Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction

• Published in: PloS one 2015-11, Vol.10 (11), p.e0143523-e0143523
• Main Authors: Heckler, Erin J, Kholodovych, Vladyslav, Jain, Mohit, Liu, Tong, Li, Hong, Beuve, Annie
• Format: Article
• Language: English
• Subjects: Adenoviruses; Amino Acid Sequence; Amino acids; Animals; Biochemistry; Cardiovascular system; Catalytic converters; Catalytic Domain; Cercopithecus aethiops; Cloning; Computer applications; COS Cells; Crosslinking; Cyclic GMP; Enzymes; GTP; Guanosine triphosphate; Guanylate cyclase; Guanylate Cyclase - chemistry; Guanylate Cyclase - metabolism; Heme; Immunoprecipitation; In vivo methods and tests; Inhibition; Lysine; Mass spectrometry; Mass spectroscopy; Medical schools; Molecular Sequence Data; Mutagenesis; Neurosciences; Nitric oxide; Peptide mapping; Peptides; Pharmacology; Physiology; Protein Binding; Protein disulfide-isomerase; Protein Disulfide-Isomerases - chemistry; Protein Disulfide-Isomerases - metabolism; Proteins; Proteomics; Reagents; Receptors, Cytoplasmic and Nuclear - chemistry; Receptors, Cytoplasmic and Nuclear - metabolism; Soluble Guanylyl Cyclase; Thiols
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0143523

Soluble guanylyl cyclase (sGC) is a heterodimeric nitric oxide (NO) receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI) interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.