Structural studies of enzymes regulating bacterial life style and cell wall biogenesis
Author: Chen, Ming Wei
Date: 2013-12-06
Location: Föreläsningssal Lennart Nilsson, Nobels väg 15A, Karolinska Insitutet Solna, Stockholm
Time: 10:00
Department: Inst för medicinsk biokemi och biofysik / Dept of Medical Biochemistry and Biophysics
Abstract
Bacteria can adapt to different biotic and abiotic environments by changing their life style, switching between sessile or motile, free-living or community-bound, virulent or dormant states. Each bacterium also faces the challenges of maintaining a protective barrier while growing, replicating and responding to environmental changes. Governing these processes are enzymes involved in signalling and cell-wall biogenesis, which are the subjects of this thesis.
The second messenger cyclic di-GMP (c-di-GMP) regulates a vast array of processes such as motility, biofilm formation, virulence and cell cycle progression on transcriptional, post-transcriptional and post-translational levels. RocR from Pseudomonas aeruginosa is a response regulator protein containing an active EAL domain that breaks down c-di-GMP. The crystal structure of RocR was determined to 2.5 Å resolution, revealing a compact tetrameric structure with the subunits displaying two conformational states. The unique architecture allows two phospho-receiver domains to be adjacent to the EAL active sites while being exposed and available for phosphorylation events. Also, solution studies using SAXS and biochemical analyses suggest that the protein does not require large conformational changes to alter its phosphodiesterase activity, leading to a mechanistic model of signal propagation from the phosphorylation site to the EAL active site based on secondary structural changes. Tbd1265 from Thiobacillus denitrificans is a transmembrane protein containing a GGDEF-EAL tandem domain that can both synthesise and hydrolyse c-di-GMP. Functional studies confirmed the bifunctionality of this tandem domain and further suggested that a predicted coiled-coil region preceding the GGDEF domain is required for activity. The crystal structure of a construct comprising these two domains of Tbd1265 was determined to 3.4 Å, revealing a conformation of the GGDEF domains in the dimeric molecule that is not compatible with product-inhibition or catalysis. We propose a regulatory mechanism where Tbd1265 can adopt at least three conformations (resting, active and inhibited) based on signals from the periplasmic binding protein (PBP) domain.
MurB is an essential oxidoreductase that produces UDP-N-acetylmuramic acid, a precursor for peptidoglycan synthesis. The crystal structure of the ternary complex of P. aeruginosa MurB with NADP+ and FAD revealed that the substrate channel can accommodate two distinct substrate molecules. The study also revealed a potassium ion in the active site that directly binds the substrates and can stabilise the transition state of the reaction, thus explaining the activating effect of potassium ions on MurB catalysis. The structure of the MurB ternary complex provides a useful template for the design of novel enzyme inhibitors that might be developed into promising drug candidates.
The second messenger cyclic di-GMP (c-di-GMP) regulates a vast array of processes such as motility, biofilm formation, virulence and cell cycle progression on transcriptional, post-transcriptional and post-translational levels. RocR from Pseudomonas aeruginosa is a response regulator protein containing an active EAL domain that breaks down c-di-GMP. The crystal structure of RocR was determined to 2.5 Å resolution, revealing a compact tetrameric structure with the subunits displaying two conformational states. The unique architecture allows two phospho-receiver domains to be adjacent to the EAL active sites while being exposed and available for phosphorylation events. Also, solution studies using SAXS and biochemical analyses suggest that the protein does not require large conformational changes to alter its phosphodiesterase activity, leading to a mechanistic model of signal propagation from the phosphorylation site to the EAL active site based on secondary structural changes. Tbd1265 from Thiobacillus denitrificans is a transmembrane protein containing a GGDEF-EAL tandem domain that can both synthesise and hydrolyse c-di-GMP. Functional studies confirmed the bifunctionality of this tandem domain and further suggested that a predicted coiled-coil region preceding the GGDEF domain is required for activity. The crystal structure of a construct comprising these two domains of Tbd1265 was determined to 3.4 Å, revealing a conformation of the GGDEF domains in the dimeric molecule that is not compatible with product-inhibition or catalysis. We propose a regulatory mechanism where Tbd1265 can adopt at least three conformations (resting, active and inhibited) based on signals from the periplasmic binding protein (PBP) domain.
MurB is an essential oxidoreductase that produces UDP-N-acetylmuramic acid, a precursor for peptidoglycan synthesis. The crystal structure of the ternary complex of P. aeruginosa MurB with NADP+ and FAD revealed that the substrate channel can accommodate two distinct substrate molecules. The study also revealed a potassium ion in the active site that directly binds the substrates and can stabilise the transition state of the reaction, thus explaining the activating effect of potassium ions on MurB catalysis. The structure of the MurB ternary complex provides a useful template for the design of novel enzyme inhibitors that might be developed into promising drug candidates.
List of papers:
I. Ming Wei Chen, Masayo Kotaka, Clemens Vonrhein, Gérard Bricogne, Feng Rao, Mary Lay Cheng Chuah, Dmitri Svergun, Gunter Schneider, Zhao-Xun Liang and Julien Lescar. Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic Di-GMP signaling. J Bacteriol. 2012; 194, 4837–4846.
Fulltext (DOI)
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II. Chong Wai Liew, Ming Wei Chen, Chun Loong Ho, Surya Wahyu, Tobias Cornvik, Gunter Schneider, Zhao-Xun Liang and Julien Lescar. The structure of the diguanylate cyclase-phosphodiesterase tandem domain of a bacterial sensor protein suggests a model for its activation. [Manuscript]
III. Ming Wei Chen, Bernhard Lohkamp, Robert Schnell, Julien Lescar and Gunter Schneider. Substrate channel flexibility in Pseudomonas aeruginosa MurB accommodates two distinct substrates. PLoS ONE. 2013; 8, e66936.
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Pubmed
View record in Web of Science®
I. Ming Wei Chen, Masayo Kotaka, Clemens Vonrhein, Gérard Bricogne, Feng Rao, Mary Lay Cheng Chuah, Dmitri Svergun, Gunter Schneider, Zhao-Xun Liang and Julien Lescar. Structural insights into the regulatory mechanism of the response regulator RocR from Pseudomonas aeruginosa in cyclic Di-GMP signaling. J Bacteriol. 2012; 194, 4837–4846.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Chong Wai Liew, Ming Wei Chen, Chun Loong Ho, Surya Wahyu, Tobias Cornvik, Gunter Schneider, Zhao-Xun Liang and Julien Lescar. The structure of the diguanylate cyclase-phosphodiesterase tandem domain of a bacterial sensor protein suggests a model for its activation. [Manuscript]
III. Ming Wei Chen, Bernhard Lohkamp, Robert Schnell, Julien Lescar and Gunter Schneider. Substrate channel flexibility in Pseudomonas aeruginosa MurB accommodates two distinct substrates. PLoS ONE. 2013; 8, e66936.
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Schneider, Gunter
Issue date: 2013-11-15
Rights:
Publication year: 2013
ISBN: 978-91-7549-402-9
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