Intermediates along the NAD+ cyclisation reaction pathway of ADP-ribosyl cyclase

Abstract

Calcium is a ubiquitous, versatile intracellular signal regulating numerous cellular processes [1]. The versatility of Ca2+ signalling emerges from the use of an extensive repertoire of signalling components known as a Ca2+ signalling toolkit that can be assembled in combinations to create signals with different spatial and temporal profiles. Mobilisation of intracellular Ca2+ stores into the cytoplasm is mediated through three structurally divergent messengers, one of them being cyclic ADP-ribose (cADPR) [2]. cADPR acts on the ryanodine receptor to elicit Ca2+ release. it is ubiquitous across species and is an endogenous modulator of Ca2+ - induced Ca2+ release thereby regulating a wide range of physiological processes [3]. cADPR is a cyclic nucleotide converted from NAD+ by multifunctional enzymes of the ADP-ribosyl cyclase family [4]. Aplysia ADP-ribosyl cyclase (Aplysia cyclase) was the first enzyme identified to catalyse the cyclisation of NAD+ to cADPR, with the release of nicotinamide. Aplysia cyclase is a 30kDa protein initially isolated from Aplysia ovotestis [5]. It is also found to be present in the neurons of the Aplysia buccal ganglion, where production of cADPR can enhance the evoked synaptic transmission [6]. Recently, it has been shown that Aplysia cyclase translocates from the cytosol into the nucleus upon depolarisation of Aplysia neurons, providing a mechanism for selective and specific activation of the nuclear Ca2+ store in neurons offering versatility for the neurons to respond to a wide range of stimuli [7]. Cyclisation of NAD+ into cADPR involves a two step reaction, the elimination of the nicotinamide ring and the cyclisation of the intermediate resulting in the covalent attachment of the adenine ring to the anomeric carbon of the terminal ribose. Cyclisation of NAD+ results in the linkage of the adenine and terminal ribose moieties established via the N1-position of the adenine ring, while cyclisation of NAD+ analogues resulted in the covalent attachment to the terminal ribose via the N7 position of the purine ring of these analogues [8]. In this study, we have determined the structures of wildtype Aplysia cyclase complexed with its substrates, NAD+ and NGD+, and its products cADPR and cGDPR. In addition, we were also able to capture the reaction intermediates of the cyclisation reaction either by controlling the soaking time of the substrate or with the use of substrate analogues. Taken together, we are able to obtain snapshots of the cyclisation process of the dinucleotide resulting in either N1 or N7 linkage of the purine ring to the terminal ribose.