Calcium-binding Loop Research Articles

The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion.

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

The Synaptotagmin Calcium-Binding Loops Modulate the Rate of Fusion Pore Expansion

The Synaptotagmin Calcium-Binding Loops Modulate the Rate of Fusion Pore Expansion

Photoinactivation related dynamics of ctenophore photoproteins: Insights from molecular dynamics simulation under electric-field

Photoinactivation is a common phenomenon in bioluminescence ctenophore photoproteins (e.g mnemiopsin, berovin and BfosPP) with still unknown mechanism. The activity of coelenterate photoproteins (e.g aequorin), which has high structural similarity with ctenophore photoproteins, is not affected by light. Recently, we have characterized the effects of light on ctenophore photoprotein mnemiopsin, in different conformations, which has demonstrated light induced structural changes, uniquely secondary structures, of both apo and holo mnemiopsin. This paper is further expansion of our previous work, by applying molecular dynamics simulations to investigate photoinactivation related dynamics of berovin at atomistic level, in comparison with aequorin, under the influence of electric component of electromagnetic field. The results have indicated that the intense electric filed could influence structure of both berovin and aequorin but in different manner, whereas moderate electric field only effects on berovin's structure remarkably. In this case, increased helicity of residues E180-M193 and decreased helical contents of L38-D46 and L125-D138 segments are considerable in berovin as well as flexibility elevation of calcium binding loops. These changes cause structural expansion of berovin, especially at N-terminal domain, in direction of electric field. In conclusion, the induced structural changes of mentioned helical parts together with elevated fluctuation of their adjacent segments, N26-D46 and M193-Y206, indicate the influence of light on substrate stabilizing residues, Arg41 and Y204. This condition could presumably leads to inactivation of bioluminescence reaction due to separation of substrate from the cavity of the protein.

Disorder in Milk Proteins: α-Lactalbumin. Part C. Peculiarities of Metal Binding.

This is a concluding part of the three-part article from a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. In this paper, we describe the peculiarities of metal binding to a multifunctional milk protein, α-lactalbumin, which has two domains, a large α-helical domain and a small β-sheet domain connected by a calcium binding loop. It is known that in addition to four disulfide bonds, the native fold of this protein is stabilized by binding of a calcium ion. In fact, although in various mammals, α-lactalbumins are rather poorly conserved possessing the overall sequence identity of ~16%, the positions of all eight cysteines and a calcium binding site (residues DKFLDDDITDDI in human protein) are strongly conserved. Curiously, this conserved calcium binding loop is located within a region with increased structural flexibility. Besides canonical calcium binding, α-lactalbumin is known to interact with other metals, such as zinc (for which it has a specific binding site), and, in its apo-form, it can bind other divalent and monovalent cations. The binding of Mg2+, Na+, and K+ to the Ca2+ site increases α-lactalbumin stability against action of heat and various denaturing agents, with the higher stabilization effects being imposed by the stronger bound metal ions.

Molecular modeling of Gly80 and Ser80 variants of human group IID phospholipase A2 and their receptor complexes: potential basis for weight loss in chronic obstructive pulmonary disease.

Weight loss is a well known systemic manifestation of chronic obstructive pulmonary disease (COPD). A Gly80Ser mutation on human group IID secretory phospholipase A2 (sPLA2) enhances expression of the cytokines that are responsible for weight loss. In this study, we seek to establish a structural correlation of wild type sPLA2 and the Gly80Ser mutation with function. sPLA2 with glycine and serine at the 80th positions and the M-type receptor were modelled. The enzymes were docked to the receptor and molecular dynamics was carried out to 70ns. Structural analysis revealed the enzymes to comprise three helices (H1-H3), two short helices (SH1 and SH2), and five loops including a calcium binding loop (L1-L5), and to be stabilized by seven disulfide bonds. The overall backbone folds of the two models are very similar, with main chain RMSD of less than 1Å. The active site within the substrate binding channel shows a catalytic triad of water-His67-Asp112, showing a hydrogen bonded network. Major structural differences between wild type and mutant enzymes were observed locally at the site of the mutation and in their global conformations. These differences include: (1) loop-L3 between H2 and H3, which bears residue Gly80 in the wild type, is in a closed conformation with respect to the channel opening, while in the mutant enzyme it adopts a relatively open conformation; (2) the mutant enzyme is less compact and has higher solvent accessible surface area; and (3) interfacial binding contact surface area is greater, and the quality of interactions with the receptor is better in the mutant enzyme as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that could determine the increased potency of the mutant enzyme with macrophage receptor for cytokine secreting function, resulting in exacerbation of cachexia in COPD.

