Structural insights into the activation of phospholipase D1 by the small GTPase ARL11.

Phospholipase D1 (PLD1) is a lipid-metabolizing enzyme that produces phosphatidic acid (PA) and contributes to pathologies such as cancer, neurodegeneration, and cardiovascular diseases. PLD1 is known to be activated by Rho GTPases at a well-defined site, and by ARF GTPases at a separate, poorly defined site. Recently, the ARF-like GTPase 11 (ARL11) was identified as an activator of PLD1, but the mechanism by which ARL11 stimulates PLD1 activity remained unknown. Here, we use in vitro biochemical assays and structural predictions to provide insight into PLD1 activation by ARL11. We find that ARL11 must be loaded with GTP to stimulate PLD1 and corroborate that the loop within the catalytic domain of PLD1 is necessary for this stimulation. Leveraging AlphaFold 3, we predicted the structure of an ARL11-PLD1 complex. Point mutants of ARL11 at the predicted PLD1-ARL11 interface disrupted PLD1 stimulation, confirming the orientation of ARL11 in complex with PLD1. The N- and C-terminal ends of the PLD1-specific loop were predicted to be disordered in the absence of ARL11 but were predicted to adopt secondary structure elements that interact with ARL11 in the complex. Internal deletion mutants of PLD1 that retained these elements, but removed the remainder of the loop, were sufficient for ARL11 stimulation. These findings suggest that ARL11 interacts with PLD1 using three adjacent, biochemically distinct interfaces, and that the ends of PLD1's disordered loop form ordered structures to facilitate these interactions. Overall, this advances our structural understanding of the activation of PLD1 by the small GTPase ARL11.