PAS-mediated Dimerization of Soluble Guanylyl Cyclase Revealed by Signal Transduction Histidine Kinase Domain Crystal Structure

Xiaolei Ma,Nazish Sayed,Padmamalini Baskaran,Annie Beuve,Focco Van Den Akker

doi:10.1074/jbc.m706218200

Abstract

Signal transduction histidine kinases (STHK) are key for sensing environmental stresses, crucial for cell survival, and attain their sensing ability using small molecule binding domains. The N-terminal domain in an STHK from Nostoc punctiforme is of unknown function yet is homologous to the central region in soluble guanylyl cyclase (sGC), the main receptor for nitric oxide (NO). This domain is termed H-NOXA (or H-NOBA) because it is often associated with the heme-nitric oxide/oxygen binding (H-NOX) domain. A structure-function approach was taken to investigate the role of H-NOXA in STHK and sGC. We report the 2.1 A resolution crystal structure of the dimerized H-NOXA domain of STHK, which reveals a Per-Arnt-Sim (PAS) fold. The H-NOXA monomers dimerize in a parallel arrangement juxtaposing their N-terminal helices and preceding residues. Such PAS dimerization is similar to that previously observed for EcDOS, AvNifL, and RmFixL. Deletion of 7 N-terminal residues affected dimer organization. Alanine scanning mutagenesis in sGC indicates that the H-NOXA domains of sGC could adopt a similar dimer organization. Although most putative interface mutations did decrease sGCbeta1 H-NOXA homodimerization, heterodimerization of full-length heterodimeric sGC was mostly unaffected, likely due to the additional dimerization contacts of sGC in the coiled-coil and catalytic domains. Exceptions are mutations sGCalpha1 F285A and sGCbeta1 F217A, which each caused a drastic drop in NO stimulated activity, and mutations sGCalpha1 Q368A and sGCbeta1 Q309A, which resulted in both a complete lack of activity and heterodimerization. Our structural and mutational results provide new insights into sGC and STHK dimerization and overall architecture.

Highlights

An additional evolutionary relationship was detected between soluble guanylyl cyclase (sGC) and 2 other cyanobacterial signaling proteins [1, 2]: the heme-nitric oxide/ oxygen-binding (H-NOX) associated H-NOXA domain in sGC is present at the N terminus of a cyanobacterial signal transduction histidine kinase (STHK) and 2-component hybrid sensor and regulator (2-CHSR) (Fig. 1) postulated to have a PAS-like fold [1]
The H-NOXA domain is a central subdomain in both subunits: in sGC␤1, it is flanked by the N-terminal H-NOX domain and C-terminal predicted coiled-coil (CC) [9, 10] and catalytic guanylyl cyclase (GC) domain (Fig. 1A). sGC␣1 has a similar subunit arrangement except that its N-terminal domain does not bind heme
NpSTHK Structure Reveals a PAS Fold—The structure of the H-NOXA domain of NpSTHK was solved via single wavelength anomalous dispersion (SAD) of first a smaller ⌬7 N-terminal-truncated fragment, followed by molecular replacement to solve the full-length NpSTHK H-NOXA domain structure (Table 1 and Fig. 1C)

Summary

The abbreviations used are

H-NOXA, heme nitric oxide and oxygen binding associated; PAS, an acronym formed from three proteins: Per, Arnt, Sim; STHK, signal transduction histidine kinase; 2-CHSR, two-component hybrid sensor and regulator; sGC, soluble guanylyl cyclase; NO, nitric oxide; CC, coiled coil; SAD, single wavelength anomalous dispersion; r.m.s., root mean square. Analogous regions in sGC␣1 have been shown to be important for sGC activity via deletion studies [9]. These regions in sGC␣1 and sGC␤1 have recently been further narrowed down [18]. We carried out additional structure-function studies in sGC and find that the H-NOXA subdomains of sGC could likely form a arranged PAS-type heterodimer with an important role for functional sGC heterodimerization by the H-NOXA domains

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION