STUDIES ON A METAL UPTAKE PROTEIN FROM CANDIDATUS LIBERIBACTER ASIATICUS

Abstract

Metal ion uptake and sequestration is critical for bacterial survival and growth in the environment as well as within various hosts. Transition metals such as manganese, zinc, and iron play an important role as enzyme co-factors for a number of biological processes including DNA replication, protein synthesis, respiration, cell wall synthesis and neutralization of reactive oxygen species. Metal deficiencies greatly inhibit the growth of microorganisms. Therefore, inhibition of metal uptake can serve as a possible strategy towards developing antibacterial agents against the pathogenic bacteria. Citrus Huanglongbing (HLB) is an extremely destructive, fast-spreading disease of citrus which causes severe economic losses worldwide. The disease is caused by phloem-limited, unculturable, Gram-negative α-proteobacteria Candidatus Liberibacter spp. Three species known are „Candidtaus Liberibacter asiaticus‟ (CLas), „Ca. L. africanus‟, and „Ca. L. americanus‟. CLas is considered to be the most devastating species and is transmitted by Asian citrus psyllid, Diaphorina citri. Almost all citrus plants are susceptible to HLB and to date, there is no established cure for this century-old and yet, newly emerging disease. HLB is mainly identifies by blotchy mottling with green patches on leaves. Infected shoots are undersized, and the branches slowly die due to disease progress. Fruit from infected trees may be small and irregular, with deprived coloration. HLB seriously effects the citrus industry by reducing the lifespan of the trees and diminishing fruit yield as well fruit quality. The symptoms of HLB have been associated with those of zinc deficiency and it has been reported that CLas infection significantly reduces levels of zinc and several other mineral nutrients. Proteins are large biological molecules which perform various functions within living organisms including metabolic reactions catalysis, DNA replication, cell signaling, and transport of molecules from one location to another. In course of time, proteins evolve to perform specific functions by changing their primary sequence and hence tertiary structure. Proteins have been classified into various superfamilies on the basis of common ancestry which are further divided into families consisting of evolutionary related protein and subfamilies based on three dimensional structures. ATP binding cassette (ABC) family is one of the largest family, present in all forms of life from prokaryotes to humans, includes several hundred different membrane transport proteins. These proteins transport a variety of substrates ii including ions, sugars, amino acids, phospholipids, cholesterol, peptides, polysaccharides, proteins and other ligands. The metal ions like Zn2+, Mn2+ and Fe2+ are also transported across membranes through ABC transport systems. The bacterial ABC-type transport systems comprise of three components that are: a solute-binding protein (SBP) found in the periplasm in Gram-negative bacteria or linked to the cytoplasmic membrane in Gram-positive bacteria, trans-membrane permease and nucleotide-binding protein (ATPase). The solute binding proteins which are involved in uptake of divalent metal ions zinc, manganese and iron belongs to the Cluster A-I family of substrate binding proteins. The Znu system, a member of ABC transporter family, is critical for survival and pathogenesis of CLas. Two homologues of this system have been identified in CLas. It has been reported that only first ZnuABC gene cluster is functional and able to complement Δznu Escherichia coli and Δznu Sinorhizobium meliloti strains. In the present work, a periplasmic solute binding protein from second of the two gene clusters of Znu system (CLas-ZnuA2) have been characterized by bioinformatics analysis and other biophysical techniques which include X-ray crystallography, surface plasmon resonance (SPR) and circular dichriosm (CD). The thesis has been divided into four chapters. Chapter 1 reviews the literature describing about bacterial ABC permeases and their mechanisms of substrate translocation, the structure and mechanism of metal binding and release of Cluster A-I proteins and their role in virulence of pathogenic bacteria. The chapter also describes about history of HLB disease, host range, evolutionary relationships between CLas and other related bacteria, detection and genome analysis of CLas and its virulence mechanism and hitherto controls strategies. Chapter 2 describes the materials used and methods adopted for the present work. The