Structural studies of the RPFF protein involved in Quorum Sensing and the IGHMBP2 protein involved in Spinal Muscular Atrophy with Respiratory Distress type 1.
Lim, Siew Choo.
Date of Issue2012
School of Biological Sciences
This thesis is composed of two separate projects on the proteins RpfF and Ighmbp2, and their functional role in quorum sensing and neurodegenerative disorder, respectively. The details of the RpfF protein and the helicase domain of Ighmbp2 protein are presented in Chapters 1 to 5 and Chapters 6 to 10, respectively. Quorum sensing is a form of communication in bacterial cells to allow coordination of gene expression in response to fluctuations in cell-population density. The pathogen Xanthomonas campestris pathovar campestris (Xcc) uses a small diffusible molecule named diffusible signal factor (DSF) as its quorum sensing signal to assess the population density and regulate virulence factor production. DSF regulates a cluster of genes designated as rpfABCDEFG which are important for the production of virulence factors such as extracellular enzymes and extracellular polysaccharides in Xcc. Recent studies have indicated that the regulation of DSF biosynthesis involves the interaction of RpfC and RpfF proteins. However, the mechanism of how RpfC regulates RpfF activity is not well understood. The crystal structure of the enoyl-CoA hydratase RpfF at a resolution of 1.8 Å is presented in this study. RpfF adopts a self-associating fold such that the C-terminal helix wrap round the N-terminal α/β core domain. Structural homology search in the Protein Data Bank revealed that RpfF is structurally similar to members of the hydratase/ isomerase superfamily. Further structural and mutational analyses identified Glu141 and Glu161 as conserved glutamate residues positioned in the catalytic pocket and are critical to RpfF activity in the DSF biosynthesis pathway. Superposition with ligand-bound enoyl-CoA hydratase showed that the C-terminal helix of RpfF is positioned at the entrance of the catalytic pocket and is in steric hinderance with the Co-A moiety of the substrate. In addition, the structure of RpfF in complex with REC domain of RpfC shows the REC domain interacting with the C-terminal helix of RpfF. These findings suggest that interaction with RpfC may lock RpfF in an inactive conformation, therefore having a negative regulating effect on DSF production. The second part of the thesis examines the Ighmbp2 protein which is implicated in the neurodegenerative disease spinal muscular atrophy with respiratory distress type I (SMARD1). Most of the genetic mutations causing SMARD1 occur in the region of the Ighmbp2 gene coding for the helicase domain. Although several studies have pointed out Ighmbp2 involvement in DNA replication, transcription, pre-mRNA splicing, and RNA metabolism, its exact role remains elusive. Based on sequence alignment and biochemical studies, Ighmbp2 protein has been classified as a member of the helicase superfamily I that can unwind RNA and DNA duplexes in the 5’ → 3’ direction in an ATP-dependent manner. To have a better understanding of this protein from the structural perspective, crystal structures of the helicase domain of Ighmbp2 in the free and ssRNA-bound states are obtained. The helicase domain of Ighmbp2 has high structural similarity with the helicase domain of Upf1, a protein in the Upf1-like subfamily. Upon ssRNA binding, conformation changes were observed in domains 1A and 1B. Data from surface plasmon resonance experiments showed that the presence of a R3H domain, located at the C-terminus of Ighmbp2, enhances Ighmbp2 nucleic acid binding ability. This highlights the importance of accessory domains in regulating helicase enzymatic activities. Based on Ighmbp2 helicase structure, the implications of SMARD1 causing mutations in Ighmbp2 are addressed from the structural perspective.