Full-length p40phox structure suggests a basis for regulation mechanism of its membrane binding

Abstract

The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40phox associates via the PB1 domain with the essential oxidase activator p67phox, and is considered to function by recruiting p67phox to phagosomes; in this process, the PX domain of p40phox binds to phosphatidylinositol 3-phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P-binding activity of p40phox is normally inhibited by the PB1 domain both in vivo and in vitro. The crystal structure of the full-length p40phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40phox PB1 domain for the PX domain localizes on the opposite side of that for the p67phox PB1 domain, and thus the PB1-mediated PX regulation occurs without preventing the PB1-PB1 association with p67phox.

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

“…1A, center), as reported previously in RAW 264.7 macrophages (12) and PBL-985 granulocytes (28). We (12,27) and others (13,31) (Fig. 1B).…”

“…Cho and Stahelin (58) described a general mechanism of membrane-protein interactions in which membrane adsorption of PX domain-containing proteins such as p47 phox and p40 phox (18,56) is initially driven by nonspecific electrostatic interactions (between anionic lipids in membranes and cationic surfaces of proteins) and by diffusion, which is then followed by specific interaction with PIs and interfacial penetration (66) of hydrophobic and aromatic residues located near its respective binding site of PIs. Crystallographic studies on p40 phox , revealed that intramolecular PX-PB1 domain interactions are sterically inhibiting access of the PX domain with membraneembedded PI(3)P, rather than completely masking the PI(3)Pbinding site (13); in other words, the PX domain is able to access PI(3)P in certain conditions because three-dimensional positioning of membrane-embedded PI(3)P changes during phagosome formation. This speculation is supported by reports that full-length p40 phox binds to soluble PI(3)P to the same extent as the PX domain (39) and that full-length p40 phox binds to PI(3)P in surface plasmon resonance and lipid monolayer assays in which PI(3)P is flexible in the lipid monolayer (56).…”

“…In unstimulated phagocytes, the oxidase is dissociated and inactive; the flavocytochrome b 558 is stored on the membranes of intracellular granules (4), and the other Phox proteins associate in a separate cytoplasmic ternary complex (p47 phox -p67 phox -p40 phox ) (5) in a dephosphorylated state (6 -8). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse to newly forming phago-somes, and the cytoplasmic ternary complex binds to these membranes; p47 phox is phosphorylated (9,10), thereby inducing conformational changes that promote interaction of the ternary complex with p22 phox (11), and p40 phox also undergoes conformational changes by disruption of the intramolecular PX-PB1 domain interaction to enable the ternary complex to bind through the p40 phox PX domain to PI(3)P (12,13), which is enriched in phagosomes (14 -16).…”

Phosphatidylinositol 3-Phosphate-dependent and -independent Functions of p40phox in Activation of the Neutrophil NADPH Oxidase

“…p40 phox contains an N-terminal PtdIns(3)P-binding PX domain (19,20), a central SH3 domain capable of interacting with a proline-rich region in p47 phox in vitro (21,22), and a C-terminal PB1 domain that is required for constitutive interaction with p67 phox (23). Recently, the structure of full-length p40 phox was solved (24). The structure, together with a companion cell biology-based study, showed that p40 phox exists in a "closed" state where the PX and PB1 domains interact with each other, preventing the PX domain from interacting with PtdIns(3)P-containing membranes (25).…”

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

“…The N-terminal region of p67phox interacts with GTP-bound Rac and the activation domain of p67phox allows electron flow in the p22phox/Nox2 complex required for superoxide production [56,133]. The fully active NADPH oxidase is also under complex regulation by lipid metabolites that interact with the Phoxhomology (PX) domains of p47phox and p40phox [2,67].…”