Unlocking Biradical Character in Diborepins.

Systems that possess open- and closed-shell behavior attract significant attention from researchers due to their inherent redox and charge transport properties. Herein, we report the synthesis of the first diborepin biradicals. They display tunable biradical character based on the steric and electronic profile of the stabilizing ligand and the resulting geometric deviation of the diborepin core from planarity. While there are numerous all-carbon-based biradical systems, boron-based biradical compounds are comparatively rare, particularly ones in which the radical sites are disjointed. Calculations using density functional theory (DFT) and multireference methods demonstrate that the fused diborepin scaffold exhibits high biradical character, up to 95%. Use of a nonsterically demanding diaminocarbene promotes the planarization of the pentacyclic framework, resulting in the synthetic realization of a diborepin containing a dibora-quinoidal core, which possesses a closed-shell ground state and thermally accessible triplet state. The biradicals were structurally authenticated and characterized by both solution and solid-state electron paramagnetic resonance (EPR) spectroscopy. Half-field transitions were observed at low temperatures (about 170 K), confirming the presence of the triplet state. Initial reactivity studies of the biradicals led to the isolation and structural characterization of bis(borepin hydride) and bis(borepin dianion).

A disulfide chaperone knockout facilitates spin labeling and pulse EPR spectroscopy of outer membrane transporters.

Pulse EPR measurements provide information on distances and distance distributions in proteins but require the incorporation of pairs of spin labels that are usually attached to engineered cysteine residues. In previous work, we demonstrated that efficient in vivo labeling of the Escherichia coli outer membrane vitamin B12 transporter, BtuB, could only be achieved using strains defective in the periplasmic disulfide bond formation (Dsb) system. Here, we extend these in vivo measurements to FecA, the E. coli ferric citrate transporter. As seen for BtuB, pairs of cysteines cannot be labeled when the protein is present in a standard expression strain. However, incorporating plasmids that permit an arabinose induced expression of FecA into a strain defective in the thiol disulfide oxidoreductase, DsbA, enables efficient spin-labeling and pulse EPR of FecA in cells. A comparison of the measurements made on FecA in cells with measurements made in reconstituted phospholipid bilayers suggests that the cellular environment alters the behavior of the extracellular loops of FecA. In addition to these in situ EPR measurements, the use of a DsbA minus strain for the expression of BtuB improves the EPR signals and pulse EPR data obtained in vitro from BtuB that is labeled, purified, and reconstituted into phospholipid bilayers. The in vitro data also indicate the presence of intermolecular BtuB-BtuB interactions, which had not previously been observed in a reconstituted bilayer system. This result suggests that in vitro EPR measurements on other outer membrane proteins would benefit from protein expression in a DsbA minus strain.

Complexin-1 and synaptotagmin-1 compete for binding sites on membranes containing PtdInsP2.

Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P2). In the presence of PI(4,5)P2, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P2. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P2 is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P2, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.

Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport.

Outer membrane TonB-dependent transporters facilitate the uptake of trace nutrients and carbohydrates in Gram-negative bacteria and are essential for pathogenic bacteria and the health of the microbiome. Despite this, their mechanism of transport is still unknown. Here, pulse electron paramagnetic resonance (EPR) measurements were made in intact cells on the Escherichia coli vitamin B12 transporter, BtuB. Substrate binding was found to alter the C-terminal region of the core and shift an extracellular substrate binding loop 2 nm toward the periplasm; moreover, this structural transition is regulated by an ionic lock that is broken upon binding of the inner membrane protein TonB. Significantly, this structural transition is not observed when BtuB is reconstituted into phospholipid bilayers. These measurements suggest an alternative to existing models of transport, and they demonstrate the importance of studying outer membrane proteins in their native environment.

Bacteria must obtain nutrients from their surrounding environment in order to survive. In Gram-negative bacteria, proteins in the outer membrane surrounding the cell actively transport carbohydrates and trace nutrients like iron into the cell's interior. Although the structures of many of these transport proteins have been determined, the mechanism they use to move molecules across the membrane is poorly understood. To better understand this process, Nilaweera, Nyenhuis and Cafiso examined the structure of BtuB, a transport protein found in the outer membrane of Escherichia coli that is responsible for absorbing vitamin B₁₂. Previous experiments analyzing the structure of BtuB, and other similar transporters, have been carried out on purified proteins that were extracted from the outer membrane. However, these isolated proteins fail to replicate the transport activity observed in bacterial cells. Nilaweera, Nyenhuis and Cafiso therefore wanted to see how the structure of BtuB changes when it is still enclosed in the membrane of E. coli. This revealed that BtuB undergoes large structural changes when it binds to vitamin B₁₂, suggesting that this is an important part of the transport process. However, when purified BtuB was placed into an artificial membrane, these structural changes did not occur. This indicates that the cellular environment in the bacteria is needed for BtuB to carry out its transport role, and explains why previous experiments using purified proteins struggled to see this structural shift. This work highlights the importance of studying bacterial membrane proteins in their native cell environment. BtuB and similar transporters represent a large family of proteins unique to Gram-negative bacteria that have an impact on human health. Since these proteins are structurally alike, the results of this study may help resolve the transport mechanisms of other proteins, ultimately leading to new ways to control bacterial growth.

Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact.

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

Ebola virus glycoprotein interacts with cholesterol to enhance membrane fusion and cell entry.

Cholesterol serves critical roles in enveloped virus fusion by modulating membrane properties. The glycoprotein (GP) of Ebola virus (EBOV) promotes fusion in the endosome, a process that requires the endosomal cholesterol transporter NPC1. However, the role of cholesterol in EBOV fusion is unclear. Here we show that cholesterol in GP-containing membranes enhances fusion and the membrane-proximal external region and transmembrane (MPER/TM) domain of GP interacts with cholesterol via several glycine residues in the GP2 TM domain, notably G660. Compared to wild-type (WT) counterparts, a G660L mutation caused a more open angle between MPER and TM domains in an MPER/TM construct, higher probability of stalling at hemifusion for GP2 proteoliposomes and lower cell entry of virus-like particles (VLPs). VLPs with depleted cholesterol show reduced cell entry, and VLPs produced under cholesterol-lowering statin conditions show less frequent entry than respective controls. We propose that cholesterol-TM interactions affect structural features of GP2, thereby facilitating fusion and cell entry.

Native Cell Environment Constrains Loop Structure in the Escherichia coli Cobalamin Transporter BtuB.

The extracellular loops of bacterial outer membrane (OM) transporters are thought to sample a range of conformations in the apo state but to undergo a gating motion and assume a more defined conformation upon the binding of substrate. Here, we use pulse electron paramagnetic resonance to examine the conformations of the extracellular loops of BtuB, the Escherichia coli TonB-dependent vitamin B12 transporter, in whole cells. Unlike previous measurements carried out in vitro, the loops assume well-defined configurations in situ that closely match the in surfo crystal structures. Moreover, there is no evidence that the loops undergo significant gating motions upon the binding of substrate. The results demonstrate that the structure of BtuB is dependent upon an intact native OM environment, in which a critical component is likely to be the extracellular lipopolysaccharide. In general, this work indicates that measurements on OM proteins in reconstituted membrane systems may not reflect the native state of the protein in vivo.

Evidence for the Supramolecular Organization of a Bacterial Outer-Membrane Protein from In Vivo Pulse Electron Paramagnetic Resonance Spectroscopy.

In the outer membrane of Gram-negative bacteria, membrane proteins are thought to be organized into domains or islands that play a role in the segregation, movement, and turnover of membrane components. However, there is presently limited information on the structure of these domains or the molecular interactions that mediate domain formation. In the present work, the Escherichia coli outer membrane vitamin B12 transporter, BtuB, was spin-labeled, and double electron-electron resonance was used to measure the distances between proteins in intact cells. These data together with Monte Carlo simulations provide evidence for the presence of specific intermolecular contacts between BtuB monomers that could drive the formation of string-like oligomers. Moreover, the EPR data provide evidence for the location of the interacting interfaces and indicate that lipopolysaccharide mediates the contacts between BtuB monomers.

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

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca2+ 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 Pb2+, a structural and functional surrogate of Ca2+, 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 Pb2+-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.

Disulfide Chaperone Knockouts Enable In Vivo Double Spin Labeling of an Outer Membrane Transporter.

Recent advances in the application of electron paramagnetic resonance spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer membrane (OM) proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli OM B12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here, we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic-facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated OMs or reconstituted systems, and the ability to label and perform electron paramagnetic resonance in cells is expected to be applicable to a range of other bacterial OM proteins.

Phosphatidylinositol 4,5 Bisphosphate Controls the cis and trans Interactions of Synaptotagmin 1.

Synaptotagmin 1 acts as the Ca2+ sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here, we describe measurements on full-length membrane-reconstituted synaptotagmin 1 using site-directed spin labeling in which we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate, even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+ binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS-containing bilayers and will bind in a cis configuration to membranes containing PS even if a phosphatidylinositol 4,5 bisphosphate membrane is presented in trans. The data are consistent with a bridging activity for synaptotagmin 1 in which the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

In situ observation of conformational dynamics and protein ligand-substrate interactions in outer-membrane proteins with DEER/PELDOR spectroscopy.

Observation of structure and conformational dynamics of membrane proteins at high resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer-membrane proteins (OMPs) in intact Escherichia coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in situ labeling of engineered cysteines with a methanethiosulfonate spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OMs) under selective conditions. The interspin distances are measured in situ, using pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. The residual background signals, which are problematic for in situ structural biology, contribute specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5-7 d, and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and data analysis typically take 4-5 d. The described protocol provides a general strategy for observing protein ligand-substrate interactions, oligomerization, and conformational dynamics of OMPs in their native OM and intact E. coli.

