STUDIES ON SOLUTE BINDING PROTEINS FROM CANDIDATUS LIBERIBACTER ASIATICUS

Abstract

Huanglongbing or citrus greening is the most destructive disease of citrus orchids all over the world. This disease causes the substantial losses in citrus production. HLB is caused by three species of phloem-limited, unculturable, Gram-negative fastidious α-proteobacteria; Candidatus Liberibacter asiaticus (Las), Candidatus Liberibacter africanus (Laf) and Candidatus Liberibacter americanus. Of the three species, Candidatus Liberibacter asiaticus is widely distributed and most virulent strain. It is transmitted by Asian citrus psyllid (Diaphorina citri Kuwayama). HLB was mainly identified by blotchy chlorosis /mottling of leaves, yellow shoot, stunted growth, vein corking. HLB affected fruits are small in size, of lower quality, often lopsided and contain aborted seeds. HLB disease is seriously affecting the citrus world by decreasing the lifespan of the citrus trees and lowering the yield as well as the quality of fruits. Currently, no strategies have been developed to manage the HLB disease and to stop the spreading of this disease to new citrus areas. The recommended control strategy is to chemically control the psyllids and removal of the infected trees. Transition metals such as manganese, zinc and iron sequestration or uptake are essential for bacterial survival and proliferation in the environment as well as within host. They are essential for the activity of a wide range of enzymes, involved in DNA replication, protein synthesis, cell wall synthesis, and oxidative stress management. Metal deficiency greatly inhibits the growth of the bacteria. Therefore, inhibition of the uptake of metal can be a possible strategy for the development of antibacterial agents against the pathogenic bacteria. ATP binding cassette (ABC) family is one of largest family, found in all kingdoms of life. These proteins transport a large range of substrates such as metals, sugars, amino acids, cholesterol, phospholipids, peptides, proteins, polysaccharides and other metabolites. Metal ions such as Zn2+, Mn2+ and Fe+2 uptake across the membrane is facilitated by the ATP- binding cassette (ABC-type) transport system. This system works by using the solute binding protein (SBPs) present in periplasm in Gram-negative bacteria and linked to the cytoplasmic membrane in Gram-positive bacteria, to capture the molecule and deliver the substrate for translocation by trans membrane domain of ABC transporter powered by the hydrolysis of ATP by nucleotide binding domain of the membrane. The SBPs involved in the ii capture of divalent metal ions like Zn2+, Mn2+ , and Fe+2 belongs to Cluster A-1 family of substrate binding proteins. The Znu system, a member of ABC transporter family, is critical for survival and pathogenesis of Candidatus Liberibacter asiaticus (CLA). The two gene clusters have been identified as homologues of the znuABC transporter in CLA. The studies demonstrated that only genes from one of the two znuABC clusters were able to functionally complement the system in these ΔznuABC E. coli and ΔznuABC S. meliloti. The ZnuA gene from the znuABC cluster, which is unable to complement ΔznuABC E. coli and S. meliloti, encodes for a periplasmic solute-binding protein (CLas-ZnuA2), which show homology to Mn/Fe-specific rather than Zn-specific proteins in Cluster A-I SBPs. Previously, in our lab, crystal structure analysis of CLas-ZnuA2, a periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA in metal-free state, intermediate state of metal binding, Mn2+ -bound state and Zn2+ -bound state revealed that the mechanistic resemblance of CLas-ZnuA2 seems to be closer to the Zn-specific rather than Mn-specific SBPs of cluster A-I family. Biophysical characterization of CLas-ZnuA2 suggested that it is a low metal binding affinity protein. The subtle communications within and between domains from crystal structure analysis revealed that protein seems to prefer a metal-free state. The unique features of CLas-ZnuA2 included a highly restrained loop L3 and presence of a proline in linker helix. In the present work, we further extended our work by mutation studies, particularly in L3 and linker helix, to understand the nature of interactions and their overall effect. Also, in-silico studies on CLas-ZnuA1, a Zn2+ solute binding protein from first of the two gene clusters of Znu system and Esbp, an extracellular solute binding proteins involved in uptake of iron, in CLA were carried out. The 3-dimensional structures were predicted through homology modeling and further virtual screening with molecular docking was used to identify the small molecule inhibitor(s) against these proteins. The thesis has been divided into four chapters. CHAPTER 1 reviews the literature describing the bacterial ABC transporter system and their mechanism to transport the substrate across the membrane, importance of metal in biological processes and mechanism of metal binding and release of Cluster A-I proteins and their role in virulence of pathogenic bacteria. The chapter also describes the history of HLB disease, causal Organism, identification, diagnosis, vector and also detection and genome analysis of CLas and it’s virulence mechanism and strategies to manage the disease. iii CHAPTER 2 deals with the mutational studies of CLas-ZnuA2 and their characterization using X-ray crystallography, surface plasmon resonance (SPR) and circular dichroism (CD). The ZnuA gene from second of the two gene clusters encodes for a periplasmic solute binding protein (CLas-ZnuA2) which shows homology to Mn/Fe-specific rather than Zn-specific cluster A-I SBPs. The subtle internal communications through an intricate network of interactions play a key role in metal-binding and release and maintaining structural integrity in periplasmic metal uptake proteins. Six mutations Ser38Ala; Tyr68Phe; Pro153Ala; Glu159Ala; Asn193Ala and Pro153Ala/Glu159Ala has been created in CLas-ZnuA2 using site-directed mutagenesis and expressed in E. coli BL21-DE3 host cell and purified by Ni-NTA chromatography. All mutations except for S38A and Y68F resulted in destabilization/degradation of