Multivalent lipid targeting by the calcium-independent C2A domain of Slp-4/granuphilin

Alnaas, A. A.; Watson-Siriboe, A.; Tran, S.; Negussie, M.; Henderson, J. A.; Osterberg, J. R.; Lee Chon, N.; Oviedo, J.; Lyakhova, T.; Michel, C.; Reisdorph, N.; Reisdorph, R.; Shearn, C. T.; Lin, H.; Knight, J. D.

doi:10.1101/2020.05.29.123596

bioRxiv · 10.1101/2020.05.29.123596

Multivalent lipid targeting by the calcium-independent C2A domain of Slp-4/granuphilin

Alnaas, A. A.·Watson-Siriboe, A.·Tran, S.·Negussie, M.·Henderson, J. A.·Osterberg, J. R.·Lee Chon, N.·Oviedo, J.·Lyakhova, T.·Michel, C.·Reisdorph, N.·Reisdorph, R.·Shearn, C. T.·Lin, H.·Knight, J. D.

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Abstract

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS), but much weaker when either the background anionic lipids or PIP2 are removed. Through computational and experimental approaches, we show that this high affinity membrane interaction arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, there exists a larger cationic surface surrounding the cluster which contributes substantially to the affinity for physiologically relevant lipid compositions. While mutations at the PIP2-selective site decrease affinity for PIP2, multiple mutations are needed to decrease binding to physiologically relevant lipid compositions. Docking and molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant in the bacterially expressed protein, which arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and has a low membrane affinity. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

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Alnaas, A. A., Watson-Siriboe, A., Tran, S., Negussie, M., Henderson, J. A., Osterberg, J. R., Lee Chon, N., Oviedo, J., Lyakhova, T., Michel, C., Reisdorph, N., Reisdorph, R., Shearn, C. T., Lin, H., Knight, J. D.. 2020-05-31. Multivalent lipid targeting by the calcium-independent C2A domain of Slp-4/granuphilin. https://doi.org/10.1101/2020.05.29.123596

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