Enhanced Immunoaffinity Purification of Human Neutrophil Flavocytochrome B for Structure Determination by Electron Microscopy

Jesaitis, Algirdas J.; Riesselman, Marcia H.; Taylor, R. M.; Brumfield, Susan K.

doi:10.1007/978-1-4939-9424-3_3

Cited by 5 publications

(2 citation statements)

References 39 publications

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“…Peak fractions containing the complex were pooled for cryo-EM sample preparation. Concentration of NOX2 was estimated with absorbance at 415 nm (ε 415 =0.262 μM −1 cm −1 ) ( Jesaitis et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

Structure of human phagocyte NADPH oxidase in the resting state

Liu

Song

et al. 2022

eLife

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Phagocyte oxidase plays an essential role in the first line of host defense against pathogens. It oxidizes intracellular NADPH to reduce extracellular oxygen to produce superoxide anions that participate in pathogen killing. The resting phagocyte oxidase is a heterodimeric complex formed by two transmembrane proteins NOX2 and p22. Despite the physiological importance of this complex, its structure remains elusive. Here we reported the cryo-EM structure of the functional human NOX2-p22 complex in nanodisc in the resting state. NOX2 shows a canonical 6-TM architecture of NOX and p22 has four transmembrane helices. M3, M4, and M5 of NOX2 and M1 and M4 helices of p22 are involved in the hetero-dimer formation. DH domain of NOX2 in the resting state is not optimally docked onto the transmembrane domain (TMD), leading to inefficient electron transfer and NADPH binding. Structural analysis suggests that the cytosolic factors might activate the NOX2-p22 complex by stabilizing the dehydrogenase domain (DH) in a productive docked conformation.

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Section: Methodsmentioning

confidence: 99%

Structure of human phagocyte NADPH oxidase in the resting state

Liu

Song

et al. 2022

eLife

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show abstract

“…Peak fractions containing the complex were pooled for cryo-EM sample preparation. Concentration of NOX2 was estimated with absorbance at 415 nm (ε 415 =0.262 μM −1 cm −1 ) 26 .…”

Section: Discussionmentioning

confidence: 99%

Structure of human phagocyte NADPH oxidase in the resting state

Song

et al. 2022

Preprint

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Phagocyte oxidase plays an essential role in the first line of host defense against pathogens. It oxidizes intracellular NADPH to reduce extracellular oxygen to produce superoxide anions for pathogen killing. The resting phagocyte oxidase is a heterodimeric complex formed by two transmembrane proteins NOX2 and p22. Despite the functional importance of this complex, its structure remains elusive. Here we reported the cryo-EM structure of the human NOX2-p22 complex in nanodisc in the resting state. The structure shows that p22 is formed by four transmembrane helices and interacts with NOX2 through its M1 and M4 helices. Hydrophobic residues on M3, M4, and M5 of NOX2 contribute to the complex formation. Structural analysis suggests that the cytosolic factors activate the NOX2-p22 complex by stabilizing the dehydrogenase domain (DH) in a productive docked conformation which is efficient for electron transfer between DH and the transmembrane domain.

show abstract