Hybrid Refinement of Heterogeneous Conformational Ensembles Using Spectroscopic Data.

Multistructured biomolecular systems play crucial roles in a wide variety of cellular processes but have resisted traditional methods of structure determination, which often resolve only a few low-energy states. High-resolution structure determination using experimental methods that yield distributional data remains extremely difficult, especially when the underlying conformational ensembles are quite heterogeneous. We have therefore developed a method to integrate sparse, multimultimodal spectroscopic data to obtain high-resolution estimates of conformational ensembles. We have tested our method by incorporating double electron-electron resonance data on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin-1a into biased molecular dynamics simulations. We find that our method substantially outperforms existing state-of-the-art methods in capturing syntaxin's open-closed conformational equilibrium and further yields new conformational states that are consistent with experimental data and may help in understanding syntaxin's function. Our improved methods for refining heterogeneous conformational ensembles from spectroscopic data will greatly accelerate the structural understanding of such systems.

Exploration of the TRIM Fold of MuRF1 Using EPR Reveals a Canonical Antiparallel Structure and Extended COS-Box.

MuRF1 (TRIM63) is a RING-type E3 ubiquitin ligase with a predicted tripartite TRIM fold. TRIM proteins rely upon the correct placement of an N-terminal RING domain, with respect to C-terminal, specific substrate-binding domains. The TRIM domain organization is orchestrated by a central helical domain that forms an antiparallel coiled-coil motif and mediates the dimerization of the fold. MuRF1 has a reduced TRIM composition characterized by a lack of specific substrate binding domains, but contains in its helical domain a conserved sequence motif termed COS-box that has been speculated to fold independently into an α-hairpin. These characteristics had led to question whether MuRF1 adopts a canonical TRIM fold. Using a combination of electron paramagnetic resonance, on spin-labeled protein, and disulfide crosslinking, we show that TRIM63 follows the structural conservation of the TRIM dimerization domain, observed in other proteins. We also show that the COS-box motif folds back onto the dimerization coiled-coil motif, predictably forming a four-helical bundle at the center of the protein and emulating the architecture of canonical TRIMs.

A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis.

The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.

Choice of reconstitution protocol modulates the aggregation state of full-length membrane-reconstituted synaptotagmin-1.

Synaptotagmin-1 (Syt1) functions as the Ca2+ sensor in neuronal exocytosis, and it is routinely incorporated into lipid bilayers along with other components of the fusion machinery in order to reconstruct the in vivo fusion process. Here, we demonstrate that the detergent used to reconstitute full-length Syt1 has a significant effect on the state of the protein in bilayers. When octyl-ß-d-glucopyranoside is used to reconstitute the protein, Syt1 is present in an aggregated state that is mediated by the long juxta-membrane linker. EPR spectra from spin labels in the two C2 domains of Syt1 no longer resemble those obtained from a soluble construct containing these domains, and the C2B domain no longer exhibits a Ca2+ -dependent membrane insertion. In contrast, when reconstituted using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Syt1 is largely monomeric and the EPR spectra from C2A and C2B resemble those of the soluble construct. This result demonstrates that the choice of detergent used to reconstitute Syt1 can modulate the state of the neuronal Ca2+ -sensor.

A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.

TRIM5α SPRY/coiled-coil interactions optimize avid retroviral capsid recognition.

Restriction factors are important components of intrinsic cellular defense mechanisms against viral pathogens. TRIM5α is a restriction factor that intercepts the incoming capsid cores of retroviruses such as HIV and provides an effective species-specific barrier to retroviral infection. The TRIM5α SPRY domain directly binds the capsid with only very weak, millimolar-level affinity, and productive capsid recognition therefore requires both TRIM5α dimerization and assembly of the dimers into a multivalent hexagonal lattice to promote avid binding. Here, we explore the important unresolved question of whether the SPRY domains are flexibly linked to the TRIM lattice or more precisely positioned to maximize avidity. Biochemical and biophysical experiments indicate that the linker segment connecting the SPRY domain to the coiled-coil domain adopts an α-helical fold, and that this helical portion mediates interactions between the two domains. Targeted mutations were generated to disrupt the putative packing interface without affecting dimerization or higher-order assembly, and we identified mutant proteins that were nevertheless deficient in capsid binding in vitro and restriction activity in cells. Our studies therefore support a model wherein substantial avidity gains during assembly-mediated capsid recognition by TRIM5α come in part from tailored spacing of tethered recognition domains.

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity.

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca2+-dependent manner. In these cases, membrane association is triggered by Ca2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd2+, in lieu of Ca2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd2+-complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca2+ ion binding to the C2 domain loop regions.