Activation-resistant homozygous protein C R229W mutation causing familial perinatal intracranial hemorrhage and delayed onset of thrombosis

IntroductionWe describe a family with two first-degree cousins who presented with similar phenotypes characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis. Patients/methodsWe enrolled the two affected patients, five unaffected family members and fifty-five normal controls. Clinical, laboratory, and radiological characteristics of patients were obtained. Exome sequencing was performed for the older affected child. PROC c.811 C>T was genotyped by PCR in patients, family members, and controls. Protein C amidolytic activity and antigen were measured using the STACHROM® protein C kit and ELISAs. To define functional abnormalities caused by the patients' mutation, recombinant wildtype protein C and its mutants R229W, R229Q and R229A were studied. ResultsFor the two cousins, protein C amidolytic activity was 61% and 59% and antigen was 57% and 73% (nl 70–140%), respectively. Exome sequencing revealed a homozygous variant in exon 9 of the protein C (PROC) gene c.811 C>T (R229W). The R229W mutation is located in the calcium binding loop of protein C's protease domain that mediates thrombomodulin interactions. Recombinant R229W-protein C mutant was strikingly defective in rate of activation by thrombin: thrombomodulin, suggesting an in vivo deficit in these children for generation of activated protein C. ConclusionsThese cases emphasize that protein C and activated protein C are important in maintaining the integrity of the brain vascular endothelium in humans. Moreover, routine protein C assays utilizing snake venom protease fail to detect protein C mutants that are resistant to thrombin:thrombomodulin activation.

Disorder in Milk Proteins: α -Lactalbumin. Part A. Structural Properties and Conformational Behavior.

This is a first part of the two-part article that continues a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. We introduce here α-lactalbumin, a small (Mr 14 200), simple, acidic (pI 4-5), Ca(2+)-binding protein that might constitute up to 20% of total milk protein. Although function (it is one of the two components of lactose synthase that catalyzes the final step of the lactose biosynthesis in the lactating mammary gland), structure (protein has two domains, a large α -helical domain and a small β -sheet domain connected by a calcium binding loop), and folding mechanisms (α-lactalbumin is well-known as a classic example of the molten globule state) of this model globular protein are relatively well understood, α-lactalbumin continues to surprise researchers and clearly continues to have high discovery potential. The goal of this review is to summarize some recent advances in the field of α-lactalbumin research and to analyze the peculiarities of the "intrinsic disorder code" of this protein.

Molecular Dynamics Simulations of Synaptotagmin Binding to Lipid Bilayers

Synaptotagmin 1 (Syt1) is a protein associated with synaptic vesicles that serves as a neuronal calcium sensor. In response to an action potential, calcium ions inflow into the nerve terminal, bind Syt1, and trigger fusion of vesicles with the plasma membrane. Although the structure, dynamics, and calcium binding properties of Sy1 have been extensively studied, it is still debated how specifically Syt1 triggers fusion. Syt1 includes a transmembrane region and two cytosolic domains, C2A and C2B, connected by a flexible linker. Both domains have calcium binding loops, and calcium binding is thought to promote the insertion of domain tips into lipid bilayers. To elucidate this mechanism, we performed molecular dynamics (MD) simulations of calcium free and calcium bound forms of Syt1 in water/ion environment and took advantage of periodic boundary conditions to model the dynamics of Syt between bilayer bilayers. We found that calcium binding loops of C2A domain have strong affinity to lipids even in the absence of calcium, and calcium biding strongly promotes their penetration into bilayers. In contrast, C2B binding loops did not interact with lipids in the absence of calcium, and even upon calcium binding only a week interaction of C2B calcium binding loops with lipids was observed. Apparently, this interaction may depend upon lipid composition and may require specific lipids which were not included in our simulations. Calcium binding pockets of C2 domains had a strong preference for facing opposing membranes during the entire length of the simulation. These results suggest a scenario whereby the tip of Syt1 C2A domain preferentially interacts with the vesicle membrane and immerges into lipids upon calcium binding, while C2B domain interacts with the plasma membrane, possibly being anchored by membrane-specific lipids, such as PIP2.