CLas-ZnuA2 gene has been cloned in pET 28c expression vector. The recombinant protein has been produced in E. coli and purified to homogeneity by affinity and size exclusion chromatography methods. The multiple sequence alignment and phylogenetic analysis of CLas-ZnuA2 and other related proteins has been done. The CD experiments of the protein in metal-free state as well as in presence of different concentration of metal ions have been carried out at different temperatures and the effect of different metals on the thermal stability of protein has been elucidated. The SPR experiments have been performed to elucidate the binding affinity of the protein towards different metal ions. The crystallization of the purified protein was iii performed and the crystal structure was determined in metal-free, intermediate and Mn2+ and Zn2+ bound states. Chapter 3 describes the results obtained in the present work. The sequence similarity search showed that CLas-ZnuA2 shares significant sequence similarity with Cluster A-I proteins which are involved in uptake of divalent metal ions (Mn/Fe/Zn) in PDB database. The phylogenetic analysis of proteins present in PDB database showed that CLas-ZnuA2 clusters with Mn-specific proteins. Multiple sequence alignment revealed presence of conserved metal binding residues His39, His106, Glu172 and Asp247 specific for Mn/Fe binding. The CD studies showed that metal bound states of CLas-ZnuA2 are more thermally stable than metal-free state. The SPR studies showed that CLas-ZnuA2 have almost equal order of affinity for Mn2+ and Zn2+. The three dimensional structure of CLas-ZnuA2 in metal-free state, intermediate state and metal bound states showed the presence of similar fold like other related Cluster A-I proteins containing two α/β domains connected by a rigid linker helix. One unique feature is presence of curved linker helix in all states of CLas-ZnuA2 due to the presence of a proline residue within linker helix. This Pro was found to be absent in other reported related structures, however closely related proteins in non-reduntant database possess the Pro in linker helix. The major difference observed in metal-free and metal-bound states was the position of loop hosting His39 which was moved outside and side chain of His39 was flipped away in metal-free state while in metal-bound states this loop was moved inside and His39 side chain was flipped towards the metal ion. In intermediate state, this loop and thus His39 occupies similar position like metal-free state having occupancy of 0.8 and metal ion was present with half occupancy. Also metal ion was not in ideal coordination distances from other three metal binding residues. The Mn2+ and Zn2+ bound structures are almost similar with some variations in the intra-molecular interactions. Both Mn2+ and Zn2+ coordinate pentavalently with square pyramidal geometry rather than ideal octahedral and tetrahedral geometry respectively. The coordination chemistry observed is different from tetrahedral geometry for Mn2+ and Zn2+ ions observed in other Mn-specific SBPs. Chapter 4 includes the discussion. The comparison of CLas-ZnuA2 structure in three states showed that metal binding and release is facilitated by a large displacement along with a change in orientation of the side chain for one of the metal binding residue (His39) flipped away from metal binding site in metal-free form. The sequence and structure of CLas-ZnuA2 iv has been compared with metal-free and metal-bound states of related proteins and unique mechanism of metal binding and release has been proposed which is different from other related Mn-specific metal binding proteins. The crystal structure captured in intermediate state of metal binding revealed the changes in conformation and interaction of the loop hosting His39 during the metal binding. A rigid body movement of C-domain along with partial unfolding of linker helix at its C-terminal during metal binding, as reported for PsaA, a Mn-binding protein from Streptococcus pneumonia, was not observed in CLas-ZnuA2. The present results suggested that despite showing maximum sequence identity to the Mn/Fe-specific SBPs, the mechanistic resemblance of CLas-ZnuA2 seems to be closer to Zn-specific SBPs of Cluster A-I family. In conclusion, CLas-ZnuA2 protein shows unique features and has been diverged from related Cluster A-I proteins to adopt different mechanism of metal binding achieved by single amino acid change possessing Pro in linker helix due to which it might be involved in transport of both Mn2+ and Zn2+ ions depending upon the requirement of bacteria.