Clifford E. Felder

FoldIndex©: a simple tool to predict whether a given protein sequence is intrinsically unfolded

Summary: An easy-to-use, versatile and freely available graphic web server, FoldIndex© is described: it predicts if a given protein sequence is intrinsically unfolded implementing the algorithm of Uversky and co-workers, which is based on the average residue hydrophobicity and net charge of the sequence. FoldIndex© has an error rate comparable to that of more sophisticated fold prediction methods. Sliding windows permit identification of large regions within a protein that possess folding propensities different from those of the whole protein. Availability: FoldIndex© can be accessed at http://bioportal.weizmann.ac.il/fldbin/findex Contact: Joel.Sussman@weizmann.ac.il Supplementary information: http://www.weizmann.ac.il/sb/faculty_pages/Sussman/papers/suppl/Prilusky_2005

Proteomic signatures: Amino acid and oligopeptide compositions differentiate among phyla

Availability of complete genome sequences allows in‐depth comparison of single‐residue and oligopeptide compositions of the corresponding proteomes. We have used principal component analysis (PCA) to study the landscape of compositional motifs across more than 70 genera from all three superkingdoms. Unexpectedly, the first two principal components clearly differentiate archaea, eubacteria, and eukaryota from each other. In particular, we contrast compositional patterns typical of the three superkingdoms and characterize differences between species and phyla, as well as among patterns shared by all compositional proteomic signatures. These species‐specific patterns may even extend to subsets of the entire proteome, such as proteins pertaining to individual yeast chromosomes. We identify factors that affect compositional signatures, such as living habitat, and detect strong eukaryotic preference for homopeptides and palindromic tripeptides. We further detect oligopeptides that are either universally over‐ or underabundant across the whole proteomic landscape, as well as oligopeptides whose over‐ or underabundance is phylum‐ or species‐specific. Finally, we report that species composition signatures preserve evolutionary memory, providing a new method to compare phylogenetic relationships among species that avoids problems of sequence alignment and ortholog detection. Proteins 2004.

A server and database for dipole moments of proteins

An Internet server at http://bip.weizmann.ac.il/dipol calculates the net charge, dipole moment and mean radius of any 3D protein structure or its constituent peptide chains, and displays the dipole vector superimposed on a ribbon backbone of the protein. The server can also display the angle between the dipole and a selected list of amino acid residues in the protein. When the net charges and dipole moments of approximately 12 000 non-homologous PDB biological units (PISCES set), and their unique chains of length 50 residues or longer, were examined, the great majority of both charges and dipoles fell into a very narrow range of values, with long extended tails containing a few extreme outliers. In general, there is no obvious relation between a proteins charge or dipole moment and its structure or function, so that its electrostatic properties are highly specific to the particular protein, except that the majority of chains with very large positive charges or dipoles bind to ribosomes or interact with nucleic acids.An Internet server at http://bip.weizmann.ac.il/dipol calculates the net charge, dipole moment and mean radius of any 3D protein structure or its constituent peptide chains, and displays the dipole vector superimposed on a ribbon backbone of the protein. The server can also display the angle between the dipole and a selected list of amino acid residues in the protein. When the net charges and dipole moments of ∼12 000 non-homologous PDB biological units (PISCES set), and their unique chains of length 50 residues or longer, were examined, the great majority of both charges and dipoles fell into a very narrow range of values, with long extended tails containing a few extreme outliers. In general, there is no obvious relation between a proteins charge or dipole moment and its structure or function, so that its electrostatic properties are highly specific to the particular protein, except that the majority of chains with very large positive charges or dipoles bind to ribosomes or interact with nucleic acids.

