Crystal structure of the guanylyl cyclase Cya2

Abstract

Cyclic GMP (cGMP) is an important second messenger in eukaryotes. It is formed by guanylyl cyclases (GCs), members of the nucleotidyl cyclases class III, which also comprises adenylyl cyclases (ACs) from most organisms. To date, no structures of eukaryotic GCs are available, and all bacterial class III proteins were found to be ACs. Here we describe the biochemical and structural characterization of the class III cyclase Cya2 from cyanobacterium Synechocystis PCC6803. Cya2 shows high specificity for GTP versus ATP, revealing it to be the first bacterial GC, and sequence similarity searches indicate that GCs are also present in other bacteria. The crystal structure of Cya2 provides first structural insights into the universal GC family. Structure and mutagenesis studies show that a conserved glutamate, assisted by an interacting lysine, dominates substrate selection by forming hydrogen bonds to the substrate base. We find, however, that a second residue involved in substrate selection has an unexpected sterical role in GCs, different from its hydrogen bonding function in the related ACs. The structure identifies a tyrosine that lines the guanine binding pocket as additional residue contributing to substrate specificity. Furthermore, we find that substrate specificity stems from faster turnover of GTP, rather than different affinities for GTP and ATP, implying that the specificity-determining interactions are established after the binding step.bacterial ͉ cGMP T he second messenger cGMP plays a central role in regulating bodily function such as the cardiovascular system and vision (1). In mammals, cGMP is formed by a family of guanylyl cyclases comprising a soluble GC (sGC) and several transmembrane receptor enzymes (rGCs). Mammalian GCs have a catalytic domain fused, via a central domain, to either an extracellular ligand binding domain (rGCs) or a nitric oxide binding heme domain (sGC) (1-3). In lower eukaryotes, guanylyl cyclase domains are fused to a variety of regulatory domains and function in processes such as cell motility control or chemotaxis (4).All known guanylyl cyclases belong to class III of the purine nucleotidyl cyclase family, which was subdivided in six phylogenetically separated classes, I-VI, based on sequence homologies within the catalytic cores (5, 6). Class III also comprises adenylyl cyclases (ACs) from almost all domains of life (no member has been confirmed in plants), including most bacteria (7). GCs, in contrast, have been described only in Dictyostelium and higher organisms, and their existence in prokaryotes has only been speculated upon (8). Despite their physiological importance, no structural data on GC enzymes are available to date. For class III ACs, in contrast, several crystal structures have been reported, revealing a conserved basic architecture of the catalytic cores (6, 9-11), which is assumed to be shared by GCs (1, 12).Class III cyclases require dimerization of two catalytic domains for activity, which leads to formation of shared active sites in ACs (6, 7). Modeling st...

Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

“…1C; CyaC Asp1061 is shown as a reference). An Asp rotation opening the ion sites was observed in the guanylyl cyclase (GC) Cya2 (22), but in sAC, the whole Asp99-containing β2/3 loop is pulled away, toward Arg176, through an Asp99/ Arg176 salt bridge. Asp99 and the β2/3 loop are thereby held in an inactive position overlapping the ion site region.…”

“…S1C and Table S2), showing that crystallized sAC-cat is catalytically active and that this Lys334 conformation is functional. Other AC/ATP-analog complexes show a Lys/Ade interaction (23), but with reaction-competent substrates, this interaction appears to form late during catalysis and is relevant for turnover rather than substrate binding (22,36). The AC activity of sAC is inhibited by GTP ( Fig.…”

“…Its identification as the BBS confirms speculations that the degenerate site accommodates physiological regulators and communicates with the active site (3). Presumably, this degenerated site with regulatory function represents an evolutionary remnant from homodimeric ancestor class III cyclases that can show cooperativity or half-of-the-site activity (22,37).…”

“…Their catalytic cores are formed through symmetrical or pseudosymmetrical association of two identical or highly similar catalytic domains, C 1 and C 2 (22)(23)(24); in mammalian ACs, both domains reside on a single polypeptide chain. Such C 1 C 2 pseudoheterodimers form two pseudosymmetrical sites at the dimer interface: the active site and a degenerated, inactive pocket (3,23).…”

tRNA His guanylyltransferase (THG1), a unique 3′-5′ nucleotidyl transferase, shares unexpected structural homology with canonical 5′-3′ DNA polymerases

“…3B) (29). In contrast, adenylyl and guanylyl cyclases contain only two catalytic aspartates and a cysteine, alanine or glycine in lieu of the third carboxylate (21,30). The highly conserved hTHG1 carboxylates D29, D76, and E77 correspond to D475, D654, and E655 in T7 DNA polymerase.…”