Guanylyl cyclase sees the light

Cyclic guanosine monophosphate (cGMP) is a critical second messenger that regulates cardiovascular function and vision in humans. Two recent papers, including one in BMC Structural Biology, have revealed atomic structures of the enzymes that catalyze the synthesis of cGMP providing new clues about the molecular basis of substrate specificity and allosteric regulation in nucleotide cyclases.

D Do om ma ai in n a ar rc ch hi it te ec ct tu ur re e a an nd d r re eg gu ul la at ti io on n Mammalian adenylyl and guanylyl cyclases belong to the class III nucleotide cyclase family [4,5]. Atomic structures of mammalian adenylyl cyclase [6][7][8] showed that the catalytic core of class III cyclases consists of either a homodimer or a heterodimer of homologous domains that associate to form a wreath-like structure ( Figure 1, top left). One or two active sites are formed at the interface between the domains. In mammalian adenylyl cyclases, the catalytic core is composed of homologous domains (C1 and C2) that form only one functional active site. The non-functional active site is the binding site for the activator forskolin, a well-known hypotensive agent.
Most mammalian adenylyl cyclases are large transmembrane proteins and subject to complex regulation, most importantly through direct interactions with heterotrimeric GTP-binding (G) proteins (Figure 2, left-hand side). In contrast, mammalian guanylyl cyclases are modular receptor enzymes: they exist as homo-or heterodimers with one catalytic domain in each polypeptide chain, and their activity can be modulated directly by hormones or other factors that bind to their accessory domains (Figure 2, righthand side) [9]. S Sn na ap ps sh ho ot ts s o of f t th he e g gu ua an ny yl ly yl l c cy yc cl la as se e c ca at ta al ly yt ti ic c c co or re e Not surprisingly, the catalytic core of both the Synechocystis Cya2 guanylyl cyclase (which has approximately 30% sequence identity to mammalian adenylyl cyclase and guanylyl cyclase isoforms) and the C. reinhardtii enzyme (which has 40-50% sequence identity to mammalian guanylyl cyclases) adopt the same wreath-like structure that was described for adenylyl cyclase (Figure 1). What then can be learned from these structures? Discrimination between GTP and ATP among class III nucleotide cyclases is critical for the fidelity of signaling by cGMP and cAMP. This specificity is determined, at least in part, by a pair of activesite residues (Glu and Cys) in guanylyl cyclases and their counterparts (Lys and Asp) in adenylyl cyclases. For example, when the guanylyl cyclase Glu-Cys pair is replaced with a Lys-Asp pair, the guanylyl cyclase is converted into a highly specific adenylyl cyclase [10,11]. Although the adenylyl cyclase Lys-Asp pair interacts directly with the purine base [7], it is not known for certain whether their equivalents in guanylyl cyclases contribute to specificity in the same manner.
Unfortunately, neither of the guanylyl cyclase structures contains a bound ligand to help define the mechanism of specificity in the active site. The C. reinhardtii enzyme [3] contains the Glu-Cys pair typical of mammalian guanylyl cyclases at their expected topological positions, but covalent modification of the active-site cysteine (a consequence of the conditions necessary for crystallization) precludes further interpretation. Interestingly, the bacterial Cya2 guanylyl cyclase [2] has a non-canonical Glu-Gly pair, with the critical Glu side chain being supported by a third basic residue, conserved as Arg or Lys in guanylyl cyclases. Unexpectedly, guanylyl cyclase activity is reduced and adenylyl cyclase activity increased when the Glu-Gly pair of Cya2 is replaced by a Glu-Cys pair [2]. Thus, the specific features  O On nc ce e a a w wr re ea at th h, , a al lw wa ay ys s a a w wr re ea at th h? ?
The molecular mechanism of mammalian adenylyl cyclase activation has been elusive because there is as yet no structure representing a physiologically relevant basal, inactive state of the catalytic core (a C1:C2 heterodimer in the absence of activating ligands). The structure of the C2 homodimer of adenylyl cyclase has been used as a surrogate, but its conformation is probably influenced by the presence of two molecules of forskolin, which is used to stabilize the dimer interface, and the fact that it lacks a C1 domain, which is required for catalytic activity [6].
The guanylyl cyclase structures provide new images of basal conformations that may be more physiologically relevant. The catalytic core of the Synechocystis guanylyl cyclase enzyme [2] assumes a conformation most similar to catalytically competent states of adenylyl cyclases [8,12], although, surprisingly, it adopts a closed conformation that apparently needs to open before nucleotides can access the active site. This could represent a regulatory difference from the C. reinhardtii guanylyl cyclase and mammalian cyclases. The C. reinhardtii guanylyl cyclase [3] adopts a conformation similar to that of the adenylyl cyclase C2 homodimer structure, in that the α1 helix, which binds to the βand γ-phosphates of the nucleotide (Figure 1), is not properly aligned with the rest of the active site. The structure may therefore serve as a useful model for the inactive state of mammalian cyclases. However, the fact that crystals could not be obtained without covalent modification of cysteines leaves open the possibility that this structure still does not represent the true ground-state conformation.
[13] described the active and inactive structures of a pH-sensing mycobacterial class III adenylyl cyclase. Importantly, this structure included the regulatory domains of the enzyme. In the active state, the catalytic domains assume the familiar wreath-like dimer. In the inactive state, they adopt a dramatically different conformation in which they interact extensively with the regulatory domains. Although this enzyme is more distantly related to mammalian adenylyl cyclases than the two guanylyl cyclases described here (approximately 23% identity) [2,3], it highlights the fact that accessory domains, such as those found in the two guanylyl cyclase enzymes and in mammalian adenylyl cyclases, can have a dramatic impact on the conformation of the catalytic core in its inactive state.
There is also evidence that the ground-state structures of some class III nucleotide cyclases may consist simply of loosely associated, and therefore minimally active, catalytic domains. Although the C. reinhardtii guanylyl cyclase enzyme [3] was a homodimer under all conditions tested (J Winger, personal communication), the Cya2 catalytic core exists in an equilibrium between monomeric, dimeric and oligomeric forms [2]. Forskolin and G proteins are known to dramatically enhance the affinity of the independently