BIOCHEMICAL AND STRUCTURAL STUDIES ON CheR METHYLTRANSFERASE FROM BACILLUS SUBTILIS

Abstract

Two-component signaling systems (TCSS) enable bacteria to sense, respond and adapt to changes in the environment. The prototypical TCSS is comprised of a Histidine protein kinase and a response regulator. TCSS control a wide variety of processes in bacteria such as nitrogen regulation, osmolarity, chemotaxis, sporulation etc. The most widely studied TCSS i.e. chemotaxis enables bacteria to respond to the chemicals in their environment, by moving towards the chemo-attractants, and away from the repellents. The swimming motion of bacteria alternates between two modes, smooth runs and re-orientating tumbles that randomly change the directional movement. Runs result when flagella rotates in counter-clockwise (CCW) direction whereas clockwise (CW) rotation of flagella results in tumble. In the presence of chemo-attractants (or chemo-repellents), the frequency of tumbles is decreased, resulting in longer runs, enabling the bacteria to proceed in favorable direction. However, despite the sustained presence of chemo-effector molecules, the pre-stimulus behavior is regained after a certain time. This phenomenon is known as adaptation. Chemotaxis enables bacteria to respond to their chemical environment by controlling the direction of flagellar rotation. However, the location of the chemotaxis receptors and the flagella is different. This prevents direct interaction between the receptors and the flagella, and the communication between them is carried out by a sophisticated signal transduction system. The end result of this signal transduction is a change in the direction of flagellar rotation. The chemicals are sensed by the methyl-accepting chemotaxis protein receptor (MCPR) that has a periplasmic ligand-binding domain, transmembrane segments and a cytoplasmic signaling domain. The docking of a binding protein or a small-molecule attractant to the periplasmic sensory domain of a receptor generates an intramolecular conformational change that is transmitted across the bilayer to the bound cytoplasmic histidine kinase. The kinase, which is up-regulated by either the repellent-occupied or empty (apo) receptor, but is down-regulated by the attractant-occupied receptor, phosphorylates itself on a specific histidine sidechain. This same phosphoryl group is then transferred from the histidine to an aspartate in the active site of a response regulator protein, CheY or CheB, each of which is an autocatalytic aspartate kinase. Phospho-CheY dissociates from the signaling complex and diffuses to the rotary motor where it docks and increases the probability of the clockwise motor rotation, thereby favoring the formation of the tumbling swimming state. The steady state ii level of phospho-CheY thus serves as a diffusible tumble signal that controls the overall frequency of tumbling. This tumble signal is modulated by two opposing reactions: creation of phospho-CheY by the receptor-kinase complex, and destruction of phospho-CheY by hydrolysis of its acyl phosphate. CheZ speeds the latter hydrolysis reaction, acting as a phosphatase. Repellents stimulate the histidine kinase activity and speed the production of phospho-CheY, whereas attractants inhibit the histidine kinase and slow phospho-CheY formation, thereby raising or lowering the steady state tumble signal, respectively. MCPRs are reversibly methylated at the conserved glutamate residues, located in the coiled-coil region of its cytoplasmic domain. CheR, a SAM-dependent methyltransferase (S-adenosyl methionine–dependent MTase), and CheB, a methylesterase (MEase), are the two antagonistic enzymes that catalyze the reversible methylation-demethylation reaction of the transmembrane MCPRs, which is accountable for chemotactic adaptation. In this study, we have carried out structural, biochemical and functional characterization of chemotaxis protein methyltransferase CheR from gram-positive bacterium Bacillus subtilis. The thesis has been divided into four chapters. Chapter 1 reviews the literature. It describes two component signaling system, bacterial chemotaxis, the chemotaxis signal transduction pathway and the functions of various proteins involved. Further, it discusses various classes of methyltransferases. ClassI methyltransferases have been described in detail. It also describes CheR methyltransferase , the mechanism of receptor recognition by CheR and, its structure. Chapter 2 focuses on identifying the functional roles of CheR, from a gram-positive bacterium, Bacillus subtilis (BsCheR). In silico structural analysis and molecular docking of BsCheR have been performed. Our study showed that the N-terminal domain played an important role in SAH binding. Also, BsCheR has been cloned and expressed to yield purified protein which was analyzed for phosphorylation using immunoblot and mass spectrometry. Immunoblot analysis showed that the purified BsCheR was phosphorylated. Further, mass spectrometry studies detected the phosphorylation at Thr3 position in the N-terminal domain of BsCheR. Phosphorylation of BsCheR suggested a regulatory role of the N-terminal domain, analogous to its antagonistic iii enzyme, the chemotaxis-specific methylesterase (CheB). In addition, comparative sequence analysis has also been carried out to investigate if the CheR MTase of the gram-positive Bacillus sp. has distinct receptor recognition site and a positively charged α2 helix, for interaction with the negatively charged substrate methylation sequence, as in the gram-negative bacteria. Sequence analyses revealed that the α2 helix of the N-terminal domain was involved in the recognition of the methylation site of the chemotactic receptor. Chapter 3 focuses on structural analysis of BsCheR. BsCheR, in complex with SAH, has been crystallized by vapour diffusion in sitting drop and its structure has been determined by X-ray crystallography using molecular replacement method. Crystal structure analyses have shown BsCheR to contain four structural components as in StCheR: N-terminal domain, linker, C-terminal domain, and a beta-subdomain appended to C-terminal domain. Interactions of BsCheR and StCheR with SAH cofactor have also been compared. However, the primary goal of study was to investigate any differences in pentapeptide-dependent and pentapeptide-independent chemotactic methyltransferase. Structural comparison of StCheR and BsCheR indicated different length as well as arrangement of helices in their N-terminal domain. Structural insight into BsCheR also revealed a novel V-shaped helix α4 with a kink at Asp53 in the N-terminal domain. The kink is speculated to have a role in transferring conformational changes within and/or between the domains upon/for receptor interaction. Comparison of the beta-subdomains revealed beta-loop connecting β4 and β5 to be double the length in StCheR as compared to that in BsCheR. We anticipate that further studies of CheR in conjunction with CheD, CheY and CheC will provide insight into the role of beta-subdomain in BsCheR. The structural differences in BsCheR and StCheR might reflect the differences in mechanism of receptor recognition and interaction in pentapeptide-dependent and pentapeptide-independent methylation systems. Chapter 4 describes site-directed mutagenesis of a key Asp residue. The mutant Asp130→Lys was over-expressed and was found to be completely insoluble. We concluded that Aspartate 130 plays a role in folding and/or solubility of the protein. Additionally, methyltransferase assay for CheR was carried out using methyltransferase colorimetric assay kit from Cayman chemicals. Glutathione was used as a substrate. The activity of the enzyme was calculated at each of the two iv concentrations of the substrate used. Further, ITC studies were carried out to calculate the binding affinity of BsCheR with SAM.