the protein. The bioinformatics analysis revealed the creation of hot spot due to P153A mutation in linker helix making CLas-ZnuA2 susceptible to destabilization and degradation. The crystal structure analysis of S38A and Y68F mutants in metal-free and metal-bound forms showed variations in interactions, an increase in the number of alternate conformations and distortions in secondary structure elements. The S38A mutation in CLas-ZnuA2 showed major changes in structure and interactions at the domain interface in loop L3 at the opening of the metal-binding cleft. The metal-free state of S38A CLas-ZnuA2 showed the major sideward shift of part of L3 as compared to metal-free wild-type CLas-ZnuA2 where L3 is displaced away from metal-binding cleft exhibiting an open conformation. Due to mutation of Ser38 to Ala, the particular hydrogen bond between the side-chains of Ser38 and Tyr68 ceases to exist and now the Tyr68 forms hydrogen bond with the main-chain oxygen of Ala38. This conformation is partly similar to the one which occurs on metal-binding in wild-type structure where a larger inward shift of part of the L3 loop (residues 38-40) is observed. The mutation of S38A demonstrated that the sliding of Ser38 present on restrained L3 during metal-binding is part of the metal-binding mechanism for this low metal-binding affinity protein. The mutation to Ala leads to disruption of that mechanism along with disruption of the intricate network of interactions affecting the overall fine-tuned structure. While no sideward shift of L3 was observed in Y68F mutations. The Y68F mutation completely abolished any interaction between mutated Phe68 and Ser38. In the metal-bound form of Y68F CLas-ZnuA2, the inward shift of L3 is not complete to bring His39 within coordinating distance with metal. There were notable changes in interactions of metal coordinating residues with second shell residues in both mutant structures as compared to wild-type metal-free and metal-bound structures of CLas-ZnuA2 were observed. The results suggested that any change in critical residues could alter the subtle iv internal communications and result in disturbing the fine-tuned structure required for optimal functioning. Both S38A and Y68F mutations in CLas-ZnuA2 resulted in the notable alterations in the network of interactions which might affect the mechanism of action of the protein. Although the thermal and binding affinity studies did not show significant change as compared to wild-type CLas-ZnuA2 may be due to very low metal-binding affinities can be explained by the mutational study in PsaA (Mn-binding protein from Streptococcus pneumonia), where, two engineered disulphide bond at the C-terminal helix restricts the flexibility through cross-linking and resulted in the significant decrease in the binding capacity of the mutant PsaA for Mn2+ and Zn2+. CHAPTER 3 deals with the computational approach to identify the small molecule inhibitor(s) against CLas-ZnuA1, which can block the binding of Zn and might be able to inhibit the CLA growth. ZnuA1 is the periplasmic component of ZnuABC transporter system, involved in uptake of Zn metal ion in CLA. Thus, inhibiting this process may be a promising approach to design a drug against CLA. CLas-ZnuA1 has been cloned in the pET-28c expression vector. In order to select the novel lead molecules, the model of CLas-ZnuA1 was used for virtual screening of drug-like molecules from the ZINC database by utilizing virtual screening tool PyRx 0.8. 50 drug-like molecules were identified having higher binding energy as the comparison to the binding energy of reference molecule RDS51 (PDB ID: 4BBP) which showed concomitantly binding with Zn (II) and inhibit the growth of Salmonella enterica. Five molecules were selected for further analysis on the basis of comparison of the binding affinity energy of the AutoDock Tools and AutoDock Vina. Molecular dynamics were performed for determined the dynamics and stability of CLas-ZnuA1-RDS51 and CLas-ZnuA1-inhibitor(s) complexes. MMPBSA method has been employed for binding free energy calculations. The results reveal that ZINC15670529, ZINC92774705, ZINC06510089, ZINC79841324, and ZINC69594834 were found to bind at the active site of CLas-ZnuA1 and inhibit its binding to Zn metal ion. CHAPTER 4 deals with the computational approach to identify the small molecule against Esbp, which can block the binding of Fe and might be able to inhibit the CLA growth. Gram-negative pathogenic microorganisms have developed a range of various high-affinity iron uptake systems for survival like small iron chelators “siderophores”. Another uptake system is also used, which is directly involved in the acquisition of iron from transferrin and/or lactoferrin. Further transport across the membrane is facilitated by the ATP- binding cassette (ABC-type) transport system. v Therefore, affecting the binding process of iron by identifying the small molecule inhibitor at the metal binding site will interrupt the function of CLas-Esbp and thus inhibit the iron uptake process, and this strategy could be used in the discovery of antimicrobial agent against the pathogenic microorganism. CLas-Esbp has been cloned in pET-28c expression vector. The 3-dimensional structure of CLas-Esbp was predicted by homology modeling. The Drug like molecules was retrieved from ZINC database and used for virtual screening. 50 molecules were identified on the basis of binding affinity, fulfilling the range of Lipinski rule of five. Three molecules were selected for further analysis based on the comparison of binding affinity from AutoDock Tools and AutoDock Vina. Furthermore, the protein-ligand complexes were subjected to molecular dynamics simulation to understand the dynamics and stability of the complex(s). Molecular Mechanic/Poisson-Boltzmann Surface Area (MMPBSA) was employed for binding free energy calculation. The results revealed that ZINC03143779, ZINC05491830, and ZINC19210425 were found to be bind at the binding site of CLas-Esbp protein. This computational approach provides an idea in the further development of inhibitor designing against essential proteins of pathogenic microorganisms.