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Crystal structure of human PLD1 provides insight into activation by PI(4,5)P2 and RhoA

Abstract

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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Fig. 1: Structure of human PLD1.
Fig. 2: Structural comparison of PLD family phosphodiesterases with human PLD1.
Fig. 3: Active-site cavity in PLD1.
Fig. 4: A phosphohistidine intermediate helps define the catalytic cycle of PLD1.
Fig. 5: A polybasic pocket defines a new PIP2 activation site at the membrane.
Fig. 6: Model of RhoA activation of PLD1.

Data availability

Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 6U8Z. Source data for Figs. 1 and 35 are presented with the paper.

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Acknowledgements

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.).

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Contributions

F.Z.B. performed all protein purifications, crystallization experiments, liposome sedimentation assays, docking experiments and in vitro activity assays. C.M.S. performed all cell-based activity assays. J.A.B. and T.S.H. constructed key plasmids. F.Z.B. and M.V.A. determined and refined the final crystal structure. F.Z.B., M.A.F. and M.V.A. contributed intellectual and strategic input. M.A.F. and M.V.A. supervised work. F.Z.B., M.A.F. and M.V.A. wrote and edited the final manuscript. All authors approved the final manuscript.

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Correspondence to Michael V. Airola.

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Supplementary information

Supplementary Information

Supplementary Table 1 and Figs. 1 and 2.

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RhoA and lipid modeled structures

Structural CIF files

Source data

Source Data

Source data for figs. 1 and 3–5

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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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