Dimerization of nitric oxide-sensitive guanylyl cyclase requires the alpha 1 N terminus.
The enzyme nitric oxide-sensitive guanylyl cyclase is an obligate heterodimer consisting of an alpha and a beta subunit. Whereas the C-terminal parts of the subunits have been shown to be sufficient for catalysis, regulation was assigned to the N termini. The central domains have been postulated to be responsible for the formation of alphabeta heterodimers. Here, we have analyzed dimerization by precipitation of various N- and C-terminally truncated alpha(1) mutants with beta(1) wild type or deletion mutants thereof after coexpression in the baculovirus/Sf9 system. In contrast to the current hypothesis, our analysis revealed that an N-terminal region of the alpha(1) subunit (amino acids 61-128) is mandatory for quantitative dimerization. The central domain (amino acids 367-462) contributes but is not sufficient to mediate robust alphabeta interaction. Wild type-like binding of the identified minimum dimerization region of alpha(1) (amino acids 61-462) requires the N-terminal and central region of beta(1) (amino acids 1-385). Furthermore, we observed an unequal stability of the alpha(1) and beta(1) subunit. Whereas beta(1) forms heme containing homodimers and is stable, alpha(1) appears to be prone to misfolding and degradation when heterodimerization is impaired by deletion of important sequences.
Wagner
Corina
C
Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, MA N1, Ruhr-Universität-Bochum, 44780 Bochum, Germany.
Russwurm
Michael
M
Jäger
Ronald
R
Friebe
Andreas
A
Koesling
Doris
D
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D019281:Dimerization; D006162:Guanylate Cyclase; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase; D018411:Spodoptera / Q000235:genetics
D010446:Peptide Fragments; D021122:Protein Subunits; D018160:Receptors, Cytoplasmic and Nuclear; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase