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

Abstract

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.

NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology

“…A combined SANS and molecular modeling study of SpNOX provided a first lowresolution structural characterization of a full-length NOX enzyme [226]. This investigation revealed a distinctly less compact structure than the docking of the CsNOX domains implied, and the SpNOX SANS data strongly suggested a flexible linker between the TM and DH domains (Figure 12c) as well as the potential for substrate and cofactor binding to contribute to an active conformation [226].…”

“…This study obtained two structures of mouse DUOX1, one in complex with DUOXA1 and one in an inactive dimer of dimers configuration. Similar to SpNOX [226], the inactive dimer-of-dimers state shows flexibility in the positioning of the cytosolic DH domain of DUOX1 (Figure 13a). This flexibility would seem to arise from the linker between the TM and DH domains, but this presents a paradox.…”

“…The FAD cofactor co-crystallized with the protein is la-belled and the unstructured EF-hand-binding loop is depicted in dotted gray (D611-T634). (c) Some of the conformations generated by Pepsi-SANS along Non-linear Normal Mode Analysis for SpNOX in a semi-transparent ribbon style[226]. The two most distant conformations have been represented in opaque ribbon; between these two configurations, the gap between the D-loop of the TM domain and the FAD-binding site varies from 34 to 43 A, highlighting the flexibility of the inter-domain linker.…”

Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD

“…To assess the validity of the structural insights gained from the heterodimeric deAMPylation complex crystal (obtained with monomeric FICD L258D-H336A and a lid-truncated BiP-AMP), the properties of the intact protein complex was analysed by a solution-based structural method. Low-resolution structures of biomacromolecules can be resolved by small-angle X-ray and neutron scattering (SAXS/SANS) 26 – 29 . SAXS is sensitive to electron density, while SANS is sensitive to atomic nuclei.…”

“…By analysing the data over the entire scattering q -range, through flex-fitting, it is also possible to capture some of the dynamics of the protein complex in solution (as demonstrated recently 29 ). Although no individual flex-fit structure produced a significantly reduced average χ 2 across all datasets, a number of flex-fit output structures did have significantly different and reduced χ 2 variance (Supplementary Fig.…”