Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier

• Published in: Biophysical journal Vol. 119; no. 3; pp. 605 - 618
• Main Authors: Vermot, Annelise, Petit-Härtlein, Isabelle, Breyton, Cécile, Le Roy, Aline, Thépaut, Michel, Vivès, Corinne, Moulin, Martine, Härtlein, Michael, Grudinin, Sergei, Smith, Susan M.E., Ebel, Christine, Martel, Anne, Fieschi, Franck
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.08.2020; Biophysical Society; The Biophysical Society
• Subjects: Biochemistry, Molecular Biology; Life Sciences; Membrane Proteins; NADPH Oxidases; Neutron Diffraction; Oxidation-Reduction; Scattering, Small Angle; Structural Biology
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2020.06.025

Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a “stealth” carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.