Molecular Mechanism of the Synaptotagmin-SNARE Complex that is Essential for Synchronous Synaptic Neurotransmitter Release

Synaptotagmin-1 and neuronal SNARE proteins have key roles in evoked synchronous neurotransmitter release. However, it is still unknown how they cooperate to trigger synaptic vesicle fusion at the molecular level. We determined atomic-resolution crystal structures of calcium- and magnesium-bound complexes between synaptotagmin-1 and the neuronal SNARE complex. One of these structures was determined to atomic resolution with diffraction data from an X-ray free-electron laser (XFEL), leading to accurate rotamer assignments for many side chains, including those at the interfaces between molecules. This is one of the first new crystal structures determined using XFEL diffraction data. Moreover, in contrast to the tens of thousands to millions of crystals typically used in XFEL-based crystallography experiments, we obtained a reasonably complete data set from a few hundred images captured from 72 of the 148 crystals exposed to the XFEL beam. The electron density maps obtained from the XFEL diffraction data were notably superior to those of synchrotron data sets. The structures reveal several interfaces, including a large, specific, calcium independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for calcium triggered neurotransmitter release in mouse hippocampal neuronal synapses and for calcium triggered vesicle fusion in a reconstituted single vesicle fusion system. We propose that this interface forms before calcium triggering and moves en bloc as calcium influx promotes the interactions between synaptotagmin-1 and the plasma membrane. The simultaneous membrane interactions of the calcium binding loops, of the polybasic region, and of membrane-proximal region of the SNARE complex would deform the plasma membrane. This morphological change of the plasma membrane juxtaposes the membranes closer than the critical distance (0.9 nm) to promote stalk formation, and subsequently leads to fusion pore opening.

Chasing the Functional Asymmetry between C2A and C2B in Full-Length Synaptotagmin 1 during Ca2+-Dependent Membrane Binding

Synaptotagmin 1 (Syt1) acts as the major calcium sensor in neuronal exocytosis. Syt1 is a synaptic vesicle-anchored membrane protein that contains two tandem Ca2+ binding C2 domains, named C2A and C2B. There is evidence that Syt1 interacts with membrane lipids and SNARE proteins simultaneously to facilitate two key steps during Ca2+-triggered membrane fusion: vesicle binding and content release. Although the overall function of Syt1 has been extensively investigated, the detailed molecular behavior of the single C2A and C2B domains in the neural regulatory process remains unclear. In particular, the differential function of the two C2 domains and how they cooperate in membrane fusion has not been determined.Here we employed EPR spectroscopy, fluorescence interference contrast (FLIC) microscopy and total internal reflection fluorescence (TIRF) microscopy to dissect the state of C2A and C2B domains in full-length Syt1 during Ca2+-dependent membrane binding. CW-EPR lineshapes and power saturation of spin-labeled positions in calcium binding loops of C2A in full-length Syt1 (1-421) and truncated Syt1 (1-266) without C2B domain suggest that C2A domain alone in Syt1 has a stronger membrane binding ability. A TIRF liposome capture assay further reveals that truncated Syt1 has a significant higher initial rate and extent in trans-binding with liposomes than full-length Syt1. Our data indicate that the C2B domain might be involved in some cis-binding thereby reducing total liposome binding. FLIC microscopy validates the strong involvement of C2A during Ca2+-dependent liposome binding.Our detailed structural and functional information thus provides a clue to differential regulatory mechanisms employed by C2A and C2B domains in full-length Syt1 interacting with Ca2+ and key membrane lipids, such as PS and PIP2.

The best and the brightest: exploiting tryptophan-sensitized Tb(3+) luminescence to engineer lanthanide-binding tags.

Consider the lanthanide metals, comprising lanthanum through lutetium. Lanthanides form stable cations with a +3 charge, and these ions exhibit a variety of useful physical properties (long-lifetime luminescence, paramagnetism, anomalous X-ray scattering) that are amenable to studies of biomolecules. The absence of lanthanide ions in living systems means that background signals are generally a nonissue; however, to exploit the advantageous properties it is necessary to engineer a robust lanthanide-binding sequence that can be appended to any macromolecules of interest. To this end, the luminescence produced by tryptophan-sensitized Tb(3+) has been used as a selection marker for peptide sequences that avidly chelate these ions. A combinatorial split-and-pool library that uses two orthogonal linkers-one that is cleaved for selection and one that is cleaved for sequencing and characterization-has been used to develop lanthanide-binding tags (LBTs): peptides of 15-20 amino acids with low-nM affinity for Tb(3+). Further validating the success of this screen, knowledge about LBTs has enabled the introduction of a lanthanide-binding loop in place of one of the four native calcium-binding loops within the protein calcineurin B.

