Wassmann, Paul. Elucidation of the regulatory mechanisms of the diguanylate cyclases PleD, DgcA and DgcB by structural and biophysical analysis. 2009, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_8738
To get insights in the environmental cues dependent regulatory mechanism of dimerization, the Rec domains bearing DGC PleD was crystallized in its pseudophosphorylated/activated form. The crystal structure of BeF3!•Mg2+ activated PleD is the first structure showing a full-length response regulator in its activated state. Comparison with the structure of non-activated PleD resulted in the elucidation of the molecular mechanisms of the dimerization process. Additionally, the formation of a two-fold symmetric, charged pocket at the (Rec1-Rec2)2 stem interface was observed, which might represent the long-sought ‘pole-localization’-signal for PleD. Besides giving insights in the substrate binding mode of the DGCs, the obtained structures shed light on the catalytic mechanism of DGCs. In combination with biochemical data the structures verified the ‘two-metal assisted’ catalysis mechanism for the DGCs. A new, c-di-GMP dimer dependent domain-cross-linking mode was revealed. It is generally applicable to DGCs, involving in the process merely the GGDEF domains. It turned out that the successful inhibition of the DGC PleD relies on the presence of primary- and secondary I-sites, whereas the initial binding of c-di-GMP depends solely on the primary I-site. PleD was shown to need R390 besides the RxxD motif (the whole I-site) to bind c-di-GMP. Finally, cross-linking of proteins by c-di-GMP, intradimeric like in PleD and intermolecularly like in DgcB, was shown and might represent the main regulatory function of this second messenger.
|Committee Members:||Jenal, Urs|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Schirmer)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||130|
|Last Modified:||30 Jun 2016 10:41|
|Deposited On:||26 Aug 2009 13:28|
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