Update 11/30/2023: Congratulations to Nara on her paper being accepted into Protein Science! You can access the pre-print either on the Protein Science website or at BioRxIV.
The synaptotagmin-like protein (Slp) family comprises five mammalian proteins (Slp-1 through Slp-5) that function as Rab27 effectors to tether secretory vesicles to the plasma membrane prior to calcium-triggered exocytosis. They have an N-terminal Slp homology domain (SHD) that binds Rab27, a small GTPase on the secretory vesicle surface that regulates membrane trafficking. They also have two C2 domains (C2A and C2B) on their C-termini that bind to lipids in the plasma membrane. Unlike synaptotagmins, Slp proteins do not require calcium for membrane binding – in fact, their C2 domains lack a full complement of aspartate residues that bind Ca2+ in synaptotagmins and other C2 domains. The mechanisms of calcium-independent membrane binding for Slp C2 domains are far less well studied than the calcium-dependent synaptotagmins.
Schematic of the role of Slp-4 within pancreatic β-cells. A: Diagram of a β-cell illustrating the canonical pathway of glucose-stimulated insulin secretion. B: Close-up of a docked secretory vesicle. Slp-4 docks to Rab proteins on the secretory vesicle via its N-terminal SHD domain (green) and to plasma membrane lipids via its two C-terminal C2 domains (purple). The SNARE complex (orange) and other proteins also participate in docking. C: Domain structure of synaptotagmin (Syt) and synaptotagmin-like proteins (Slp). Both contain two C-terminal C2 domains, but their N-terminal regions differ.
The best-studied Slp family member is Slp-4, also called granuphilin, which is involved in trafficking of large dense-core secretory granules in β-cells as well as several other cell types. We showed in 2014 that both C2 domains of Slp-4 can bind to lipids in the plasma membrane, including the signaling lipid phosphatidylinositol-(4,5)-bisphosphate (PIP2). But C2A binds much more strongly than C2B. We measured on- and off-rates of the C2A domain using different lipid compositions and found that the rate constants accurately predict the membrane binding equilibrium constant (Kx) measured using a different approach. This finding supports a simple two-state mechanism of membrane binding for the Slp-4 C2A domain.
Kinetic measurements of Slp-4 C2 domains. A: Time course of Slp-4 C2A and C2B domains binding to liposomes whose lipid composition approximates the plasma membrane. B: Time course of Slp-4 C2A and C2B domains releasing from liposomes whose lipid composition approximates the plasma membrane. Note that C2A has a faster on-rate and slower off-rate than C2B, indicating that C2A binds more strongly to these liposomes. From Lyakhova and Knight, Chem. Phys. Lipids 2014, 182, 29-37.
In an extensive follow-up study published in the Journal of Biological Chemistry in 2021, we collaborated with the computational group of Prof. Hai Lin to probe the structural and biophysical mechanism of strong Slp-4 C2A domain membrane binding. We found that the protein domain has two features that contribute nearly equally to its membrane affinity: a conserved cluster of lysine residues that binds PIP2, and a large electropositive surface comprising 10 lysine and arginine residues surrounding the lysine cluster that bind nonspecifically to other anionic lipids. While it is easy to inhibit PIP2 binding by mutating the lysine cluster, it takes at least 3 mutations to noticeably inhibit binding to other anionic lipids. The findings were confirmed through a combination of computer simulations, experiments with purified proteins and liposomes, and experiments in insulin-secreting cells.
Basic residues on the membrane-binding surface of the Slp-4 C2A domain. The protein domain structure (PDB: 3FDW) is shown as a green ribbon. Individual lysine (K), arginine (R), and histidine (H) residues on the membrane-binding surface are shown as sticks. From Alnaas et al, J. Biol. Chem. 2021, 296, 100159.
Mutations to membrane-binding residues disrupt the plasma membrane affinity of the Slp-4 C2A domain in insulin-secreting cells. The wild-type (WT) Slp-4 C2A domain and the indicated mutants were expressed in MIN6 cells fused to mCherry and imaged using fluorescence microscopy. Triangles point to bright regions at the edge of cells, indicating fluorescent protein bound to the plasma membrane. Scale bars: 10 μm. From Alnaas et al, J. Biol. Chem. 2021, 296, 100159.
Because so much of the membrane binding strength of the Slp-4 C2A domain comes from nonspecific electrostatic interactions, we wanted to gauge how biologically important these interactions are by using approaches of evolutionary bioinformatics and computational biophysics. In a paper in Protein Science we demonstrate that the polybasic membrane-binding surface of the Slp-4 C2A domain is highly conserved throughout the evolution of vertebrates. Moreover, a similar polybasic surface is found in all five C2A domains of the Slp family, suggesting that binding to anionic membrane surfaces (such as the plasma membrane) is a crucial part of how Slp family proteins work.
A large electropositive surface is conserved among human Slp proteins. The Slp-4 C2A domain crystal structure (gray) and homology models of other Slp family C2A domains (orange) are shown as ribbon diagrams. Electrostatic surfaces were computed and shown as blue (positive) and red (negative) surface maps. While the exact locations of the positively charged residues vary, all family members contain a strong electropositive surface. From Chon et al, BioRxIV 2023, 548768.
We are also interested in other Slp family members. All of them bind membranes even in the absence of calcium, but Ca2+ ions are also reported to inhibit the membrane binding of Slp-2 and to enhance the membrane binding of Slp-3 and Slp-5. How does that work? And is it important for biology? None of the mechanisms have been clearly delineated. And the precise roles of Slp family proteins – how they work together with other proteins that dock secretory vesicles to the plasma membrane, and why there are five of them – remain unclear. The Knight lab continues to be interested in this protein family, particularly in understanding how Slp proteins can be manipulated to alter insulin secretion.