STRUCTURAL STUDIES OF FMTA FROM STAPHYLOCOCCUS AUREUS

Abstract

Infectious diseases are the main cause of death in the world and microbial infections cause a high rate of mortality. Bacteria develop resistance to antibiotics or drugs due to the expression or acquiring of new genes or mutation in genes. The development of resistance in bacteria depends on the efflux the drugs, inactivation of drugs, or modifications of drug targets. Staphylococcus aureus is a round-shaped, gram-positive bacteria that cause skin infections, endocarditis, pneumonia, osteomyelitis, bacteremia, carbuncles, folliculitis, wound infections, furuncles, mastitis, urinary tract infections, abscesses, fasciitis, toxic shock syndrome, and food poisoning in humans. The exposure of S. aureus to β-lactam antibiotics results in the emergence of penicillin and methicillin-resistant S. aureus (MRSA). Penicillin resistance is due to blaZ encoded β-lactamase that can cleave and hydrolyze the β-lactam ring of penicillin. While, methicillin-resistant has emerged due to the acquiring of PBP2a or PBP2’, which has a very low affinity for methicillin. Various factors such as FmtA, femB, femC, llm, femE, femF, sigB, femA, femD, lytH, and fmhB, etc. are reported as crucial proteins for the development of methicillin-resistant in S. aureus. FmtA exhibits the esterase activity as it can remove the D-Ala from teichoic acids. Staphylococcus aureus modifies their cell envelops in different ways to protect against harmful antimicrobial molecules. Teichoic acids (TAs) are essential for the survival of S. aureus against toxic compounds and environmental stress. Teichoic acid is required for cell division, cell autolysis, metal homeostasis, virulence, attachment to artificial surfaces, biofilm formation, resistance to cationic antimicrobial peptides, and cell wall active antibiotics. TAs have zwitterionic properties because of both negative and positive charges due to phosphate groups and amino groups in D-Ala, respectively. In S. aureus, TAs consist of repetitive monomeric units of ribitol phosphate (Rbo-P) or glycerol phosphate (Gro-P). Cell wall inhibitors such as phosphomycin, bacitracin, cefoxitin, and methicillin can upregulate the FmtA. The inactivation of FmtA affects the peptidoglycan structure. The FmtA mutants S. aureus is susceptible to penicillin and methicillin and lacks the teichoic wall acids (WTA). The primary structure of FmtA showed similarities with penicillin-binding proteins (PBPs), β-lactamase, and penicillin-recognizing protein (PRP) such as D-amino acid amidase (DAP), D-esterases, D, D- endopeptidase, D, L-endopeptidase, and D-aminopeptidase. The current study focuses on the structural and computational studies to explore the catalysis of WTA by FmtA. Further, potent drug molecules were screened and identified against FmtA. ii Chapter 1 starts with the introduction and review of the literature about the development of resistance in bacteria. It further illustrates the emergence of resistance for penicillin and methicillin in Staphylococcus aureus. Later, the chapter introduces the importance of lipoteichoic acid (LTA) and wall teichoic acid (WTA). This chapter discusses already work done for FmtA, an important drug target of S. aureus. Chapter 2 of the thesis is dedicated to the structural study of FmtA from S. aureus. This study involves purification, crystallization, mutation, and computational work. FmtA protein was purified by cation exchange chromatography using 0- 1.0 M NaCl in 50 mM sodium phosphate buffer (pH 7.2). The pure protein was eluted using gel filtration chromatography and kept for crystallization in 0.2 M NaCl, 0.1 M Tris (pH 8.5 buffer), and 25% PEG 3350 at 20 °C. Needle shaped crystal was diffracted, and data were processed using HKL2000. The structure was solved using molecular replacement and the structure is homodimer consist of 14 α-helices and 15 β-strands in each monomer. Three loops, Loop I (179 to 187), Loop Ω (267 to 272), and Loop II (319 to 331), impact the architecture of the active site of FmtA. The structural comparison of FmtA with penicillin-recognizing proteins (PRPs) such as D-Ala-carboxypeptidase from Streptomyces sp. strain R61 (DDCP), alkaline D-peptidase (ADP) from Bacillus cereus DF4-B, D-amino acid amidase (DAA), D-aminopeptidase (DAP), and carboxylesterase (EstB) from Burkholderia gladioli revealed that FmtA lacks the four structural