External and internal electrostatic potentials of cholinesterase models

The electrostatic potentials for the three-dimensional structures of cholinesterases from various species were calculated, using the Delphi algorithm, on the basis of the Poisson-Boltzmann equation. We used structures for Torpedo californica and mouse acetylcholinesterase, and built homology models of the human, Bungarus fasciatus, and Drosophila melanogaster acetylcholinesterases and human butyrylcholinesterase. All these structures reveal a negative external surface potential, in the area around the entrance to the active-site gorge, that becomes more negative as the rim of the gorge is approached. Moreover, in all cases, the potential becomes increasingly more negative along the central axis running down the gorge, and is largest at the base of the gorge, near the active site. Ten key acidic residues conserved in the sequence alignments of AChE from various species, both in the surface area near the entrance of the active-site gorge and at its base, appear to be primarily responsible for these potentials. The potentials are highly correlated among the structures examined, down to sequence identities as low as 35%. This indicates that they are a conserved property of the cholinesterase family, could serve to attract the positively charged substrate into and down the gorge to the active site, and may play other roles important for cholinesterase function.

Origin of the iron(III) binding and conformational properties of enterobactin.

Enterobactin (1) is one of the most efficient natural binders of ferric ions known to date. Structural analogues of enterobactin have been synthesized, including the tribenzamide (TBA) 7, which differs from enterobactin only by lacking the catechol hydroxyl groups. The analogues have been studied by a combination of IR, NMR, and CD spectroscopy, X-ray diffraction, and empirical-force-field calculations. These studies elucidated the origin of enterobactin’s unique binding properties and of its complex’s right-handed chirality. TBA 7 in its most stable conformation, preferred in nonpolar solvents, possesses C3 symmetry, its benzamide side chains are in axial positions, hydrogen bonds are formed between the amide hydrogen and the ring oxygen, and the phenyl rings are arranged in a right-handed (A) orientation. Uncomplexed enterobactin (1) is shown to resemble closely TBA 7. The relation of the preferred A chirality of TBA 7 to the observed A chirality of ( F e e ~ ~ t ) ~ is discussed. A comparison between enterobactin and the hitherto best synthetic binder, a tricatecholamide derivative of mesitylene 3, is presented. Enterobactin’s superiority is partly due to its lower molecular strain upon binding and partly due to the lower conformational freedom of uncomplexed enterobactin. The binding strain of (Fe~ent)~? resides more in the catecholamides than in the trilactone ring, while in the synthetic analogue 3 the mesitylene ring is more strained than the catecholamides. Metal ions are essential for the maintenance of living systems. The alkali metal ions such as sodium control the transmission of nerve impulses, the alkaline earth metal ions such as calcium act as secondary messengers, and the transition-metal ions such as iron and copper are involved in enzymatic redox processes.’ A large variety of ion carriers exist in nature to control the metal ion balance.* Carriers for the transition-metal ions are rare, with the exception of iron, for which protein and non-protein chelates exist. For iron, two families of low molecular weight carriers, or siderophores, are known: those utilizing hydroxamate groups and those utilizing catechol groups as binding sites.3 Among the latter carriers, enterobactin (1) assumes a unique p ~ s i t i o n . ~ Produced by enteric bacteria when grown in iron-deficient media, enterobactin is one of the most efficient binders and carriers known. It has a binding constant of log (Kbind) = 52 for the reaction Fe3+ + en@(Feent)3where “ent6-” is the sixfold deprotonated en ter~bac t in .~ Chemically, enterobactin is a tripodlike molecule. It consists of a trilactone ring, composed of three L-serine residues, each with an attached catechol ligand. Information on the conformation of enterobactin is limited to NMR studies in MezSO at elevated temperatures5 or to IR studies in water.6

Structure of a complex of the potent and specific inhibitor BW284C51 with Torpedo californica acetylcholinesterase.

The X-ray crystal structure of Torpedo californica acetylcholinesterase (TcAChE) complexed with BW284C51 [CO[-CH(2)CH(2)-pC(6)H(4)-N(CH(3))(2)(CH(2)-CH=CH(2))](2)] is described and compared with the complexes of two other active-site gorge-spanning inhibitors, decamethonium and E2020. The inhibitor was soaked into TcAChE crystals in the trigonal space group P3(1)21, yielding a complex which diffracted to 2.85 A resolution. The structure was refined to an R factor of 19.0% and an R(free) of 23.4%; the final model contains the protein, inhibitor, 132 water molecules and three carbohydrate moieties. BW284C51 binds similarly to decamethonium and E2020, with its two phenyl and quaternary amino end-groups complexed to Trp84 in the catalytic site and to Trp279 in the peripheral binding site, and its central carbonyl group hydrogen bonded very weakly to Tyr121. Possible reasons for decamethoniums weaker binding are considered. The relative strength of binding of bisquaternary inhibitors to acetylcholinesterase and the effect of several mutations of the enzyme are discussed in the context of the respective X-ray structures of their complexes with the enzyme.

