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Probing the role of murine neuroglobin CDloop–D-helix unit in CO ligand binding and structural dynamics

DOI: 10.1021/acschembio.2c00172 DOI Help

Authors: Cécile Exertier (Sapienza Università di Roma) , Federico Sebastiani (Università di Firenze) , Ida Freda (Sapienza Università di Roma) , Elena Gugole (Sapienza Università di Roma) , Gabriele Cerutti (Columbia University) , Giacomo Parisi (stituto Italiano di Tecnologia) , Linda Celeste Montemiglio (nstitute of Molecular Biology and Pathology) , Maurizio Becucci (Università di Firenze) , Cristiano Viappiani (University of Parma) , Stefano Bruno (University of Parma) , Carmelinda Savino (National Research Council (Italy)) , Carlotta Zamparelli (Università di Roma) , Massimiliano Anselmi (Saarland University) , Stefania Abbruzzetti (University of Parma) , Giulietta Smulevich (Università di Firenze) , Beatrice Vallone (Università di Roma)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Chemical Biology

State: Published (Approved)
Published: July 2022
Diamond Proposal Number(s): 21741

Open Access Open Access

Abstract: We produced a neuroglobin variant, namely, Ngb CDless, with the excised CDloop- and D-helix, directly joining the C- and E-helices. The CDless variant retained bis-His hexacoordination, and we investigated the role of the CDloop–D-helix unit in controlling the CO binding and structural dynamics by an integrative approach based on X-ray crystallography, rapid mixing, laser flash photolysis, resonance Raman spectroscopy, and molecular dynamics simulations. Rapid mixing and laser flash photolysis showed that ligand affinity was unchanged with respect to the wild-type protein, albeit with increased on and off constants for rate-limiting heme iron hexacoordination by the distal His64. Accordingly, resonance Raman spectroscopy highlighted a more open distal pocket in the CO complex that, in agreement with MD simulations, likely involves His64 swinging inward and outward of the distal heme pocket. Ngb CDless displays a more rigid overall structure with respect to the wild type, abolishing the structural dynamics of the CDloop–D-helix hypothesized to mediate its signaling role, and it retains ligand binding control by distal His64. In conclusion, this mutant may represent a tool to investigate the involvement of CDloop–D-helix in neuroprotective signaling in a cellular or animal model.

Journal Keywords: Bioinorganic chemistry; Conformation; Crystals; Kinetics; Ligands

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-Macromolecular Crystallography

Added On: 11/07/2022 08:22


Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)