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.

Figure 1

Role of the PB1 domain in p40^phox binding to PtdIns(3)P. (A) A schematic diagram of p40^phox. The PX, SH3, and PB1 domain are colored in yellow, green, and magenta. (B) Subcellular distribution of p40^phox and its truncated proteins in transiently transfected HeLa cells. Left panel, distribution of GFP-tagged p40^phox in fixed HeLa cells (green); right panel, distribution of endogenous EEA-1 (red) and Hoechst staining of the nucleus (blue) in the same field of fixed HeLa cells. The insets show the magnified views. Scale bar, 5 μm. (C) Localization of ectopically expressed p40^phox and its truncated proteins in RAW264.7 cells ingesting IgG-coated beads. Scale bar, 5 μm. (D) SDS–PAGE analysis of GST-tagged p40^phox proteins used in the following lipid-binding assays (E, F). Proteins were stained with Coomassie brilliant blue. (E) ELISA-format lipid-binding assay. Each well of a titer plate coated with phospholipids containing 10% of PI or PtdIns(3)P was incubated with the indicated p40^phox proteins. The bound proteins were stained as described under Materials and methods. Each value represents the mean of data with bars representing s.d. in more than three independent experiments. (F) Co-sedimentation assay with liposomes. Liposomes containing 5% of PI or PtdIns(3)P were incubated with the indicated p40^phox proteins. After centrifugation, proteins in the supernatant (S) and precipitate (P) fractions were analyzed by SDS–PAGE. The experiments have been repeated more than three times with similar results.

Figure 2

The overall structure of p40^phox. (A) Structure of the two molecules of p40^phox in the asymmetric unit. Molecule A, which has a clear electron density, is colored blue, and molecule B, which has a poor electron density, is colored red. (B) A stereo pair of a ribbon diagram of p40^phox. Molecule B is superimposed onto Molecule A by the PX domain. Each molecule is color coded as in (A). (C) Experimental and calculated small-angle X-ray scattering curves for p40^phox. The smooth curve in red corresponds to the scattering curve calculated from the dummy atom model derived using the program DAMMIN, which is superposed on the experimental curve (dots with error bars). (D) The crystal structure of p40^phox (Molecule A, ribbon diagram) is superimposed onto the low-resolution model restored by the DAMMIN, shown as green surface representation. The right model is rotated by 90° around the y-axis and the upper model is rotated by 90° around the x-axis according to the arrows.

Figure 3

The interface between the PX domain and the PB1 domain. (A) A ribbon diagram of Molecule A. Each domain of p40^phox is colored coded as in Figure 1A. (B) Stereo diagram of a close-up view (the region corresponds to the rectangle in (A)) of the interface between the PX domain and the PB1 domain. Amino-acid residues involved in the interaction are shown in a stick representation. (C) Superposition of the p40^phox PX domain complexed with PtdIns(3)P (blue; PDB code 1H6H) onto that of crystal structure of p40^phox (yellow). The SH3 domain is removed for clarity. PtdIns(3)P, R58, R60, and R105 of the each PX domain, and E259 and D269 of the PB1 domain, which interact with R58 and R60 respectively, are shown in a stick representation. (D) The model of the interaction between the full-length p40^phox and membrane-bound PtdIns(3)P. SH3 domain is connected by dashed line, since relative position of the SH3 domain does not seem to be restricted.

Figure 4

Role of E259, D269, and F320, residues in the PB1 interface for the PX domain, in the PtdIns(3)P-binding activity. (A) Subcellular distribution of p40^phox and its mutant proteins carrying the indicated amino-acid substitution in transiently transfected HeLa cells. Left panel, distribution of GFP-tagged p40^phox in fixed HeLa cells (green); right panel, distribution of endogenous EEA-1 (red) and Hoechst staining of the nucleus (blue) in the same field of fixed HeLa cells. The insets show the magnified views. Scale bar, 5 μm. (B) Localization of ectopically expressed p40^phox and its mutant proteins in RAW264.7 cells ingesting IgG-coated beads. Scale bar, 5 μm. (C) SDS–PAGE analysis of GST-tagged p40^phox and its mutant proteins carrying the indicated amino-acid substitution, which were used in the following lipid-binding assays (D, E). (D) The PtdIns(3)P-binding activity estimated by the ELISA-format phosphoinositide-binding assay as in Figure 1E. (E) The PtdIns(3)P-binding activity estimated by the liposome co-sedimentation assay as in Figure 1F.

Figure 5

Dual roles for the PB1 domain of p40^phox. (A) Ribbon diagram of the superposition of p40^phox/p67^phox PB1 heterodimer (blue is p67^phox PB1 and orange is p40^phox PB1; PDB code 1OEY) onto that of the full-length p40^phox (color coded as in Figure 1A). A C-terminal extension region is shown in dashed circle. (B) Subcellular distribution of GFP-tagged p40^phox and HA-tagged p67^phox in transiently transfected HeLa cells. Left panel, distribution of GFP-tagged p40^phox in fixed HeLa cells (green); right panel, distribution of endogenous HA-tagged p67^phox (red) and Hoechst staining of the nucleus (blue) in the same field of fixed HeLa cells. The insets show the magnified views. Scale bar, 5 μm. (C) Proteins of lysates prepared from HeLa cells expressing both p40^phox and p67^phox were immunoprecipitated (IP) with the anti-GFP or control IgG, and then analyzed by immunoblot (Blot) with the anti-HA or anti-GFP antibodies. (D) In vitro interaction between purified GST-p40^phox and His-tagged p67^phox in the presence or absence of PtdIns(3)P.