Estudo da dinâmica e caracterização estrutural da interface proteica em Quimeras Xilanase-XBP por simulação molecular

Abstract

In the field of molecular biology and biotechnology, proteins owning two or more combined activities, coupled with an appropriate structural stability, have been widely applied. A few years ago three hybrid enzymes / chimeras were produced by a semi-rational protein fusion strategy, resulting in the insertion of a Xylose Binding Protein (XBP) to a Xylanase GH11, whose function is to degrade xylan, the main component of the plant cell wall. The enzymes produced showed a catalytic efficiency in the degradation of xylan two to three times higher than the isolated xylanase, besides increasing its activity in the presence of xylose, a substance that would naturally inhibit xylanase alone. Xylose activation occurs via allosteric mechanism. Currently, simulation techniques such as molecular dynamics (DM) are the best ones to bring forward detailed information at the atomic level regarding structural protein behavior, but they present limited timescale to investigate the problem fully, because the structures generated can be trapped in a local energy minimum. In this work, we explore alternative ways of circumventing these limitations, with a computationally less demanding technique for generation of new ensemble of structures, the CONCOORD, for the case of chimeric proteins with allostery. More robust statistical techniques (PCA and FMA) were used to analyze the results in order to provide a more complete analysis of the functional motions in these proteins. In addition, a detailed structural characterization of the protein interfaces between the enzymatic domains was performed since these interfaces play a fundamental role in the allosteric effect. The FMA analysis indicated that the stability of the interface is well related to a specific movement, the rotation of the xylanase and XBP domains in opposite directions, which showed good correlation with the interfacial energy for chimeras 209, 262 and 271. In the case of chimera 271 the correlation was slightly worse due to a more intense and extensive movement of the thumb region (xylanase) compared to chimeras 209 and 262. The presence of intermittent interfacial residues, especially in chimeras 209 and 262, was identified. Chimera 271 has the most stable interface, with the least amount of intermittent interfacial residues. It was observed that the occurrence of a high conformational variation during the simulations is associated to a change in the interfacial energy, mainly for chimeras 209 and 262, that is, when the protein undergoes some considerable conformational transition, the interfacial energy responds, increasing or decreasing, which is in agreement with the existence of intermittent interfacial residues. Detection of such residues is fundamental to understand the structural allosterism and stabilization mechanism determined by the XBP insertion position in xylanase. The results obtained here may guide a rational proposition of point mutations in the chimeras in order to improve the stability of the protein interface and, consequently, the allosteric effect, such as the insertion of disulfide bridges in the interface prioritizing the residues identified in the protein interface. Mutant hybrid enzymes produced in silico can be tested experimentally by the collaborating groups.