Two structural motifs within canonical EF-hand calcium-binding domains identify five different classes of calcium buffers and sensors.

Proteins with EF-hand calcium-binding motifs are essential for many cellular processes, but are also associated with cancer, autism, cardiac arrhythmias, and Alzheimer's, skeletal muscle and neuronal diseases. Functionally, all EF-hand proteins are divided into two groups: (1) calcium sensors, which function to translate the signal to various responses; and (2) calcium buffers, which control the level of free Ca2+ ions in the cytoplasm. The borderline between the two groups is not clear, and many proteins cannot be described as definitive buffers or sensors. Here, we describe two highly-conserved structural motifs found in all known different families of the EF-hand proteins. The two motifs provide a supporting scaffold for the DxDxDG calcium binding loop and contribute to the hydrophobic core of the EF hand domain. The motifs allow more precise identification of calcium buffers and calcium sensors. Based on the characteristics of the two motifs, we could classify individual EF-hand domains into five groups: (1) Open static; (2) Closed static; (3) Local dynamic; (4) Dynamic; and (5) Local static EF-hand domains.

Natural and Artificial Mutations in αIIb Integrin Lead to a Structural Deformation of a Calcium-Binding Site

The platelet integrin αIIbβ3 is widely accepted as a structural and a functional model of the broad integrin protein family. The four calcium-binding sites in the αIIb subunit contribute to biogenesis and stability of the protein. Mansour et al. (J Thromb Haemost 9:192-200, 2011) showed that the natural Asn2Asp mutation causing Glanzmann thrombasthenia, prevented surface expression of αIIbβ3, whereas the artificial Asn2Gln mutation only decreased its level. Molecular dynamics simulations and EDTA chelation assay were used here to explore the mechanism of these structural deformations. We show a considerable expansion of the calcium-binding site 3 in Asn2Asp mutation, whereas the Asn2Gln toggles between normal and expanded conformations. The αIIbβ3 surface expression level correlates to the relative spending time in the expanded conformation. By a comparison to other calcium-binding sites of αIIb and of other α integrins we show that the size of a calcium-binding loop is conserved. EDTA chelation assay shows a sensitivity to calcium removal, which correlates with the reduction in αIIbβ3 surface expression and with the calcium binding site expansion, thus verifying the simulation data. Here we indicate that Asn2 mutation affects a calcium-binding site 3 of αIIb, which structural deformation is proposed to deprive calcium binding and interfere with an integrin intracellular trafficking and its surface expression.

Characterization of the lipid binding properties of Otoferlin reveals specific interactions between PI(4,5)P2 and the C2C and C2F domains.

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30–50 μM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25–95 μM) and low affinities (Kd = 400–700 μM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B–C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1′-myoinositol-4′,5′-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.

Allosteric Activation of Bordetella pertussis Adenylyl Cyclase by Calmodulin: MOLECULAR DYNAMICS AND MUTAGENESIS STUDIES

Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly.

The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys-Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys-Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.

Crystallography based structure elucidation of the complex of C-terminal fragment of Aβ polypeptide of Alzheimer’s disease with phospholipase A2

Background Aggregation of Ab polypeptide plays an important role in pathogenesis of Alzheimer’s disease, the most common form of dementia [1]. Therapies based on rationally designed aggregation inhibitors require knowledge of molecular structure [2]; because of high aggregation tendency of Ab, it has not been possible to obtain complete structural information by X-ray crystallography. The hydrophobic C-terminal part of the Ab peptide is critical in triggering transformation from a-helical to b-sheet structure. Phospholipases are an important enzyme involved in the inflammatory cascade mechanism having a conserved globular structure with active site, calcium binding loop, and hydrophobic channel. It has been speculated that phospholipase A2 (PLA2) inhibits the aggregation of Ab peptide by interacting with the peptide and keeping the two peptide chains apart.