A MODULAR TREATMENT OF MOLECULAR TRAFFIC THROUGH THE ACTIVE SITE OF CHOLINESTERASE

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.

SPINE bioinformatics and data-management aspects of high-throughput structural biology

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high‐throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high‐throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high‐throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.

Enniatin B and Valinomycin as Ion Carriers: An Empirical Force Field Analysis

The alkali-ion binding properties of two natural depsipeptide ion carriers, enniatin B (EnB) and valinomycin (VM), are examined and compared by the empirical force field method. While VM has been shown to bind preferentially K+, Rb+, and Cs+ over Na+ in most solvents, EnB is considerably less specific. We find that EnB forms two kinds of complexes, internal and external. In internal complexes, the ion binds to all six carbonyl oxygens, while in external ones, only three oxygens, preferentially those of the D-hydroxy-isovaleryl residues, are bound. The size of the internal cavity is best suited for Na+, while K+ and Rb+ squeeze in asymmetrically by distorting the molecule, and Cs+ not at all. External binding is much less specific. Since internal complexes possess much higher strain energies than external ones, the latter may be at least as stable as the former, even in fairly non-polar solvents. VM is calculated to bind only internally, and with much less strain energy than EnB. The size of its internal cavity is well suited for binding the ions K+, Rb+, and Cs+, but is too big for Na+. The difference between the binding energies of Na+ and K+ is much smaller than that between the corresponding hydration enthalpies, thus explaining the binding preference for the latter ion.

Bidentate Ligation of Heme Analogues; Novel Biomimetics of the Peroxidase Active Site

The multifunctional nature of proteins that have iron-heme cofactors with noncovalent histidine linkage to the protein is controlled by the heme environment. Previous studies of these active-site structures show that the primary difference is the length of the iron-proximal histidine bond, which can be controlled by the degree of H-bonding to this histidine. Great efforts to mimic these functions with synthetic analogues have been made for more than two decades. The peroxidase models resulted in several catalytic systems capable of a large range of oxidative transformations. Most of these model systems modified the porphyrin ring covalently by directly binding auxiliary elements that control and facilitate reactivity; for example, electron-donating or -withdrawing substituents. A biomimetic approach to enzyme mimicking would have taken a different route, by attempting to keep the porphyrin ring system unaltered, as close as possible to its native form, and introducing all modifications at or close to the axial coordination sites. Such a model system would be less demanding synthetically, would make it easy to study the effect of a single structural modification, and might even provide a way to probe effects resulting from porphyrin exchange. We introduce here an alternative model system based on these principles. It consists of a two component system: a bis-imidazolyl ligand and an iron-porphyrin (readily substituted by a hemin). All modifications were introduced only to the ligand that engulfs the porphyrin and binds to the irons fifth and sixth coordination sites. We describe the design, synthesis, and characterization of nine different model compounds with increased complexity. The primary tool for characterizing the environment of each complex Fe(III) center was the Extended X-ray Absorption Fine Structure (EXAFS) measurements, supported by UV/Vis, IR, and NMR spectroscopy and by molecular modeling. Introduction of asymmetry, by attaching different imidazoles as head groups, led to the formation of two axial bonds of different length. Addition of H-bonds to one of the imidazoles in an advanced model increased this differentiation and expanded the porphyrin ring. These complexes were found to be almost identical in structure to peroxidase active sites. Similarly to the peroxidases and other synthetic models, these compounds stabilize the green, compound I-like intermediate, and catalyze the oxidation of organic substrates.