In silico induced effect of N-ε-lysine acetylation on microtubule stability and subsequent interaction of microtubule-associated proteins.

Plant systems have been considered valuable models for addressing fundamental questions of microtubule (MT) organization due to their considerable practical utility. Protein acetylation is a very common protein modification, and therate of acetylation can be modulated in cells in different biological states, and these changes can be detected at a molecular level. Here, we focused on K40, K112, and K394 residues as putative acetylation sites, which were shown to exist in both plants and mammals. Such residual effect of acetylation causes critical but unclear effect on MT stability. In turn, it was shown that acetylation indirectly affects the probability of interaction with different MAPs (Microtubule-associated proteins). In a multiscale study using an all-atom force field to reproduce several lattice-forming elements found on the surface the microtubule, we assembled a fragment of a plant microtubule composed of nine tubulins and used it as a model object along with the existing human complex. Triplets of tubulins assembled in a lattice cell were then simulated for both human and plant protein complexes, using a coarse-grained force field. We then analyzed the trajectories and identified some critical deformations of the MAP interaction surface. The initial coordinates were used to investigate the structural scenario in which autophagy-related protein 8 (ATG8) was able to interact with the MT fragment.

Structural and functional features of lysine acetylation of plant and animal tubulins.

The study of the genome and the proteome of different species and representatives of distinct kingdoms, especially detection of proteome via wide-scaled analyses has various challenges and pitfalls. Attempts to combine all available information together and isolate some common features for determination of the pathway and their mechanism of action generally have a highly complicated nature. However, microtubule (MT) monomers are highly conserved protein structures, and microtubules are structurally conserved from Homo sapiens to Arabidopsis thaliana. The interaction of MT elements with microtubule-associated proteins and post-translational modifiers is fully dependent on protein interfaces, and almost all MT modifications are well described except acetylation. Crystallography and interactome data using different approaches were combined to identify conserved proteins important in acetylation of microtubules. Application of computational methods and comparative analysis of binding modes generated a robust predictive model of acetylation of the ϵ-amino group of Lys40 in α-tubulins. In turn, the model discarded some probable mechanisms of interaction between elements of interest. Reconstruction of unresolved protein structures was carried out with modeling by homology to the existing crystal structure (PDBID: 1Z2B) from B. taurus using Swiss-model server, followed by a molecular dynamics simulation. Docking of the human tubulin fragment with Lys40 into the active site of α-tubulin acetyltransferase, reproduces the binding mode of peptidomimetic from X-ray structure (PDBID: 4PK3).

Protein phosphatases potentially associated with regulation of microtubules, their spatial structure reconstruction and analysis.

According to the sequence and profile comparison with known catalytic domains, where identified protein phosphatases potentially involved in regulation of microtubule dynamics and structure from Arabidopsis thaliana, Nicotiana tabacum, Medicago sativa, Oryza sativa subsp. japonica, Zea mays, and Triticum aestivum. Selected proteins were related to classical non-receptor, serine/threonine-specific and dual protein phosphatases. By application of template structures of human protein phosphatases, it was performed homology modelling of the catalytic domains of 17 plant protein phosphatases. Based on the results of the structural alignment, molecular dynamics, and conservatism in positions of functionally importance, it was confirmed homology of selected plant proteins and known protein phosphatases regulating structure and dynamics of microtubules.