peptide through comparison of the interaction of Phe or Trp with Lys or Arg. The diagonal interactions ranged from -0.20 to -0.48 kcal/mole. Our experimental values for the diagonal cation-pi interactions are similar to those found in alpha-helical studies. Upfield shifting of the Lys and Arg side chains indicates that the geometries of cation-pi interactions adopted in the 12-residue beta-hairpin are comparable to those found in protein structures. The Lys was found to interact through the polarized Cepsilon, and the Arg is stacked against the aromatic ring of Phe or Trp. Folding of these peptides was found to be enthalpically favorable (DeltaH degrees equals approximately -3 kcal/mole) and entropically unfavorable (DeltaS degrees equals approximately -8 cal mole(-1) K(-1)).     

Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level

To assess whether there are universal rules that govern amino acid–base recognition, we investigate hydrogen bonds, van der Waals contacts and water-mediated bonds in 129 protein–DNA complex structures. DNA–backbone interactions are the most numerous, providing stability rather than specificity. For base interactions, there are significant base–amino acid type correlations, which can be rationalised by considering the stereochemistry of protein side chains and the base edges exposed in the DNA structure. Nearly two-thirds of the direct read-out of DNA sequences involves complex networks of hydrogen bonds, which enhance specificity. Two-thirds of all protein–DNA interactions comprise van der Waals contacts, compared to about one-sixth each of hydrogen and water-mediated bonds. This highlights the central importance of these contacts for complex formation, which have previously been relegated to a secondary role. Although common, water-mediated bonds are usually non-specific, acting as space-fillers at the protein–DNA interface. In conclusion, the majority of amino acid–base interactions observed follow general principles that apply across all protein–DNA complexes, although there are individual exceptions. Therefore, we distinguish between interactions whose specificities are ‘universal’ and ‘context-dependent’. An interactive Web-based atlas of side chain–base contacts provides access to the collected data, including analyses and visualisation of the three-dimensional geometry of the interactions.     

Hypotheses and trends on how body size affects trophic interactions in a guild of South American killifishes

A chief structuring force in food webs is the hierarchy of trophic interactions, where bigger animals feed on smaller ones. The anatomic and physiological explanations of why body size determines this hierarchy are embodied within the concept of gape limitation. The relaxation of gape limitation and an increase in energetic demands due to predators' larger body size determine the size and diversity of prey species. However, these patterns may be related to further trends in trophic interactions with body size, which have been less considered. Specifically, the passive incorporation of prey should involve a nested distribution of prey among predator size classes. However, predators avoid smaller resources because of their low energy return, with a clumped distribution of prey potentially generating modular organization with qualitative changes in prey identity (e.g. zooplankton, macroinvertebrates and fishes). Finally, size‐mediated interactions (such as direct and indirect competition) may cause predators of similar body size to differentiate among prey organisms, resulting in a checkerboard distribution of prey identity. Consequently, nestedness, modularity and checkerboard distributions of prey among predators of different size classes should form emergent network structures that are directly related to clear ecological mechanisms. We analyse these predictions in a killifish guild, where trends in trophic positions, prey richness, evenness and the number of energy sources systematically scale with body size. We found significant nestedness and segregation in diet among different size classes, supporting the progressive incorporation of prey items coupled with prey differentiation among similar classes. However, we also detected an ‘anti‐modular’ trend, which contradicts theoretical expectations and previous results. We hypothesize that this anti‐modularity is determined by the high biodiversity of the system and the continuous representation of prey size classes. These results reinforce the concept of size‐mediated interactions and its connection with community biodiversity as a main structuring force of food webs.