The protein complexin plays a critical role in the regulation of SNARE mediated synaptic vesicle exocytosis at presynaptic nerve terminals in the brain. Complexin functions both to inhibit spontaneous synaptic vesicle fusion and to enhance synchronized fusion. The central helix domain of complexin is required for both its inhibitory and its facilitatory functions, but additional regions of the protein, in particular the C- terminal domain (CTD), which follows the central helix and the accessory helix, which immediately precedes the central helix, are also required for complexin's full inhibitory function. Our objective is to understand the molecular basis for the functional roles of the CTD and the accessory helix. The CTD of complexin interacts with phospholipid membranes, and this interaction serves to localize complexin to synaptic vesicles in vivo and is required for complexin's inhibitory function. Two motifs within the CTD act to both localize complexin specifically to highly curved synaptic vesicle membranes and to activate complexin's inhibitory activity only when bound to synaptic vesicles, but important questions remain regarding the mechanisms by which membrane-binding contributes to complexin's inhibitory function. Specifically, the structural basis for the curvature-dependent interactions of the two CTD motifs with membranes remain unclear. In addition, many complexins feature a C-terminal CAAX box motif that leads to farnesylation, and the membrane-binding properties of farnesylated complexins have not been explored. This proposal aims at filling key gaps in our knowledge of how the structural features of membrane-bound complexin relate to the function of the protein. To do this, two specific aims will be pursued. (1) To characterize the structural basis for the membrane curvature-dependent structural transition of the amphipathic helix motif of complexin's CTD, to assess the role of membrane curvature dependent helix formation in regulating complexin's inhibitory function and to assess the influence of this transition on membrane binding affinity and kinetics. (2) To characterize the structural basis for membrane interactions by both farnesylated and unmodified C-terminal motifs in complexin's CTD, and to asses the functional implications of these interactions. The accessory helix of complexin also contributes significantly to the inhibition of neurotransmitter release. Recent results suggest that the accessory helix acts to nucleate and stabilize helical structure in the central helix, and thereby facilitates the interactions of the central helix with SNARE proteins. In a third specific aim (3) we will test this hypothesis by directly characterizing alterations in the helicity of the accessory and central helices, measuring how such alterations influence SNARE binding in vitro, and correlating these measurements with effects on complexin's inhibitory function in vivo. Together these three aims will serve to elucidate the molecular basis for the roles of complexin's CTD and accessory helix in the inhibition of neurotransmitter release.

Public Health Relevance

The protein complexin plays an important role in the ability of neurons to communicate with each other via the release of neurotransmitters, a process that is critical for proper nervous system function and that is often perturbed in neurological and psychiatric disorders. Although a specific complexin region is known to interact with the core machinery that mediates neurotransmission, we do not understand how other parts of the protein contribute to its function. Understanding the mechanistic basis for the role of different complexin regions in its function will clarify a fundamental aspect of how communication between neurons is regulated.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM117518-01A1
Application #
9185473
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Chin, Jean
Project Start
2016-09-15
Project End
2020-07-31
Budget Start
2016-09-15
Budget End
2017-07-31
Support Year
1
Fiscal Year
2016
Total Cost
$331,185
Indirect Cost
$133,685
Name
Weill Medical College of Cornell University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Jentsch, Julian-Alexander; Kiburu, Irene; Pandey, Kalpana et al. (2018) Structural basis of sterol binding and transport by a yeast StARkin domain. J Biol Chem 293:5522-5531
Sung, Yoon-Hui; Eliezer, David (2018) Structure and dynamics of the extended-helix state of alpha-synuclein: Intrinsic lability of the linker region. Protein Sci 27:1314-1324
Lv, Guohua; Kumar, Ashutosh; Huang, Yun et al. (2018) A Protofilament-Protofilament Interface in the Structure of Mouse ?-Synuclein Fibrils. Biophys J 114:2811-2819
Eliezer, David (2017) Proteins acting out of (dis)order. Elife 6:
Wragg, Rachel T; Parisotto, Daniel A; Li, Zhenlong et al. (2017) Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins. Front Mol Neurosci 10:146
Snead, David; Lai, Alex L; Wragg, Rachel T et al. (2017) Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function. Front Mol Neurosci 10:154