STUDIES ON PERIPLASMIC SOLUTE BINDING PROTEINS OF CANDIDATUS LIBERIBACTER ASIATICUS

Abstract

Citrus greening or huanglongbing (HLB), one of the most devastating disease of citrus worldwide, is caused by a phloem-limited unculturable Gram negative fastidious α- proteobacteria. It is classified on the basis of its geographical origin and 16S rDNA sequence into three species, Candidatus Liberibacter asiaticus (Las), Candidatus Liberibacter africanus (Laf), Candidatus Liberibacter americanus (Lam). Among three species, Candidatus Liberibacter asiaticus is most virulent strain and is transmitted by Asian citrus psyllid (Diaphorina citri Kuwayama). An early symptom of HLB in citrus is blotchy mottling with green islands on leaves. As the disease progresses, infected shoots are undersized and the branches slowly die. The Fruit from infected trees may be small, lopsided, dark aborted seeds, with deprived coloration and tends to drop prematurely. Till date no effective control strategies have been developed for management of HLB disease and stop it from spreading to disease free new citrus-production areas. The current disease control strategies include the minimizing the psyllid population chemically and biologically and prevent trees from becoming infected by removing the infected tree. However, targeting some of the crucial proteins which are essential for survival of the bacterium holds a promising strategy to combat the bacterium. Proteins are the most versatile biological macromolecules in living systems and perform diverse array of functions in essentially all biological processes. They function as transporters, catalysts, provide mechanical support, immune protection, transmit nerve impulses, storage of other molecules such as oxygen and control growth and differentiation. ATP-Binding Cassette (ABC) transporters are integral membrane proteins. It facilitates the transfer of diverse substrates across the membrane using energy provided by ATP hydrolysis. The ABC transporters consist of two transmembrane domains (TMDs) that form a specific ligand transport channel. The two cytosolic ATP-binding domains (ABDs) that bind and hydrolyze ATP to provide the energy for the translocation of substrate across the membrane. In addition to these transmembrane subunits, prokaryotic ABC-importers having periplasmic solute binding protein (SBP), which traps the substrate in the periplasmic space and delivers it to the external surface of the transport complex. The SBPs have evolved to recognize a wide variety of solutes with high affinity and specificity, and have also been involved in signal transduction, transcriptional regulation and catalysis. The Cluster F-IV and A-I family of ii substrate binding proteins are involved in transport of amino acids (cystine, cysteine, arginine, glutamine, histidine, glutamate/aspartate) and divalent metal ions (Zn2+, Mn2+ and Fe2+) respectively. The amino acid transporters are important for bacteria to establish infection. In human pathogenic bacteria L. monocytogenes, the SBP of cysteine transporter (CtaP) was shown to be involve in virulence mechanism in a murine model of intravenous infection. Proteome analysis showed that the Candidatus Liberibacter asiaticus (CLA) possess essentially two amino acid binding receptors, one specific for cationic amino acids and other is a putative cysteine binding protein. The expression of periplasmic cationic amino acid ABC transporter mainly occurs in citrus psyllid host while the second one having a maximum sequence homology to cystine-binding receptors mainly expresses in the planta. In this study, we have determined high resolution crystal structure of putative cystine-binding periplasmic ABC transporter (CLasTcyA) of CLA from cluster F-IV in complex with cysteine, cystine, arginine and citrate. The crystal structure analysis revealed the unique features of the protein suggesting a new adaptive mechanism of ligand binding and release. Surface plasmon resonance (SPR), MicroScale Thermophoresis (MST), Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) analysis was carried out to ascertain binding affinity and thermal stability of the protein with different ligands. Also, several mutation studies was carried out to understand the role of residue involved in stabilization of substrate molecule. The bioinformatics-based virtual screening was employed to screen the potential inhibitor molecules. The Znu system, a member of A-I family of substrate binding proteins is essential for pathogenesis and survival of CLA. The CLas having two ZnuABC systems, out of which only one is functional. Previously, in our lab, crystal structure of periplasmic metal binding receptor (CLas-ZnuA2) from CLas was determined . It’s showed, CLas-ZnuA2 is a low affinity metal binding protein with a unique mechanism of metal binding and release. In the present work, we have extended our study to analyze the binding of divalent transition heavy metals like Cd2+, Ba2+, and Hg2+ to CLas-ZnuA2 using SPR, CD, intrinsic fluorescence and DSC studies. The thesis has been divided into four chapters. CHAPTER 1 reviews the