Structural snapshots of nitrosoglutathione binding and reactivity underlying S-nitrosylation of photosynthetic GAPDH
Edoardo Jun Mattioli,
Jacopo Rossi,
Maria Meloni,
Marcello De Mia,
Christophe H. Marchand,
Andrea Tagliani,
Silvia Fanti,
Giuseppe Falini,
Paolo Trost,
Stéphane D. Lemaire,
Simona Fermani,
Matteo Calvaresi,
Mirko Zaffagnini
Affiliations
Edoardo Jun Mattioli
Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy
Jacopo Rossi
Department of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, Italy
Maria Meloni
Department of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, Italy
Marcello De Mia
CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, France
Christophe H. Marchand
CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, France; CNRS, Institut de Biologie Physico-Chimique, Plateforme de Protéomique, FR550, F-75005, Paris, France
Andrea Tagliani
Department of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, Italy; CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, France
Silvia Fanti
Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy
Giuseppe Falini
Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy
Paolo Trost
Department of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, Italy
Stéphane D. Lemaire
CNRS, Sorbonne Université, Institut de Biologie Physico-Chimique, UMR8226, F-75005, Paris, France; Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, Laboratory of Computational and Quantitative Biology, UMR7238, F-75005, Paris, France
Simona Fermani
Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy; CIRI Health Sciences & Technologies (HST), University of Bologna, I-40064, Bologna, Italy; Corresponding author. Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy.
Matteo Calvaresi
Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy; CIRI Health Sciences & Technologies (HST), University of Bologna, I-40064, Bologna, Italy; Corresponding author. Department of Chemistry “G. Ciamician”, University of Bologna, I-40126, Bologna, Italy.
Mirko Zaffagnini
Department of Pharmacy and Biotechnologies, University of Bologna, I-40126, Bologna, Italy; Corresponding author.
S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.
