Structure-Function Characterization of a Fic Protein From The Respiratory Pathogen, Bordetella Bronchiseptica

Hee-jeong Kim, Purdue University

Abstract

Fic (filamentation induced by cAMP) are a recently discovered family of enzymes that predominantly utilize ATP to post-translationally modify their protein targets to alter cellular signaling. Specifically, these proteins use ATP to covalently add an AMP to their targets, by a process called Fic-mediated adenylylation or AMPylation. In addition, some Fic proteins have also been shown to utilize UTP, CDP-choline, or ATP to modify their targets with UMP, phosphocholine, or phosphate moieties. Fic proteins are evolutionarily conserved and are defined by an HxFx(G/A)N(G/K)RxxR motif, where the His is a conserved residue needed for catalytic activity. The Mattoo lab discovered that the human Fic HYPE (Huntingtin yeast-interacting protein E) also functions as an adenylyltransferase that regulates ER (endoplasmic reticulum) homeostasis and the unfolded protein response by adenylylating the ER chaperone, BiP. Analysis of additional Fic proteins is needed to better understand the functional repertoire of Fic proteins, a key goal of the Mattoo lab. In this study, the function of the sole Fic protein in Bordetella bronchiseptica, a respiratory pathogen with a broad host range, including humans is explored. By optimizing the purification of wild type (WT) and catalytically active or inactive B.bronchiseptica Fic (called BbFic), enzymatic activity of BbFic was assessed and characterized. Here, we report that unlike most Fic proteins, BbFic is the first known Fic protein that preferentially utilizes GTP over ATP. The binding and kinetics parameters by which BbFic uses GTP for auto-modification were measured, and we provided a structural analysis to explain the GTP specificity. Our data provided key insight for target recognition of nucleotide specificity. This is the first report of a GTP-specific Fic protein, thus adding greater diversity to the enzymatic activity of Fic proteins.

Degree

M.S.

Advisors

Mattoo, Purdue University.

Subject Area

Biology

COinS