Mohammed Ibrahim - Probing Domain Interactions in Soluble Guanylate Cyclase

Version 1

      Publication Details (including relevant citation   information):

      Biochemistry, 2011, 50 (20), pp 4281–4290

      DOI: 10.1021/bi200341b

      Publication Date (Web): April 14, 2011

      Abstract:

      Eukaryotic nitric oxide (NO) signaling involves modulation of   cyclic GMP (cGMP) levels through activation of the soluble   isoform of guanylate cyclase (sGC). sGC is a heterodimeric   hemoprotein that contains a Heme–Nitric oxide and OXygen binding   (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a   coiled-coil (CC) domain, and a catalytic domain. To evaluate the   role of these domains in regulating the ligand binding properties   of the heme cofactor of NO-sensitive sGC, we constructed chimeras   by swapping the rat β1 H-NOX domain with the homologous region of   H-NOX domain-containing proteins from Thermoanaerobacter   tengcongensis, Vibrio cholerae, and   Caenorhabditis elegans (TtTar4H, VCA0720, and   Gcy-33, respectively). Characterization of ligand binding by   electronic absorption and resonance Raman spectroscopy indicates   that the other rat sGC domains influence the bacterial and worm   H-NOX domains. Analysis of cGMP production in these proteins   reveals that the chimeras containing bacterial H-NOX domains   exhibit guanylate cyclase activity, but this activity is not   influenced by gaseous ligand binding to the heme cofactor. The   rat–worm chimera containing the atypical sGC Gcy-33 H-NOX domain   was weakly activated by NO, CO, and O2, suggesting   that atypical guanylate cyclases and NO-sensitive guanylate   cyclases have a common molecular mechanism for enzyme activation.   To probe the influence of the other sGC domains on the mammalian   sGC heme environment, we generated heme pocket mutants (Pro118Ala   and Ile145Tyr) in the β1 H-NOX construct (residues 1–194), the β1   H-NOX-PAS-CC construct (residues 1–385), and the full-length α1β1   sGC heterodimer (β1 residues 1–619). Spectroscopic   characterization of these proteins shows that interdomain   communication modulates the coordination state of the heme–NO   complex and the heme oxidation rate. Taken together, these   findings have important implications for the allosteric mechanism   of regulation within H-NOX domain-containing proteins.

      Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi200341b