signatures, i.e., long Loop I, the interaction between Loop I and Ω-Loop, the folding of Loop II over the active site, and the tilting of the β12 and β13 strands. The absence of these structural signatures creates the active site of FmtA to shallow, enlarged, and solvent-exposed. Structural analysis showed that 1st motif residues (Ser127 and Lys130), 2nd motif residues (Tyr211 and Asp213), and 3rd motif residues (Asn343 and Gly344) are present at the active site of FmtA and hydrogen bond network among Ser127, Lys130, and Asp213 resembles the catalytic triad of a typical serine protease. FmtA Y211A and D213A variants exhibited 48% and 37% of esterase activity of wild type FmtA. While the removal of side chains of Lys130 and Tyr211 in FmtA K130AY211A mutant results in complete loss of esterase activity. Molecular docking results predict that WTA bind with a good binding affinity at the active site of FmtA. Molecular dynamics simulation confirmed that WTA bind at the active site of FmtA and results in the formation of a stable FmtA-WTA complex. Overall, this study concludes that Ser127 acts as a nucleophile and Lys130 might serve as a general base for acylation/deacylation. Tyr211 is required to hold the Ser127 and D-Ala group of WTA for catalysis. iii Chapter 3 of the thesis describes the work carried out to investigate the catalytic mechanism of WTA by FmtA. Here, we have used the solved crystal structure of FmtA to generate the FmtA conformational changed structure (FmtA Conf). FmtA (Conf)-WTA Michaelis complex was generated and validated by molecular docking and molecular simulation. The dynamics and stability of FmtA (Conf)-WTA and FmtA (Conf) mutants-WTA complexes were examined using molecular docking, molecular dynamics, and molecular mechanics generalized Born surface area (MM-GBSA) to determine the role of active site residues (Ser127, Lys130, Tyr211, Asp213, Asn343, and Gly344) for binding of substrate. An integrated two-layered our Own N-layered Integrated molecular Orbital and molecular mechanics (ONIOM) with (B3LYP/6-311G (d,p): AMBER) computational model was employed to compute the standard Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) to assess the role of oxyanion hole residues (Ser127 and Gly344) for the stability of FmtA-WTA complex. Further, combined quantum mechanics/molecular mechanics method (B3LYP/6-311G(d,p): AMBER) was used to explore the catalytic pathway from FmtA (Conf)-WTA Michaelis complex to the acyl-enzyme complex by potential energy surface scan. Overall, in the current study, usage of an extensive computational techniques reveals that Ser127 acts as a nucleophile, Lys130 performs the acylation/deacylation, and Tyr211 plays a key role in binding of the substrate in FmtA-WTA complex. In addition, Ser127 and Gly344 present at 1st and 3rd motif have been identified as an oxyanion hole residues to stabilize and neutralize the tetrahedral intermediate and acyl-enzyme complex during the catalysis of WTA by FmtA. Chapter 4 focuses on the screening and identification of drug molecules against FmtA of S. aureus. Here, in this study, the crystal structure of FmtA (PDB ID: 5ZH8) was used for structure-based virtual screening against drug molecules from e-LEA3D: Cheminformatics Tools and database. Molecular docking results predicted that screened drug molecules (ofloxacin, roflumilast, and furazolidone) interact with active site residues (Ser127, Lys130, Tyr211, Asp213, and Asn343) of FmtA. Molecular dynamics simulation was employed to determine the atomic stability of FmtAΔ42-drug(s) complexes. Several molecular simulation analysis such as RMSD, RMSF, Rg, SASA, hydrogen bond, PCA, and FEL confirmed that binding of drugs with FmtAΔ42 form a stable FmtAΔ42- drug(s) complexes. Molecular mechanics generalized Born surface area (MM-GBSA) and Quantum mechanics/molecular mechanics (QM/MM) conclude that active site residues (Ser127, Lys130, Tyr211, Asp213, and Asn343) of FmtA played an essential role in the binding of drugs with protein to form a lower energy stable protein-drug(s) complexes. Circular dichroism and fluorescence iv spectroscopy showed that structural and conformational changes occurred in FmtAΔ42 in the presence of drugs. Chapter 5 gives the brief details of the summary and future perspective for FmtA from S. aureus. Finally, Chapter 6, Bibliography enlists all the references which were used during the course of this thesis.