The impact of crystallization conditions on structure-based drug design: A case study on the methylene blue/acetylcholinesterase complex.

Structure-based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but <6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active-site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are ∼3.0Å further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct π-cation interactions with the indole. These findings have implications for structure-based drug design, since data for ligand-protein complexes with polyethylene glycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design.

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 protein's 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.

A molecular keypad lock: a photochemical device capable of authorizing password entries.

This paper describes a new concept in the way information can be protected at the molecular scale. By harnessing the principles of molecular Boolean logic, we have designed a molecular device that mimics the operation of an electronic keypad lock, e.g., a common security circuit used for numerous applications, in which access to an object or data is to be restricted to a limited number of persons. What distinguishes this lock from a simple molecular logic gate is the fact that its output signals are dependent not only on the proper combination of the inputs but also on the correct order by which these inputs are introduced. In other words, one needs to know the exact passwords that open this lock. The different password entries are coded by a combination of two chemical and one optical input signals, which can activate, separately, blue or green fluorescence output channels from pyrene or fluorescein fluorophores. The information in each channel is a single-bit light output signal that can be used to authorize a user, to verify authentication of a product, or to initiate a higher process. This development not only opens the way for a new class of molecular decision-making devices but also adds a new dimension of protection to existing defense technologies, such as cryptography and steganography, previously achieved with molecules.

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

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.

Chemical input multiplicity facilitates arithmetical processing.

We describe the design and function of a molecular logic system, by which a combinatorial recognition of the input signals is utilized to efficiently process chemically encoded information. Each chemical input can target simultaneously multiple domains on the same molecular platform, resulting in a unique combination of chemical states, each with its characteristic fluorescence output. Simple alteration of the input reagents changes the emitted logic pattern and enables it to perform different algebraic operations between two bits, solely in the fluorescence mode. This system exhibits parallelism in both its chemical inputs and light outputs.

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.

Application of the empirical force field to macrocyclic ion carriers, siderophores, and biomimetic analogs.

The empirical force field (EFF), developed by Prof. Lifson, was applied to the study of macrocyclic alkali ion carriers and to di- and tripodal and open chain siderophores and synthetic biomimetic molecules binding transition metals. The highly symmetric nature of these structures facilitated a favorable coordination geometry of the ligating groups about the metal, which helped organize the entire molecule into a fairly rigid structure. In our combined experimental-theoretical approach, EFF calculations were used to help predict likely candidates to synthesize, and provided a wealth of structural data to complement what we learned from the spectroscopic measurements, while feedback from these measurements allowed us to continue improving the EFF itself. The simple, highly modular design of the biomimetic analogs allowed rapid synthesis and systematic examination of a large number of related structures, as well as facilitating an efficient, piecewise conformational scanning for the theoretical calculations. In the early years, we focused on macrocyclic polylactones and lactams binding monovalent alkali ions, particularly the natural products enniatin and valinomycin, including inside a crystal lattice. Later we switched to bi- and tridentate siderophores, natural microbial iron carriers, and synthetic biomimetic analogs-in particular, of enterobactin, ferrichrome, and ferrioxamine B. Over the years a large number of biomimetic siderophores have been prepared, some active in a broad range of microorganisms while others are highly species specific. The results of this work have broad applications in many areas, including the design of novel drugs and antimicrobial agents, helical polymeric structures, and polynuclear metal complexes.

Bidentate ligation of heme analogues; novel biomimetics of 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 iron's 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.

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 decamethonium's 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.