Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions

Abstract

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca 2þ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb 2þ , a structural and functional surrogate of Ca 2þ , to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb 2þ -complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, ''prime'' Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.

Structural anatomy of Protein Kinase C C1 domain interactions with diacylglycerol and other agonists

“…Chloroform solutions of long-chain 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) and short-chain 1,2-dihexanoyl- sn -glycero-3-phosphocholine (DHPC) (Avanti Polar Lipids), or their deuterated versions d 54 -DMPC (Avanti Polar Lipids) and d 40 -DHPC (Cambridge Isotope Laboratories), were aliquoted and dried extensively under vacuum. The bicelles of q = 0.5 (defined by the DMPC to DHPC molar ratio of 1:2) were prepared by suspending the dried lipid films in the NMR buffer, as previously described 42 . Additional lipid components: 1,2-dimyristoyl- sn -glycero-3-phospho-L-serine (DMPS) and di-octanoyl- sn -1,2-glycerol (DAG) were incorporated for all DAG-binding experiments, to produce the final molar ratios of DMPC:DMPS:DAG = 75:15:10.…”

Solid-State NMR Study to Probe the Effects of Divalent Metal Ions (Ca2+ and Mg2+) on the Magnetic Alignment of Polymer-Based Lipid Nanodiscs

“…One of the limitations of many of the polymer-based nanodiscs is their instability against divalent metal ions, for example, Ca 2+ and Mg 2+ . However, Ca 2+ and Mg 2+ are two of the most important cations present in the human body, which are critical for the function of a variety of biomolecules. − For example, a concentration of 1–10 mM Mg 2+ is essential for some of the RNAs to fold and for the ribozyme activities . There are some proteins that require divalent cations for their activities, for example, Ca 2+ -dependent calmodulin and other proteins, Mg 2+ -dependent bovine heart glycogen synthase D, and glucose-6-phosphate dehydrogenase enzyme. , The concentration of divalent cations can also influence the membrane binding, folding, structure, and membrane orientation of membrane proteins such as phospholamban and annexins. − For these reasons, it is important to make sure that the membrane mimetics are tolerant to the presence of divalent metal ions.…”

Electrostatic regulation of thecis- andtrans-membrane interactions of synaptotagmin-1

“…However, in the pre-fusion state for vesicle docking and priming, the C2AB domain of synaptotagmin-1 is most likely bound to the plasma membrane through the PIP 2 -interacting polybasic region of the C2B domain and the SNARE complex 49 in a Ca 2+ -independent manner. Ca 2+ can induce a re-orientation of the C2AB domain on the plasma membrane by changing the binding mode with the SNARE complex 49 and PIP 2 45 . This change in orientation may act as a switch to trigger synaptotagmin-1-dependent vesicle fusion in neurons and neuroendocrine cells.…”