The signal transduction enzyme phospholipase D1 (PLD1) hydrolyzes phosphatidylcholine to generate the lipid second-messenger phosphatidic acid, which plays roles in disease processes such as thrombosis and cancer. PLD1 is directly and synergistically regulated by protein kinase C, Arf and Rho GTPases, and the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we present a 1.8 Å-resolution crystal structure of the human PLD1 catalytic domain, which is characterized by a globular fold with a funnel-shaped hydrophobic cavity leading to the active site. Adjacent is a PIP2-binding polybasic pocket at the membrane interface that is essential for activity. The C terminus folds into and contributes part of the catalytic pocket, which harbors a phosphohistidine that mimics an intermediate stage of the catalytic cycle. Mapping of PLD1 mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors, enabling rationale inhibitor design for this well-studied therapeutic target.
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We thank the staff at the FMX and GM/CA-CAT beamlines for assistance during data collection. Beamline FMX (17-ID-2) is operated by LSBR, supported by NIH/NIGMS (P41GM111244) and DOE/BER (KP1605010). GM/CA@APS has been funded in whole or in part with federal funds from the NCI (ACB-12002) and the NIGMS (AGM-12006). The Eiger 16M detector was funded by an NIH–Office of Research Infrastructure Programs High-End Instrumentation Grant (S10 OD012289). This research used resources of the APS, a US Department of Energy (DOE) Office of Science User Facilities operated for the DOE Office of Science by Argonne National Laboratory (under contract no. DE-AC02-06CH11357). This work was also supported by NIH awards R35GM128666 (M.V.A.), T32GM092714 (F.Z.B.) and R01GM084251 (M.A.F.), NSF award 1612689 (C.M.S.), a Carol Baldwin Breast Cancer Award (M.A.F.) and a Chhabra-URECA award (J.A.B.).
The authors declare no competing interests.
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Bowling, F.Z., Salazar, C.M., Bell, J.A. et al. Crystal structure of human PLD1 provides insight into activation by PI(4,5)P2 and RhoA. Nat Chem Biol 16, 400–407 (2020). https://doi.org/10.1038/s41589-020-0499-8
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