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Functional and structural characterization of ZapA protein, involved in bacterial cell division

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Author(s):
Maria Luiza Caldas Nogueira
Total Authors: 1
Document type: Doctoral Thesis
Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
Defense date:
Examining board members:
José Ricardo Murari Pires; Juliana Fattori; Juliana Ferreira de Oliveira; Marcio Vinicius Bertacine Dias
Advisor: Ana Carolina de Mattos Zeri
Abstract

The ZapA protein is involved in bacterial cell division. Several cellular elements are organized at the right time of the cell cycle in order to divide a single bacterial cell into two daughter cells. For this division to occur, the cell wall and cell envelope create a septum, guided by a macromolecular complex called divisome. The separation of the daughter cells is assisted by the constriction of a ring-shaped structure, the Z ring. This ring is formed by FtsZ polymers, a tubulin homolog that polymerize into protofilaments, which in turn, are organized in bundles, which are connected in a circular structure. The Z ring formation is a key event, and interconnects several proteins involved in cell division. The place and the time of this division have to be finely regulated, in order to prevent the formation of nonfunctional daughter cells. It must occur in the medial region, only after all the cellular components have already doubled and segregated to their respective poles. Among the proteins that interfere in this process, there are those involved in the regulation / synthesis of cell wall and proteins that modulate the Z ring. These modulators act by stimulating or inhibiting the Z ring formation, according to the cell requirement. ZapA (Z ring associated protein A) stabilizes lateral interactions of protofilaments of FtsZ, assisting in the Z ring formation. Although ZapA was discovered in 2002, the molecular mechanisms of their interaction with FtsZ and how it stabilizes its protofilaments are not clear. To better understand the role of ZapA in this process, we conducted structural studies of ZapA protein from G. stearothermophilus, by solving its structure by NMR and performing computational molecular docking of the FtsZ-ZapA complex. We studied the nature of the interaction by techniques such as calorimetry (ITC), which indicated the low association constant, and by co-purification assays between FtsZ and ZapA (wild type and mutants), aiding in the understanding of the importance of each amino acid residue mutated. We have identified a possible interaction area between these proteins in which the helix H3 and N-terminal region (serine 13) of FtsZ have an important role as well as the H1 ZapA helix. The understanding of the whole process of cell division is of great value, and can be used as the basis for the development of a new class of antibiotics capable of preventing bacterial division (AU)