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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.

Show MeSH
Proposed computational model of the sGC-PDI interaction Representative models of sGC and PDI interaction with possible distances between cross-linked residues are shown.Color-coding: α subunit of sGC is depicted as red ribbon, β subunit of sGC is light blue ribbon, PDI are dark blue and black ribbons. Average positions from four 100ns MD simulations of sGC residues α K672 (magenta) and β K615 (green) and the corresponding interacting residues of PDI K370 and K409, and the pair of cross-linked residues α K606 and β K559 of sGC are shown as van der Waals surfaces. A. The possible closest distance between sGC residues α K672 and β K615 is 61.3Å, which is twice more than the required distance to form a contact with both K370 and K409 residues of one molecule of PDI with linkers (30Å). B. One molecule of PDI interacts with one at a time molecule of sGC. PDI #1 interacts with sGC via α catalytic domain or PDI #2 interacts with sGC via β catalytic domain.
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pone.0143523.g005: Proposed computational model of the sGC-PDI interaction Representative models of sGC and PDI interaction with possible distances between cross-linked residues are shown.Color-coding: α subunit of sGC is depicted as red ribbon, β subunit of sGC is light blue ribbon, PDI are dark blue and black ribbons. Average positions from four 100ns MD simulations of sGC residues α K672 (magenta) and β K615 (green) and the corresponding interacting residues of PDI K370 and K409, and the pair of cross-linked residues α K606 and β K559 of sGC are shown as van der Waals surfaces. A. The possible closest distance between sGC residues α K672 and β K615 is 61.3Å, which is twice more than the required distance to form a contact with both K370 and K409 residues of one molecule of PDI with linkers (30Å). B. One molecule of PDI interacts with one at a time molecule of sGC. PDI #1 interacts with sGC via α catalytic domain or PDI #2 interacts with sGC via β catalytic domain.

Mentions: There is no full-length crystal structure or model of sGC available. In addition, the most recent PDB entry of the catalytic domain of sGC (PDB: 4NI2) [19] has both α and β subunits truncated at C-termini. Coincidentally, these truncated regions are where the cross-links between sGC (at α Lys672 and β Lys615) and PDI occur. Thus, we employed computational modeling to extend the available crystal structure of the sGC catalytic domain and then used it for docking with PDI molecules. First, the missing C-termini amino acid residues, α G663-D690 and β T609-D619 were added to the available X-ray structure of the catalytic domain of sGC (PDB 4NI2, α 471–662, β 411–608). Because the structural organization of these flexible tails are not known, we used extended conformations of peptides and then performed molecular dynamics (MD) simulations to capture the random re-organization of the added residues. Four independent MD models were produced after 100ns simulation. The visual analysis of the models revealed that the possible closest distance between sGC residues α Lys672 and β Lys615 is 61.3Å, which is twice more than the required distance to form a contact with both Lys370 and Lys409 residues of one molecule of PDI with linkers (30Å), Fig 5A. Therefore, it is not possible for one molecule of PDI to interact with both α and β subunits of sGC at the same time. Hence we posit that one PDI molecule interacts with the α subunit or the β subunit of sGC in a mutually exclusive manner and performed protein-protein docking on the GRAMM-X website with a followed-up short MD refinement. The two exclusive interactions are shown in Fig 5B. The only other crosslink that was observed in the modeling region of the sGC catalytic domain after mass-spec analysis was an inter-subunit contact between α Lys606 and β Lys559. The distance between sGC α Lys606 and β Lys559 in our model was 11.2Å which is exactly the length of the BS3 linker (11.3Å). We used this distance as an internal control of the models after MD simulation and docking. Any model that violates this distance was discarded.


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)

Proposed computational model of the sGC-PDI interaction Representative models of sGC and PDI interaction with possible distances between cross-linked residues are shown.Color-coding: α subunit of sGC is depicted as red ribbon, β subunit of sGC is light blue ribbon, PDI are dark blue and black ribbons. Average positions from four 100ns MD simulations of sGC residues α K672 (magenta) and β K615 (green) and the corresponding interacting residues of PDI K370 and K409, and the pair of cross-linked residues α K606 and β K559 of sGC are shown as van der Waals surfaces. A. The possible closest distance between sGC residues α K672 and β K615 is 61.3Å, which is twice more than the required distance to form a contact with both K370 and K409 residues of one molecule of PDI with linkers (30Å). B. One molecule of PDI interacts with one at a time molecule of sGC. PDI #1 interacts with sGC via α catalytic domain or PDI #2 interacts with sGC via β catalytic domain.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143523.g005: Proposed computational model of the sGC-PDI interaction Representative models of sGC and PDI interaction with possible distances between cross-linked residues are shown.Color-coding: α subunit of sGC is depicted as red ribbon, β subunit of sGC is light blue ribbon, PDI are dark blue and black ribbons. Average positions from four 100ns MD simulations of sGC residues α K672 (magenta) and β K615 (green) and the corresponding interacting residues of PDI K370 and K409, and the pair of cross-linked residues α K606 and β K559 of sGC are shown as van der Waals surfaces. A. The possible closest distance between sGC residues α K672 and β K615 is 61.3Å, which is twice more than the required distance to form a contact with both K370 and K409 residues of one molecule of PDI with linkers (30Å). B. One molecule of PDI interacts with one at a time molecule of sGC. PDI #1 interacts with sGC via α catalytic domain or PDI #2 interacts with sGC via β catalytic domain.
Mentions: There is no full-length crystal structure or model of sGC available. In addition, the most recent PDB entry of the catalytic domain of sGC (PDB: 4NI2) [19] has both α and β subunits truncated at C-termini. Coincidentally, these truncated regions are where the cross-links between sGC (at α Lys672 and β Lys615) and PDI occur. Thus, we employed computational modeling to extend the available crystal structure of the sGC catalytic domain and then used it for docking with PDI molecules. First, the missing C-termini amino acid residues, α G663-D690 and β T609-D619 were added to the available X-ray structure of the catalytic domain of sGC (PDB 4NI2, α 471–662, β 411–608). Because the structural organization of these flexible tails are not known, we used extended conformations of peptides and then performed molecular dynamics (MD) simulations to capture the random re-organization of the added residues. Four independent MD models were produced after 100ns simulation. The visual analysis of the models revealed that the possible closest distance between sGC residues α Lys672 and β Lys615 is 61.3Å, which is twice more than the required distance to form a contact with both Lys370 and Lys409 residues of one molecule of PDI with linkers (30Å), Fig 5A. Therefore, it is not possible for one molecule of PDI to interact with both α and β subunits of sGC at the same time. Hence we posit that one PDI molecule interacts with the α subunit or the β subunit of sGC in a mutually exclusive manner and performed protein-protein docking on the GRAMM-X website with a followed-up short MD refinement. The two exclusive interactions are shown in Fig 5B. The only other crosslink that was observed in the modeling region of the sGC catalytic domain after mass-spec analysis was an inter-subunit contact between α Lys606 and β Lys559. The distance between sGC α Lys606 and β Lys559 in our model was 11.2Å which is exactly the length of the BS3 linker (11.3Å). We used this distance as an internal control of the models after MD simulation and docking. Any model that violates this distance was discarded.

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