Ca2+-independent and Ca2+-inhibited membrane binding by synaptotagmin-like proteins Project Summary/Abstract Protein-membrane interactions underlie most of the important steps in vesicle trafficking and exocytosis, including for hormones such as insulin. Although many of the key proteins are known, a better understanding of the structural mechanisms of membrane interaction is necessary for the future design of selective inhibitors. The broad goal of this study is to provide such a mechanistic and structural understanding for synaptotagmin- like proteins (Slp), a key family of proteins whose inhibition may be pharmaceutically useful. Two representative and yet unique members of the synaptotagmin-like protein (Slp) family will be investigated: Slp-2 and Slp-4. These have been chosen as our focus because (i) the structure of the membrane-binding C2A domain from Slp-4 is known, and (ii) Slp-2 exhibits an intriguing and unusual property of Ca2+-inhibited membrane binding. Generally, Slp proteins function to bridge between secretory vesicles and the plasma membrane, stably docking secretory vesicles prior to exocytosis. Slp proteins possess an N-terminal domain that bind Rab GTPases on secretory vesicles, and one or two C-terminal C2 domains that dock with high affinity to plasma membranes via interaction with anionic lipid molecules such as phosphatidylinositol-(4,5)- bisphosphate (PIP2) and phosphatidylserine (PS). Most Slp proteins, including Slp-4, bind membranes independently of calcium ions; however, the membrane affinity of Slp-2 is uniquely inhibited by calcium. This project is based on the hypothesis that high-affinity Slp C2A?membrane interaction involves multiple binding sites for lipids and/or other ligands, one of which acts allosterically and is the basis for calcium inhibition in Slp-2.
Specific Aim 1 asks, ?Which residues of the Slp-4 C2A domain are required for high-affinity membrane docking?? This question will be answered using a combination of experimental and computational approaches based on the known molecular structure of this protein domain. Molecular dynamics simulations will predict how different regions of the protein domain act together to interact with membrane lipids. Effects of site- directed mutagenesis will be tested experimentally using both quantitative liposome binding assays and cell- based model systems of secretion. Based on previous reports, it is predicted that mutants which substantially weaken membrane binding will enhance insulin secretion relative to the wild-type protein.
Specific Aim 2 asks, ?Does a pocket conserved among synaptotagmin-like proteins inhibit lipid binding allosterically?? For this Aim, the structural mechanism of Slp-2 C2A inhibition by calcium will be probed using fluorescence-based lipid and calcium binding measurements and electron paramagnetic resonance. The goal is to develop a structural model for how calcium binding at one site inhibits membrane binding at a distal site. If calcium binding to the Slp-2 C2A domain induces a structural change that blocks membrane association, then the corresponding vestigial pocket in Slp-4 would be a potential target for discovering inhibitory compounds. Because Slp-4 limits insulin secretion in vivo, its inhibition represents a possible avenue for future diabetes therapy.

Public Health Relevance

Ca2+-independent and Ca2+-inhibited membrane binding by synaptotagmin-like proteins Project Narrative This project will use a combination of experiments and computer modeling to answer an intriguing physical chemistry question with direct relevance to diabetes: how can binding of a protein molecule to a positively charged ion make it less attracted to a negatively charged lipid membrane? Answering this question for one unique protein, which is important for release of the pancreatic hormone glucagon, will help in deciphering the mechanism of action for a similar protein that regulates insulin release. In the long term, this understanding can lay the foundation for new diabetes therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
3R15GM102866-02S1
Application #
9496381
Study Section
Program Officer
Nie, Zhongzhen
Project Start
2014-02-01
Project End
2020-05-31
Budget Start
2017-06-01
Budget End
2020-05-31
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Schenk, Noah A; Dahl, Peter J; Hanna 4th, Michael G et al. (2018) A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling. Chem Phys Lipids 215:18-28
MacDougall, Daniel D; Lin, Zesen; Chon, Nara L et al. (2018) The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis. J Gen Physiol 150:783-807
Bendahmane, Mounir; Bohannon, Kevin P; Bradberry, Mazdak M et al. (2018) The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion. Mol Biol Cell :
Hamilton, Desmond J; Coffman, Matthew D; Knight, Jefferson D et al. (2017) Lipid-Coated Gold Nanoparticles and FRET Allow Sensitive Monitoring of Liposome Clustering Mediated by the Synaptotagmin-7 C2A Domain. Langmuir 33:9222-9230
Chon, Nara Lee; Osterberg, J Ryan; Henderson, Jack et al. (2015) Membrane Docking of the Synaptotagmin 7 C2A Domain: Computation Reveals Interplay between Electrostatic and Hydrophobic Contributions. Biochemistry 54:5696-711
Osterberg, J Ryan; Chon, Nara Lee; Boo, Arthur et al. (2015) Membrane Docking of the Synaptotagmin 7 C2A Domain: Electron Paramagnetic Resonance Measurements Show Contributions from Two Membrane Binding Loops. Biochemistry 54:5684-95
Vasquez, Joseph K; Chantranuvatana, Kan; Giardina, Daniel T et al. (2014) Lateral diffusion of proteins on supported lipid bilayers: additive friction of synaptotagmin 7 C2A-C2B tandem domains. Biochemistry 53:7904-13