Molecular analysis of mammalian adenylyl cyclases and edema factor, a bacterial adenylyly cyclase toxin

Abstract

Adenylyl cyclases (ACs) catalyze the conversion of ATP to cAMP, an important second messenger central to many signaling pathways. Nine different isoforms of mammalian ACs (mACs) are present, each with distinct localization, physiological function and regulatory mechanisms by activators and inhibitors. In addition to mACs, bacterial AC toxins such as edema factor (EF) from Bacillus anthracis and CyaA from Bordetella pertussis have also been identified. Following infection, the AC toxins cause a dramatic increase in cAMP levels, thereby disrupting several intracellular signaling pathways. This thesis is broadly divided into two parts and is aimed at validating the active-site nucleotide analogs of mACs and EF. The first part of the thesis focuses on understanding mAC regulation and the mechanism of interaction of mACs with fluorescent 2', 3'-O-(2, 4, 6-Trinitrophenyl) (TNP)- nucleotides. Using purified catalytic subunits of mAC (C1/C2) as model for mACs, we have observed the binding of TNP-nucleotides to C1/C2 and the resulting conformational changes monitored by fluorescence spectroscopy. The enzymatic assays have shown that TNP-nucleotides potently inhibit C1/C2 as well as holo-AC isoforms. An isoform-selective inhibition of AC1, AC2 and AC5 by TNP-nucleotides has been reported for the first time indicating that TNP-nucleotides can serve as models for rational design of potent isoform-specific AC inhibitors. Furthermore, biophysical and biochemical analysis of the effects of TNP- and 2' (3')-O-(N-methylanthraniloyl) (MANT) -nucleotides on the individual subunits C1 and C2 show that C1 and C2 can exist as homodimers. This homodimerization may play an important physiological role in cAMP signaling. The second part of the thesis addresses the structure-activity relationship in regulating the catalytic activity of EF and its interaction with calmodulin (CaM) using MANT derivatives of ATP and GTP as probes. Our enzymatic assays have shown that MANT-nucleotides are highly potent at inhibiting EF. MANT-nucleotides are also favorable for FRET studies indicating that our robust fluorescence assays can be used for High-Throughput Screening (HTS) of EF inhibitors. Additionally, EF-CaM interaction was probed by MANT-nucleotides. We have observed that binding and activation of EF by CaM are two independent processes in the presence and absence of calcium. Furthermore, our fluorescence assays to monitor binding of oxidized CaM to EF also indicate that methionine residues in CaM play an important role in binding to EF.