Lee, Hye Seon
(Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology)
,
Mo, Yeajin
(Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology)
,
Shin, Ho-Chul
(Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology)
,
Kim, Seung Jun
(Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology)
,
Ku, Bonsu
(Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology)
The Drosophila genome contains four low molecular weight-protein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited our understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophospha...
The Drosophila genome contains four low molecular weight-protein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited our understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophosphatases, but CG31469 was also suggested to be a putative protein arginine phosphatase. Herein, we present the crystal structures of CG31469 and Primo-1, which are the first Drosophila LMW-PTP structures. Structural analysis showed that the two proteins adopt the typical LMW-PTP fold and have a canonically arranged P-loop. Intriguingly, while Primo-1 is presumed to be a canonical LMW-PTP, CG31469 is unique as it contains a threonine residue at the fifth position of the P-loop motif instead of highly conserved isoleucine and a characteristically narrow active site pocket, which should facilitate the accommodation of phosphoarginine. Subsequent biochemical analysis revealed that Primo-1 and CG31469 are enzymatically active on phosphotyrosine and phosphoarginine, respectively, refuting their classification as pseudophosphatases. Collectively, we provide structural and biochemical data on two Drosophila proteins: Primo-1, the canonical LMW-PTP protein, and CG31469, the first investigated eukaryotic protein arginine phosphatase. We named CG31469 as DARP, which stands for Drosophila ARginine Phosphatase.
The Drosophila genome contains four low molecular weight-protein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited our understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophosphatases, but CG31469 was also suggested to be a putative protein arginine phosphatase. Herein, we present the crystal structures of CG31469 and Primo-1, which are the first Drosophila LMW-PTP structures. Structural analysis showed that the two proteins adopt the typical LMW-PTP fold and have a canonically arranged P-loop. Intriguingly, while Primo-1 is presumed to be a canonical LMW-PTP, CG31469 is unique as it contains a threonine residue at the fifth position of the P-loop motif instead of highly conserved isoleucine and a characteristically narrow active site pocket, which should facilitate the accommodation of phosphoarginine. Subsequent biochemical analysis revealed that Primo-1 and CG31469 are enzymatically active on phosphotyrosine and phosphoarginine, respectively, refuting their classification as pseudophosphatases. Collectively, we provide structural and biochemical data on two Drosophila proteins: Primo-1, the canonical LMW-PTP protein, and CG31469, the first investigated eukaryotic protein arginine phosphatase. We named CG31469 as DARP, which stands for Drosophila ARginine Phosphatase.
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제안 방법
In vitro biochemical assays were performed to verify the phosphatase activity of the two proteins. First, the dephosphorylation capability of Primo-1, which was assumed to be a canonical LMW-PTP, was examined using pNPP, a widely used phosphatase substrate. Fig.
The enzymatic property of DARP was further analyzed by comparing the specific activities of wild-type and mutant proteins toward phosphoarginine and phosphotyrosine. Compared to the wild-type, substitution of Thr11 to isoleucine caused a remarkable decrease in the dephosphorylation of phosphoarginine and a noticeable increase in the dephosphorylation of phosphotyrosine (Fig.
대상 데이터
The purified recombinant proteins were subjected to crystallization. Crystals of DARP(C7S) in the space group P212121 and wild-type Primo-1 in the space group P21 were obtained, and the structures of these two Drosophila proteins were determined at resolutions of 2.2 Å and 2.1 Å, respectively (Table 1). One DARP and two Primo-1 mole-cule(s) were contained in the asymmetric unit of each protein crystal.
이론/모형
Diffraction data were collected on the beamline 7A at the Pohang Accelerator Laboratory, Korea, and processed using the program HKL 2000 (Otwinowski and Minor, 1997). Both structures were determined by molecular replacement using the program Phaser (McCoy et al., 2007) using the Streptococcus pyogenes SP-PTP structure as a search model (Ku et al., 2016). Coot (Emsley and Cowtan, 2004) and PHE-NIX (Adams et al.
4A shows that recombinant Primo-1 was active on pNPP, confirming this protein as a catalytically active phosphatase. Kinetic constants were calculated using the Michaelis–Menten curves and the Lineweaver–Burk plots. Accordingly, the kcat, KM, and kcat/KM values of Primo-1 toward pNPP were determined as 59.
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