Group III phospholipase A2: collective unity amidst structural and functional diversity (769.1)

Secretory phospholipase A2 (PLA2) catalyses the hydrolysis of the sn‐2 position of glycerophospholipids to liberate arachidonic acid, a precursor of eicosanoids, that mediate inflammation. The group III PLA2 enzymes are present in a wide array of organisms across many species with completely different functions. For a systematic classification of the group, fifty protein sequences from different species of the group were taken for sequence, structural and phylogenetic studies. In addition to the conservation of calcium binding motif and the catalytic histidine, the sequences exhibit specific ‘amino acid signatures’. Structural modeling reveals that these enzymes have a conserved globular structure with species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C‐terminal domain and the quaternary conformational state. Phylogenetic analyses are in accordance with the structural features. The results provide a convincing platform for the classification the group III PLA2s into‐ 1A: venomous insects; IB: non‐venomous insects; II: mammals; IIIA: gila monsters; IIIB: reptiles, amphibians, fishes, sea anemones, liver fluke; IV: scorpions. This classification helps to understand structure‐function relationship, substrate specificity, designing of potent inhibitors against the drug target isoforms and understand evolutionary perspective of constituents.Grant Funding Source: Supported by FAST TRACK grant No. SR/FT/18 given by Department of Science and Technology

Zymogen Activation Confers Thermodynamic Stability on a Key Peptide Bond and Protects Human Cationic Trypsin from Degradation

Human cationic trypsinogen, precursor of the digestive enzyme trypsin, can be rapidly degraded to protect the pancreas when pathological conditions threaten, while trypsin itself is impressively resistant to degradation. For either form, degradation is controlled by two necessary initial proteolytic events: cleavage of the Leu81-Glu82 peptide bond by chymotrypsin C (CTRC) and cleavage of the Arg122-Val123 peptide bond by trypsin. Here we demonstrate that the Leu81-Glu82 peptide bond of human cationic trypsin, but not trypsinogen, is thermodynamically stable, such that cleavage by CTRC leads to an equilibrium mixture containing 10% cleaved and 90% uncleaved trypsin. When cleaved trypsin was incubated with CTRC, the Leu81-Glu82 peptide bond was re-synthesized to establish the same equilibrium. The thermodynamic stability of the scissile peptide bond was not dependent on CTRC or Leu-81, as re-synthesis was also accomplished by other proteases acting on mutated cationic trypsin. The Leu81-Glu82 peptide bond is located within a calcium binding loop, and thermodynamic stability of the bond was strictly dependent on calcium and on the calcium-coordinated residue Glu-85. Trypsinolytic cleavage of the Arg122-Val123 site was also delayed in trypsin relative to trypsinogen in a calcium-dependent manner, but for this bond cleavage was modulated by kinetic rather than thermodynamic control. Our results reveal that the trypsinogen to trypsin conformational switch modulates cleavage susceptibility of nick sites by altering both the thermodynamics and kinetics of cleavage to protect human cationic trypsin from premature degradation.

Conformational States and Dynamics of Neuronal Calcium Sensor Synaptotagmin

Release of neuronal transmitters from nerve terminals is triggered by calcium binding to presynaptic protein synaptotagmin I (Syt). Syt is thought to interact with the SNARE protein complex, which mediates the attachment of synaptic vesicles to the plasma membrane. In addition, abundant evidence suggests that calcium binding promotes interaction of Syt with the plasma membrane, thus mediating synaptic vesicle fusion. Although calcium binding sites of Syt have been identified, it remains obscure how specifically calcium binding induces conformational changes in Syt and modifies its interactionswith the SNARE complex and presynaptic membrane to promote fusion. Syt is a vesicle transmembrane protein, and its soluble fragment includes two rigid domains connected by a flexible linker. Although the three-dimensional structure of Syt has been determined by crystallography, it has been demonstrated that multiple Syt conformations are likely to co-exist in the solution. Thus, it is still unclear what conformational states of Syt determine its function. To elucidate this question, we combined molecular dynamics and Monte-Carlo approaches to investigate Syt conformational states. We identified several conformational states of Syt, and we found that one of its low-energy conformations is characterized by a parallel arrangement of the domains in which all the calcium binding loops face the same surface. This conformation would facilitate Syt attachment to the plasma membrane and could potentially promote pore opening. We also found that calcium binding may promote Syt flexibility and facilitate transitions between conformational states.