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COMMON PART

Project Number17-74-20009

Project titleStructural, functional and genetic analysis of the elongation factor EF-P and initiation complex of the pathogenic bacterium Staphylococcus aureus

Project LeadUsachev Konstantin

AffiliationKazan (Volga region) Federal University, Kazan University, KFU,

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-202 - Proteomics; structure and functions of proteins

KeywordsStaphylococcus aureus, antibiotic, translation, initiator complex, protein biosynthesis

PROJECT CONTENT

Annotation
The synthesis of protein is of paramount importance, since proteins direct all the processes of vital activity of a living cell. The ribosome is a biological machine that translates the code recorded in the matrix RNA (mRNA) into the polypeptide chain of the protein, and is thus a key element in the synthesis of the most important class of biopolymers in the cell. Due to its importance, the bacterial ribosome is one of the most important targets for antibiotic action: more than 40% of clinically used antibiotics somehow block its work. The principle of action of many antibiotics is in their selective inhibition of the protein-synthesizing apparatus of bacterial cells, without disrupting the work of the cells of the host organism and, consequently, its ribosomes. In recent decades, significant progress has been made in studying the structure and mechanism of protein synthesis in a cell. Using the method of crystallography with atomic resolution, the structure of the ribosome and several ribosome functional complexes modeling various stages of protein synthesis: initiation, elongation and termination, when the ribosome interacts with many protein factors, has been determined. Recent developments in cryoelectron microscopy make it possible to determine with high-resolution the ribosome structure and its complexes with protein factors and to interpret the mechanism of interaction and structural organization at the molecular level. And modern methods of NMR spectroscopy make it possible to study the structure and dynamics of biomolecules in solution-the natural (native) environment of living organisms. These data created the basis for modeling in the three-dimensional space of biochemical reactions occurring during protein synthesis. Modern knowledge of the structure of the ribosome and other molecular components of the translation apparatus allows one to study the regulation of these processes by means of small molecules, for example: antibiotics. This makes it important to expand this knowledge in the field of studying pathogens and to find specific differences in the organization of the apparatus of protein synthesis of pathogenic organisms.  Within the framework of this project, it is proposed to study the structure of the initiation and elongation factor of EF-P Staphylococcus aureus, one of the most dangerous pathogens for humans, most often the cause of many community and nosocomial infections. It is believed that the main role of EF-P is the provision of specialized translation, for example, maintaince of the difficulties in synthesizing protein regions with several proline residues. It is known that proteins of secretory systems such as, CadC, TonB, YafD, Rz1, AmiB, FlhC, etc., are abundant with areas with repeated proline residues that secrete pathogenicity factors, therefore, disruption of EF-P elongation factor functioning should affect Pathogenic properties of microorganisms. For example, it was shown that the lack of a normal modification in the elongation factor of EF-P reduces the virulence of Salmonella. Functional analysis based on mutants with a violation of the synthesis and modification of EF-P will reveal the role of this elongation factor in the work of systems that provide pathogenicity in S. aureus. The EF-P factor is also an initiation and translation factor involved in the formation of the initiator complex (70S, mRNA, EF-P and fMet-tRNA). The solution of the structure of this protein, as well as the initiator complex, will allow further screening of inhibitors of the translation of the pathogenic bacterium Staphylococcus aureus, on the basis of which it is possible to create new drugs.

Expected results
The aim of the project is to conduct functional analysis and to solve the spatial structure of the EF-P elongation factor of Bacterial pathogen Staphylococcus aureus by NMR and X-ray diffraction analysis, as well as determination of Initiator complex (70S, mRNA, EF-P and fMet-tRNA) structure by cryoelectron microscopy and X-ray diffraction analysis for further screening of new drugs. To achieve this goal, it is necessary to solve the following tasks:  1. Preparation and analysis of mutants incapable of synthesizing EF-P (gene silencing), as well as mutants with disturbed post-translational modification of the EF-P elongation factor in order to establish mechanisms regulating the translation of the bacterium Staphylococcus aureus  2. Cloning, isolation and purification of the EF-P protein of the bacterial pathogen Staphylococcus aureus  3. Selection of crystallization conditions for the structural solution by X-ray diffraction analysis, selection of buffer conditions for solution of protein structure in solution by high resolution NMR spectroscopy  4. Isolation and purification of the initiation complex (30S, mRNA, EF-P and fMet-tRNA) and the solution of its structure by the cryoelectron microscopy and detection the targets for new antibiotics. The results obtained will create a basis for further structural studies of protein factors in solution and in combination with the ribosome in the presence and without antibiotics, which will provide detailed information on possible areas of antibiotic binding. The information obtained in this way will make it possible to predict the structure and screening of new highly selective antibiotics.

REPORTS