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Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

Heckler EJ, Kholodovych V, Jain M, Liu T, Li H, Beuve A - PLoS ONE (2015)

Bottom Line: Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ, United States of America.

ABSTRACT
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.

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Scheme of lysine interactions between sGC and PDI.For clarity, PDI is represented twice as blue box 1 and 2, above and below α and β subunits of sGC. α and β are represented as light red and light green block arrows, respectively. The respective HNOX, PAS, coiled-coil (CC), catalytic domain (CAT) and C-terminal tail are indicated with the corresponding residue numbers (human sequence).
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pone.0143523.g002: Scheme of lysine interactions between sGC and PDI.For clarity, PDI is represented twice as blue box 1 and 2, above and below α and β subunits of sGC. α and β are represented as light red and light green block arrows, respectively. The respective HNOX, PAS, coiled-coil (CC), catalytic domain (CAT) and C-terminal tail are indicated with the corresponding residue numbers (human sequence).

Mentions: The pattern of BS3-Lys crosslinks between sGC α/β and PDI were analyzed from tandem MS data using the MassMatrix search engine as described in methods with BS3 linkage as the modification. Fig 1B shows a representative cross-linked peptide MS/MS spectrum between α and β subunits in the catalytic domain α Lys606 with β Lys559. We found that PDI molecules interact with sGC through the region that contains the second Cys-x-x-Cys active site and that sGC is crosslinked at both C-terminal catalytic domain of α and β subunits via the following lysines linkage: PDI Lys409 to sGC α Lys672 and PDI Lys370 to sGC β Lys615. Additionally, sGC inter-subunit cross-links were identified: α K5 with β Lys187, α Lys606 with β Lys559, α Lys673 with β K127, and α Lys685 with β Lys127 (Fig 2 and Table 1).


Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

Heckler EJ, Kholodovych V, Jain M, Liu T, Li H, Beuve A - PLoS ONE (2015)

Scheme of lysine interactions between sGC and PDI.For clarity, PDI is represented twice as blue box 1 and 2, above and below α and β subunits of sGC. α and β are represented as light red and light green block arrows, respectively. The respective HNOX, PAS, coiled-coil (CC), catalytic domain (CAT) and C-terminal tail are indicated with the corresponding residue numbers (human sequence).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664405&req=5

pone.0143523.g002: Scheme of lysine interactions between sGC and PDI.For clarity, PDI is represented twice as blue box 1 and 2, above and below α and β subunits of sGC. α and β are represented as light red and light green block arrows, respectively. The respective HNOX, PAS, coiled-coil (CC), catalytic domain (CAT) and C-terminal tail are indicated with the corresponding residue numbers (human sequence).
Mentions: The pattern of BS3-Lys crosslinks between sGC α/β and PDI were analyzed from tandem MS data using the MassMatrix search engine as described in methods with BS3 linkage as the modification. Fig 1B shows a representative cross-linked peptide MS/MS spectrum between α and β subunits in the catalytic domain α Lys606 with β Lys559. We found that PDI molecules interact with sGC through the region that contains the second Cys-x-x-Cys active site and that sGC is crosslinked at both C-terminal catalytic domain of α and β subunits via the following lysines linkage: PDI Lys409 to sGC α Lys672 and PDI Lys370 to sGC β Lys615. Additionally, sGC inter-subunit cross-links were identified: α K5 with β Lys187, α Lys606 with β Lys559, α Lys673 with β K127, and α Lys685 with β Lys127 (Fig 2 and Table 1).

Bottom Line: Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ, United States of America.

ABSTRACT
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.

Show MeSH