Systemic lupus erythematosus-associated neutrophil cytosolic factor 2 mutation affects the structure of NADPH oxidase complex

Abstract

In a case-control association study with 3716 North Americans of Hispanic descent and 4867 North Americans of European descent, we show that the associations of rs17849502 (NCF2 His-389 → Gln) and rs13306575 (NCF2 Arg-395 → Trp) with systemic lupus erythematosus are independent. We have shown that His-389 → Gln disrupts the binding of NCF2 to the ZF domain of VAV1, resulting in decreased NADPH oxidase activity. With respect to Arg-395 → Trp, using protein docking and structure analyses, we provide a model for the involvement of this mutation in the structure and function of the NADPH oxidase complex. This model assigns a central role to Arg-395 in the structure and stability of the quaternary NCF2/NCF4/VAV1/RAC1 NADPH oxidase complex. Arg-395 stabilizes the C-terminal tail of NCF4 and the conformation of NCF2 loop 395-402, which in turn stabilize the evolutionarily conserved interactions of NCF2/NCF4 with the DH domain of VAV1 and RAC1 region 120-137. Our findings are consistent with the high levels of conservation of all of the residues involved in these interactions.

Keywords: NADPH oxidase; NCF2; autoimmune disease; autoimmunity; computational biology; human genetics; systemic lupus erythematosus.

FIGURE 1.

Conserved interactions that stabilize the conformation of NCF4 segment 330–339 and NCF2 loop 395–402 and the effect of mutation Arg-395 → Trp. The atoms of the interacting residues are shown as spheres of van der Waals radii. A, shows the interaction between NCF4 (blue) and NCF2 (green) in the experimental structure of the complex (18). Oxygen atoms are colored red, and nitrogen atoms are in dark blue. Note the cluster on the right formed by NCF2 Arg-395 (green), Leu-402 (dark green), and NCF4 Pro-339 (blue), which is stabilized by electrostatic and hydrophobic interactions. The hydrogen bond between the guanidine nitrogen of Arg-395 and the C-terminal carboxyl of Pro-339 is indicated by a white dash. B, a model of the Arg-395 → Trp mutation. Arg was replaced with Trp without changing the backbone conformation, selecting a side-chain rotamer that does not clash with the rest of the domain (χ1 = 66.0°, χ2 = −6.3° (26, 27)). The hydrogen bond between Arg-395 and NCF4 Pro-339 cannot be formed with Trp in position NCF2 395, and the hydrophobic interactions with NCF2 Leu-402 are weakened (see “Results”).

FIGURE 2.

Evolutionary conservation of relevant regions of NCF2 (A), VAV1 (B), NCF4 (C), and RAC1 (D) in selected species. Colors are coded according to the ClustalW (15) color scheme. Numbering corresponds to the human alignment.

FIGURE 3.

Overall view (A) and details (B) of the quaternary complex NCF2/NCF4/VAV1/RAC1 showing the centrality of NCF2 loop 395–402. The solvent-accessible surfaces of NCF2, NCF4, VAV1, and RAC1 are shown in green, blue, gray, and beige, respectively, with the interacting regions in darker shades. B, details of the interactions of the NCF2/NCF4 PB1 domains with VAV1 and RAC1. The surfaces of the interacting residues were made transparent. The interacting residues are indicated, and hydrogen bonds are denoted by dashed lines.

FIGURE 4.

Schematic of the domain structures of NCF2 (A), VAV1 (B), NCF4 (C), and RAC1 (D) and quaternary interactions among NCF2, VAV1, NCF4, and RAC1 involving NCF2 loop 395–402 (E).