DJ-1 is a multifunctional protein that plays essential roles in tissues with higher order biological functions such as the testis and brain. DJ-1 is related to male fertility, and its level in sperm decreases in response to exposure to sperm toxicants. DJ-1 has also been identified as a hydroperoxide-responsive protein. Recently, a mutation of DJ-1 was found to be responsible for familial Parkinson's disease. Here, we present the crystal structure of DJ-1 refined to 1.95-Å resolution. DJ-1 forms a dimer in the crystal, and the monomer takes a flavodoxin-like Rossmann-fold. DJ-1 is structurally most similar to the monomer subunit of protease I, the intracellular cysteine protease from Pyrococcus horikoshii, and belongs to the Class I glutamine amidotransferaselike superfamily. However, DJ-1 contains an additional ␣-helix at the C-terminal region, which blocks the putative catalytic site of DJ-1 and appears to regulate the enzymatic activity. DJ-1 may induce conformational changes to acquire catalytic activity in response to oxidative stress.DJ-1 was initially identified as a novel oncogene product that transforms mouse NIH3T3 cells in cooperation with activated Ras. DJ-1 is an ϳ20-kDa protein comprising 189 amino acid residues ubiquitously expressed in various human tissues and with a particularly high level of expression in the testes (1). SP22 1 or CAP1, a rat homologue of human DJ-1, was subsequently identified as a key protein related to infertility in male rats exposed to sperm toxicants such as ornidazole and epichlorohydrin where DJ-1/CAP1/SP22 levels in the sperm and epididymis decreased with increased rat infertility (2-4). With the exception of DJ-1, no other protein decreased in response to exposure to sperm toxicants, supporting the close relationship between DJ-1 function and male fertility. Recently, Klinefelter et al. (5) revealed that DJ-1/CAP1/SP22 was located on the equatorial segment of the matured sperm head and anti-SP22

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.

The phagocyte NADPH oxidase, crucial for innate immunity, is dormant in resting cells, but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. In activation of the oxidase, the multidomain protein p67^phoxplays a central role: it translocates to the membrane as a ternary complex with p47^phoxand p40^phox, and interacts with the small GTPase Rac to assemble with the membrane-integrated catalytic protein gp91^phox, leading to superoxide production. Here we show, using small-angle X-ray scattering (SAXS) analysis, that p67^phoxadopts an elongated conformation when it exists not only as a monomer but also as the heterotrimer. Although p67^phoxharbors an N-terminal TPR domain for binding to Rac and a p40^phox-interacting PB1 domain, followed by an SH3 domain that associates with p47^phox, the present model suggests that no or few apparent associations occur between the domains. The positions of the protein-interaction domains in p67^phoxcontribute to activation of the phagocyte NADPH oxidase: the first SH3 domain that is located between the TPR and PB1 domains positively regulates oxidase activation only when it is present at the correct position; the PB1 domain placed at this SH3 domain position inhibits the oxidase by interacting with p40^phox.

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