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Fic-mediated adenylylation : catalysis and regulation

Goepfert, Arnaud. Fic-mediated adenylylation : catalysis and regulation. 2014, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10796

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Abstract

Adenylylation, also referred to as adenylation or AMPylation, is the process by which adenosine-5‘-monophosphate (AMP) is covalently attached to a protein, a nucleic acid, or a small molecule. It is a widespread post-translational modification employed by a large variety of enzymes to regulate multiple cellular functions. This modification, originally discovered in the 1960s, recently re-emerged as a modification used by bacterial effector proteins to regulate key host signaling events upon infection. The Type Three Secretion System effectors VopS and IbpA from Vibrio parahaemolyticus and Histophilus somni, respectively, harbor a FIC domain, which catalyzes adenylylation of Rho family GTPases. The covalent attachment of the bulky AMP moiety abrogates binding of downstream effectors resulting in actin cytoskeleton collapse and concomitant host cell death.
The FIC domain is not only present in a pathogenic setting but is also found in proteins that play a role in intrinsic signaling processes where its deleterious adenylylation activity needs to be tightly regulated.
In this study, I performed structural and biochemical analyses to unravel the structural determinants and mechanisms governing catalysis and regulation of Fic proteins as widespread signaling proteins.
First, by solving various structures of FIC domain-containing proteins I could show that FIC utilizes conserved active site features to favorably orientate the ATP substrate relative to the target protein, allowing AMP transfer to occur. We suggest that the catalytic mechanism can be generalized and extrapolated to all adenylylation-competent Fic proteins.
Second, I deciphered the structural mechanism controlling Fic-catalyzed adenylylation. An alpha helix characterized by a conserved [S/T]xxxE[G/N] motif tightly associates with the Fic active site and perturbs adenylylation-competent ATP binding. This alpha helix can be part of the Fic protein itself as an N- or a C-terminal extension, or can be provided by a separate antitoxin. Based on structural homology modeling, this regulation mechanism was found to be conserved from bacteria to higher eukaryotes.
The structural insights on the protein-ATP substrate binding specificity accumulated in this study will prove useful for coming efforts on rational drug design. Furtheremore, knowledge of the universal catalytic and inhibitory mechanism of Fic mediated AMP transfer will now pave the way for further studies towards the physiological roles of Fic proteins and particularly the identification of their protein targets.
Advisors:Schirmer, Tilman
Committee Members:Dehio, Christoph
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Schirmer)
UniBasel Contributors:Goepfert, Arnaud and Schirmer, Tilman and Dehio, Christoph
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:10796
Thesis status:Complete
Number of Pages:166 S.
Language:English
Identification Number:
edoc DOI:
Last Modified:22 Jan 2018 15:51
Deposited On:16 Jun 2014 06:52

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