literature describing the history and geographical distribution of HLB disease, causal organism, diagnosis, vector and its virulence mechanism and strategies to manage the disease. The chapter also describes the bacterial ABC transporter system and their mechanism of substrate binding and transport across the membrane. Importance of aminoacid iii and metal in biological processes and mechanism of substrate binding release of cluster F-IV and A-I protein and role of these protein in virulence of pathogenic bacteria. CHAPTER 2 describes the cloning, overexpression, purification and characterization using Xray crystallography, surface plasmon resonance (SPR), MicroScale Thermophoresis (MST), Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) of CLasTcyA. The purification of CLasTcyA was accomplished using two step purification method employing Ni- NTA affinity and size exclusion chromatography. The purified CLasTcyA when applied to size exclusion chromatography column showed a single peak corresponding to monomeric species. The sequence and crystal structures analysis, in ligand-bound states, revealed unique features of CLasTcyA as compared to related proteins. One of the unique features of CLasTcyA structure was found to be the positioning of C-terminal extended loop of one chain very close to substrate binding site of adjacent monomer in the asymmetric unit. The presence of a disulphide bond, unique to Candidatus Liberibacter family, holds the C-terminal extended loop in position. The analysis of substrate-binding pocket of CLasTcyA suggested a broad specificity and a completely different orientation of the bound substrates as compared to related structures. The open conformation for one of the two chains in asymmetric unit in Arg-bound structure revealed a limited open state (18.4°) for CLasTcyA as compared to open state of other related proteins (~60°). The strong interaction between Asp126 on helix-α5 of small domain and Arg82 (bigger domain) restricts the degree of opening in ligand-free open state. The dissociation constant of 1.26 μM by SPR and 3.7 μM by MST exhibited low affinity for the cystine. This is the first structural characterization of an L-cystine ABC transporter from plant pathogen and results suggest that CLasTcyA may have evolved to cater to its specific needs for its survival in the host. CHAPTER 3 describes the mutational studies of CLasTcyA and characterization using Surface Plasmon Resonance (SPR), Circular Dichroism (CD), Differential Scanning Calorimetry (DSC) and Intrinsic Fluorescence studies. Three mutations V58W; V58W/C212S/C239S; V58W/H95A has been created in CLasTcyA using site-directed mutagenesis and also c-terminal truncation has been created in double mutant CLasTcyV58W/C212S. All mutant and truncation expressed in E. coli BL21-DE3 host cell and purified by Ni-NTA chromatography. The CLasTcyAV58W showed a noticeable increase in iv binding affinity and thermal stability as compared to the native form. The mutation of two cysteines to serine in triple mutant CLasTcyAV58W/C212S/ C239S showed a marked decrease in binding affinity and thermal stability indicating the importance of disulfide bond. Likewise, a decrease in binding affinity and stability was observed on removal of C-terminal extended loop, involved in substrate binding in adjacent monomer, in truncated CLasTcyAV58W/C212S. The importance of residues from hinge region became evident where a decrease in binding affinity and stability was observed on mutation of His 95 to Ala in the double mutant CLasTcyAV58W/H95A. The bioinformatics-based virtual screening was employed to screen the potential inhibitor molecules. CHAPTER 4 describes the binding of some heavy metals to CLas-ZnuA2. The characterization has been carried out using SPR, CD, Intrinsic Fluorescence and DSC studies. Also, crystal structure analysis of CLas-ZnuA2 bound to Cd2+ was carried out. The biophysical analysis showed relatively higher binding affinity and stability of CLas-ZnuA2 in bound state with tested heavy metals as compared to Mn2+ and Zn2+. In SPR analysis, the binding affinities of 2.049 X 10-6, 7.174 X 10-6 and 5.316 X 10-5 for Hg2+, Cd2+, and Ba2+ respectively were higher as compared to earlier reported values for Mn2+ and Zn2+. The crystal structure analysis in Cd2+ bound state showed additional metal binding sites in addition to the primary binding site between the two lobes. The mutation of metal binding residues, H106C and D247C, showed relatively higher binding affinities for Ba2+ than Cd2+ as compared to native form. Molecular Docking results indicate that binding of Ba2+, Co2+, and Hg2+ to CLas-ZnuA2 is comparable to binding affinity of Cd2+ to CLas-ZnuA2. All the metals coordinate with His39, His106, Glu172, and Asp247. Molecular dynamics have been carried out to explore the dynamic movement of protein and results conclude that H106C and D247C mutants are stable and compact. Taken together with expression of permease and exporter system for heavy metals, our results demonstrate that CLas-ZnuA2 may be involved in sequestering and transport of various transition divalent metals.