Structural mechanisms of interaction of cyanolcrylates with plant tubulin

The structural mechanisms underlying the specific binding of cyanoacrylate compounds with tubulin of higher plants have been studied by the example of the interaction of ethyl-(2Z)-3-amino-2-cyano-4-ethylhex-2-enoate (CA1) and isopropyl-(2Z)-3-amino-2-cyano-4-ethylhex-2-enoate (CA2) with Arabidopsis thaliana α-tubulin. It was revealed that the cyano group of cyanoacrylates is a functional analogue of the nitrile group, which determines the processes of specific interaction with plant tubulin for dinitroaniline compounds. Based on data on spatial structure fluctuations, the dynamics of hydrogen bonds and the interaction energy of CA1 and CA2 (the most probable binding mode for these compounds with plant α-tubulin) was identified and the appropriate site of interaction was characterized. Seven out of ten residues composing this site (Gln-133, Asn-249, Val-250, Asp-251, Val-252, Asn-253, and Glu-254) are obligatory components of dinitroanilines’ binding site on the plant α-tubulin surface. Thus, the binding site on the α-tubulin surface characterized by us is able to recognize and specifically bind substances, which are cardinally different by their chemical nature and have no common pharmacophore groups, under the condition of a certain similarity of their electrostatic topology.     

Analysis of structural characteristics of alpha-tubulins in plants with enhanced cold tolerance

The uniqueness of the point substitutions in the sequences of two alpha-tubulin isotypes from psychrophilic alga Chloromonas that can determine the increased cold tolerance of this alga was analyzed. The comparison of all known amino acid sequences of plant alpha-tubulins enabled to ascertain that only M268-->V replacement is unique and may have a significant influence on spatial structure of plant alpha-tubulins. Modeling of molecular surfaces of alpha-tubulins from Chloromonas, Chalmydomonas reinhardtii and goose grass Eleusine indica showed that insertion of the amino acid replacement M268-->V into the sequence of goose grace tubulin led to the likening of this protein surface to the surface of native alpha-tubulin from Chloromonas. Alteration of local hydrophobic properties of alpha-tubulin molecular surface in interdimeric contact zone as a result of the mentioned replacement was shown that may play important role in increasing the level of cold resistance of microtubules. The crucial role of amino acid residue in 268 position for forming the interdimeric contact surface of alpha-tubulin molecule was revealed. The assumption is made about the importance of replacements at this position for plant tolerance to abiotic factors of different nature (cold, herbicides).     

Molecular and structural-biological analysis of <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> mutants for identification of the site on their β-tubulins of interaction with dinitroanilines and phosphorothioamides

The identification of the location of a point mutation on β-tubulin molecules of amiprophosmethyland trifluralin-resistant Nicotiana plumbaginifolia lines are described in this work. It was shown that in the first case, this mutation is related with the substitution of serine residue for proline in position 248; in the second case, with the substitution of phenylalanine for serine in position 317 of the β-tubulin’s amino acid sequence. Three-dimensional models of the β-tubulin molecule from Chlamydomonas with the well-known location of mutations determining dinitroaniline- and phosphorothioamides resistance (the substitution of lysine residue for methionine in position 350), and β-tubulin from Nicotiana plumbagnifolia have been reconstructed. On the basis of the analysis of the interaction site for dinitroanilines and phosphorothioamides located on the Chlamydomonas β-tubulin’s molecule it was concluded that the revealed mutations on Nicotiana plumbaginifolia β-tubulin are affected by the residues of the amino acids, participating in the formation of this site.     

Exposure of beta-tubulin regions defined by antibodies on an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>microtubule protofilament model and in the cells

Background  

The function of the cortical microtubules, composed of αβ-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited.     

Construction of three-dimensional models of Arabidopsis thaliana FtsZ-proteins on basis of crystal structure of archaebacterial FtsZ-GDP complex

Three-dimensional models of FtsZ-protein complexes with GDP from Arabidopsis thaliana L. localized in cytosol (Entrez database code NP190843) and in chloroplasts (Entrez database code AAA82068) were developed. Crystal structure of the FtsZ-GDP complex from archaea Methanococcus jannaschii (PDB-code 1FSZ) was used as a matrix. Secondary structures of computed models contain ten beta-strands. A chloroplast form of FtsZ-protein has ten alpha-helices and four 3(10)-helices, whereas cytosolic form of protein has nine and three structures correspondently and neither a0-helix before nucleotide-binding domain nor C-terminal 3(10)-helix in secondary domain. The T2-loop of nucleotide-binding pocket of chloroplast form of FtsZ-ptotein in position 111 contains non-charged alanin residue instead of the charged one which is typical for cytosolic and bacterial forms of proteins. At low sequence homology of FtsZ-proteins (approximately 47%) the developed models demonstrate high coincidence with matrix both in the structures of nucleotide-binding pocket and in the whole molecule. The models are completely suitable for further studies of possible sites of binding with dinitroaniline herbicides.     

Reconstruction of spatial structure of plant protein phosphatase type-1 and -2A in complex with okadaic acid

The homology modeling, based on known temple structures of Homo sapiens protein phosphatase type-1 and -2A was implemented. The spatial structures of the human protein phosphatases and their plant homologs from Arabidopsis thaliana was predicted. The quality of models was confirmed by conformational analysis and root mean square deviations. The sites of okadaic acid binding in molecules of plant protein phosphatases (type-1 and -2A) were proved by the data of comparative analysis and molecular dynamics.     

Bioinformatics search for plant homologues of Ste20-like serine/threonine protein kinases

Eleven plant homologuess of animal and yeast STE20-like protein kinases were identified. It was shown that unknown proteins A9RVK0 of the moss Physcomitrella patens ssp. patens and A7P2E2 of the grape Vitis vinifera were the closest plant homologues of STE20-like protein kinases. The Ste20-like protein kinase dst1 of the myxomycete Dictyostelium discoideum was the closest to the plant homologues. The spatial structure of the catalytic domain of the protein A9RVK0 from P. patens ssp. patens was predicted.