Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state
Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca2+ through the interaction of its self-contained Ca2+ binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca2+ sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMB...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-03-01
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| Series: | Biochemistry and Biophysics Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405580821002922 |
| _version_ | 1818283811023093760 |
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| author | Dustin Smith Laura Lloyd Elaine Wei Paria Radmanesh Chin-Chuan Wei |
| author_facet | Dustin Smith Laura Lloyd Elaine Wei Paria Radmanesh Chin-Chuan Wei |
| author_sort | Dustin Smith |
| collection | DOAJ |
| description | Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca2+ through the interaction of its self-contained Ca2+ binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca2+ sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMBD), in which the interaction between CaM and Nox5 is largely unclear. Here, we used the CMBD peptide and truncated DH constructs, and separately studied their interaction with CaM by fluorescence, calorimetry, and dynamic light scattering. Our results revealed that each half-domain of CaM binds one CMBD peptide with a binding constant near 106 M-1 and a binding enthalpy change of −3.81 kcal/mol, consistent with an extended 1:2 CaM:CMBD structure. However, the recombinant truncated DH proteins exist as oligomers, possibly trimer and tetramer. The oligomeric states are concentration and salt dependent. CaM binding appears to stabilize the DH dimer complexed with CaM. The thermodynamics of CaM binding to the DH is comparable to the peptide-based study except that the near unity binding stoichiometry and a large conformational change were observed. Our result suggests that the oligomeric states of Nox5, mediated by its DH domain and CaM, may be important for its superoxide-generating activity. |
| first_indexed | 2024-12-13T00:42:50Z |
| format | Article |
| id | doaj.art-9234c9a85d4b494b981b8e4089548df7 |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2024-12-13T00:42:50Z |
| publishDate | 2022-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-9234c9a85d4b494b981b8e4089548df72022-12-22T00:05:06ZengElsevierBiochemistry and Biophysics Reports2405-58082022-03-0129101198Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric stateDustin Smith0Laura Lloyd1Elaine Wei2Paria Radmanesh3Chin-Chuan Wei4Department of Chemistry, College of Arts and Sciences, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USADepartment of Chemistry, College of Arts and Sciences, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USADepartment of Biochemistry, School of Molecular and Cellular Biology, University of Illinois, Urbana Champaign, IL, 61801, USADepartment of Chemistry, College of Arts and Sciences, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USADepartment of Chemistry, College of Arts and Sciences, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA; Department of Pharmaceutical Sciences, College of Pharmacy, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA; Corresponding author. Department of Chemistry, Southern Illinois University, Edwardsville, USA.Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca2+ through the interaction of its self-contained Ca2+ binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca2+ sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMBD), in which the interaction between CaM and Nox5 is largely unclear. Here, we used the CMBD peptide and truncated DH constructs, and separately studied their interaction with CaM by fluorescence, calorimetry, and dynamic light scattering. Our results revealed that each half-domain of CaM binds one CMBD peptide with a binding constant near 106 M-1 and a binding enthalpy change of −3.81 kcal/mol, consistent with an extended 1:2 CaM:CMBD structure. However, the recombinant truncated DH proteins exist as oligomers, possibly trimer and tetramer. The oligomeric states are concentration and salt dependent. CaM binding appears to stabilize the DH dimer complexed with CaM. The thermodynamics of CaM binding to the DH is comparable to the peptide-based study except that the near unity binding stoichiometry and a large conformational change were observed. Our result suggests that the oligomeric states of Nox5, mediated by its DH domain and CaM, may be important for its superoxide-generating activity.http://www.sciencedirect.com/science/article/pii/S2405580821002922Calcium bindingNADPH Oxidase 5CalorimetrySpectroscopyGel filtration |
| spellingShingle | Dustin Smith Laura Lloyd Elaine Wei Paria Radmanesh Chin-Chuan Wei Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state Biochemistry and Biophysics Reports Calcium binding NADPH Oxidase 5 Calorimetry Spectroscopy Gel filtration |
| title | Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state |
| title_full | Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state |
| title_fullStr | Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state |
| title_full_unstemmed | Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state |
| title_short | Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state |
| title_sort | calmodulin binding to the dehydrogenase domain of nadph oxidase 5 alters its oligomeric state |
| topic | Calcium binding NADPH Oxidase 5 Calorimetry Spectroscopy Gel filtration |
| url | http://www.sciencedirect.com/science/article/pii/S2405580821002922 |
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