Crystal structure of the signaling helix coiled-coil domain of the β1 subunit of the soluble guanylyl cyclase

Abstract

BackgroundThe soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner.ResultsTo investigate the structure of the CC S-helix region, we crystallized and determined the structure of the CC domain of the sGCβ1 subunit comprising residues 348-409. The crystal structure was refined to 2.15 Å resolution.ConclusionsThe CC structure of sGCβ1 revealed a tetrameric arrangement comprised of a dimer of CC dimers. Each monomer is comprised of a long a-helix, a turn near residue P399, and a short second a-helix. The CC structure also offers insights as to how sGC homodimers are not as stable as (functionally) active heterodimers via a possible role for inter-helix salt-bridge formation. The structure also yielded insights into the residues involved in dimerization. In addition, the CC region is also known to harbor a number of congenital and man-made mutations in both membrane and soluble guanylyl cyclases and those function-affecting mutations have been mapped onto the CC structure. This mutant analysis indicated an importance for not only certain dimerization residue positions, but also an important role for other faces of the CC dimer which might perhaps interact with adjacent domains. Our results also extend beyond guanylyl cyclases as the CC structure is, to our knowledge, the first S-helix structure and serves as a model for all S-helix containing family members.

Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase

“…This organization is further supported by the fact that the length of the linkers anchoring the helical domain to the rest of the structure is insufficient to allow an antiparallel arrangement, as previously suggested (8). The helical domains were modeled as a coiled coil using homology modeling and structural prediction software (Materials and Methods).…”

“…The structure of the rat sGC β1 monomer helical domain was previously determined and is formed by a long 13-turn helix followed by a shorter 2.5-turn helix (8). Although crystallized as an antiparallel homodimer, its arrangement in native sGC is considered to be a parallel heterodimer (8,12,20). The thin stalk of density observed in our 3D reconstructions accounts for this coiled coil, and a parallel arrangement of the helices is confirmed as it is the only Fig.…”

“…The PAS and helical domains are modeled on individual domain truncations. The PAS domain is based on the PAS domain from Manduca sexta (PDB: 4GJ4) (7), and the helical domain is based on the β1 R. norvegicus structure (3HLS) (8). The catalytic domain is the Homo sapiens α1β1 crystal structure (PDB: 3UVJ) (9).…”

“…No structure of the complete holoenzyme is available to date, and its absence precludes answering key questions such as how NO occupancy of the N-terminal β H-NOX sensor domain is communicated to the C-terminal cyclase domain. Atomic models of isolated sGC domains have been obtained by X-ray crystallography or homology modeling (6)(7)(8)(9) (Fig. 1).…”

Nitric Oxide Activation of Guanylate Cyclase Pushes the α1 Signaling Helix and the β1 Heme-binding Domain Closer to the Substrate-binding Site

“…The Coiled-coil Functions as Signaling Helix-In analogy to the crystallized ␤ 1 coiled-coil, Trp-466 is located in a short helix (␣B) behind the long amphipathic helix (␣A) of the coiled-coil domain (24). Based on comparative genomics, this domain has been suggested to act as a signaling helix that transmits the flow of signals between modules in diverse multi-domain signaling proteins (25).…”