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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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BS3 cross-linking of reduced sGC and oxidized PDI and mass spectrometry analysis.Reduced sGC and oxidized PDI complex were incubated in the absence (left panel) or presence (right panel) of the lysine cross-linking reagent, BS3, then alkylated and run on 7.5% TGX gel under non-reducing conditions. Gel was stained with Coomassie blue. Representative MS/MS spectrum of a cross-linked peptide. A 6+ precursor with the m/z of 891.68 corresponds to the cross-linked peptides YCLFGNNVTLANKFESCSVPR and TETTGEKGK with K606 from sGC α subunit and K559 from sGC β subunit cross-linked. All the b- and y- series of fragments and cross-linked fragments confirmed the peptide sequence. The fragments α y9 - α y14, α y16 – α y19, β y3- β y7 indicate that α K606 and β K559 are crosslinked.
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pone.0143523.g001: BS3 cross-linking of reduced sGC and oxidized PDI and mass spectrometry analysis.Reduced sGC and oxidized PDI complex were incubated in the absence (left panel) or presence (right panel) of the lysine cross-linking reagent, BS3, then alkylated and run on 7.5% TGX gel under non-reducing conditions. Gel was stained with Coomassie blue. Representative MS/MS spectrum of a cross-linked peptide. A 6+ precursor with the m/z of 891.68 corresponds to the cross-linked peptides YCLFGNNVTLANKFESCSVPR and TETTGEKGK with K606 from sGC α subunit and K559 from sGC β subunit cross-linked. All the b- and y- series of fragments and cross-linked fragments confirmed the peptide sequence. The fragments α y9 - α y14, α y16 – α y19, β y3- β y7 indicate that α K606 and β K559 are crosslinked.

Mentions: We showed previously that the interaction between sGC and PDI takes place through a mixed disulfide exchange and is mostly transient [6]. This interaction was only observed between reduced sGC and oxidized PDI. PDI is a four domain protein with homologous N- and C-terminal WCGHC active sites, where the two redox active Cys are separated by Gly and His. When PDI is oxidized, each active site Cys-x-x-Cys forms a disulfide bond. To investigate the regions of interaction between PDI and sGC, reduced sGC and oxidized PDI were incubated in the absence or presence of the lysine cross-linking reagent, BS3. This reagent was previously used by the Montfort group to determine sGC dimer and domain interactions on a truncated construct (without the catalytic domains) [9]. The samples were next alkylated then run on an SDS-PAGE gel (Fig 1A). The BS3 linkage, as expected, increased the density of the high Molecular Weight (MW) bands and reduces the sGC α and β subunits/monomers. This switch in the band migration indicates that the BS3 reagent effectively crossed-linked PDI-sGC.


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)

BS3 cross-linking of reduced sGC and oxidized PDI and mass spectrometry analysis.Reduced sGC and oxidized PDI complex were incubated in the absence (left panel) or presence (right panel) of the lysine cross-linking reagent, BS3, then alkylated and run on 7.5% TGX gel under non-reducing conditions. Gel was stained with Coomassie blue. Representative MS/MS spectrum of a cross-linked peptide. A 6+ precursor with the m/z of 891.68 corresponds to the cross-linked peptides YCLFGNNVTLANKFESCSVPR and TETTGEKGK with K606 from sGC α subunit and K559 from sGC β subunit cross-linked. All the b- and y- series of fragments and cross-linked fragments confirmed the peptide sequence. The fragments α y9 - α y14, α y16 – α y19, β y3- β y7 indicate that α K606 and β K559 are crosslinked.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143523.g001: BS3 cross-linking of reduced sGC and oxidized PDI and mass spectrometry analysis.Reduced sGC and oxidized PDI complex were incubated in the absence (left panel) or presence (right panel) of the lysine cross-linking reagent, BS3, then alkylated and run on 7.5% TGX gel under non-reducing conditions. Gel was stained with Coomassie blue. Representative MS/MS spectrum of a cross-linked peptide. A 6+ precursor with the m/z of 891.68 corresponds to the cross-linked peptides YCLFGNNVTLANKFESCSVPR and TETTGEKGK with K606 from sGC α subunit and K559 from sGC β subunit cross-linked. All the b- and y- series of fragments and cross-linked fragments confirmed the peptide sequence. The fragments α y9 - α y14, α y16 – α y19, β y3- β y7 indicate that α K606 and β K559 are crosslinked.
Mentions: We showed previously that the interaction between sGC and PDI takes place through a mixed disulfide exchange and is mostly transient [6]. This interaction was only observed between reduced sGC and oxidized PDI. PDI is a four domain protein with homologous N- and C-terminal WCGHC active sites, where the two redox active Cys are separated by Gly and His. When PDI is oxidized, each active site Cys-x-x-Cys forms a disulfide bond. To investigate the regions of interaction between PDI and sGC, reduced sGC and oxidized PDI were incubated in the absence or presence of the lysine cross-linking reagent, BS3. This reagent was previously used by the Montfort group to determine sGC dimer and domain interactions on a truncated construct (without the catalytic domains) [9]. The samples were next alkylated then run on an SDS-PAGE gel (Fig 1A). The BS3 linkage, as expected, increased the density of the high Molecular Weight (MW) bands and reduces the sGC α and β subunits/monomers. This switch in the band migration indicates that the BS3 reagent effectively crossed-linked PDI-sGC.

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
Related in: MedlinePlus