Calcium-induced Conformational Change Research Articles

Calcium-Induced Conformational Changes in EF hands and Autoinhibitory Linker Region are Critical for Catalytic Activity of CalDAG-GEFI Towards the Small GTPase Rap1

IntroductionSignaling by the small GTPase Rap1B is critical for the rapid inside-out activation of platelet integrins, a prerequisite for platelet aggregation and hemostatic plug formation at sites of vascular injury. The guanine nucleotide exchange factor (GEF), CalDAG-GEFI (CDGI), is the predominant Rap-GEF in platelets. Cell biological studies demonstrated that agonist receptor-mediated increases in cytosolic calcium are necessary for CDGI activity, but it is currently not known if and how calcium affects CDGI activity.MethodsRecombinant CDGI (1-551) and Rap1B (1-181) proteins were purified to test nucleotide exchange activity in a fluorescence-based functional assay. Hydrogen-deuterium exchange mass spectrometry was utilized to measure dynamic differences between the active and inactive forms of CDGI.ResultsNucleotide exchange activity of CDGI was strongly diminished by substitution of calcium-binding residues E450A or E479A within EF hand 1 and EF hand 2, respectively. Addition of exogenous calcium restored catalytic activity in CDGI (E479A) but not CDGI (E450A) or CDGI (E450A+E479A). Calcium-bound active CDGI exhibited faster deuterium exchange compared to the calcium-free inactive form, in particular for residues within the EF hands and a potential autoinhibitory linker region connecting the EF hand domains and the Cdc25 catalytic domain.Consistent with regulation by an autoinhibitory linker, catalytic activity was fully restored in CDGI (E450A+E479A) by a gain-of-function substitution in valine 406.ConclusionsThese findings propose a model where calcium-induced conformational changes within the EF hands and autoinhibitory linker region are necessary for Rap1B to gain access to the catalytic binding pocket in the Cdc25 domain of CDGI. These findings will be important in our understanding of how mutations in patients impair CDGI function and in efforts to design inhibitors of CDGI signaling. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract P-17: Photophysical Properties of Freely Diffusing and Immobilized Fluorescent Conjugate Based on Calcium-Binding Protein Recoverin and Alexa647 Dye

Background: Recoverin is a calcium sensor membrane-associated protein that inhibits rhodopsin kinase thereby participating in the regulation of visual transduction. Here we examined calcium-induced conformational changes in recoverin conjugated with fluorescent dye Alexa647. Methods: Photophysical properties of immobilized and freely diffusing recoverin were investigated using fluorescence lifetime imaging microscopy and fluorescence emission spectroscopy. In solution, the formation and dissociation of the Ca2+-recoverin complex manifested as changes in Alexa647 spectra and the lifetime. In contrast, immobilization of recoverin on the microscopy glass via biotin–NeutrAvidin–biotinylated polyethylene glycol (PEG) tether inhibited changes in fluorescent signal. That can be provided by PEG as it prevented the calcium-induced changes in spectrum and lifetime of recoverin-bound Alexa647 in solution. The use of another immobilization facilitator, bovine serum albumin (BSA), did not affect calcium-induced changes in fluorescence of the conjugate in solution but produced the matrix, which was ineffective in recoverin immobilization. Results: Microscale thermophoresis demonstrated that biotinylated recoverin interacted with NeutrAvidin in solution indicating that immobilization affinity depended mainly on the geometry of the glass coating surface. Conclusion: Our results highlight the challenge of specific protein immobilization that does not affect protein functionality. By the example of recoverin, we showed that the employment of two common immobilization facilitators, PEG and BSA, yielded surfaces with different space geometry, which differently affect NeutrAvidin-based immobilization affinity as well as Ca2+-dependent conformational changes of the biotinylated protein.

Determination and evaluation of secondary structure content derived from calcium-induced conformational changes in wild-type and mutant mnemiopsin 2 by synchrotron-based Fourier-transform infrared spectroscopy

Mnemiopsin 2 from a luminous ctenophore with two functional EF-hand motifs is a calcium-regulated photoprotein that is responsible for emitting a bright blue bioluminescence upon reacting with coelenterazine and calcium ions in Mnemiopsis leidyi. Synchrotron radiation-based Fourier-transform infrared (SR-FTIR) spectroscopy was applied to analyze the distribution of secondary structures, the conformational changes resulting from calcium binding and the structural stabilities in wild-type mnemiopsin 2, as well as its mutant type that possesses three EF-hand motifs. The distribution of secondary structures of these proteins indicates that mutant apo-mnemiopsin 2 has a more stable secondary structure than the wild-type. Analyses of the SR-FTIR spectra revealed that the conformational changes at the secondary structures of both mnemiopsin 2 depend on the calcium concentrations, such that the most noticeable changes in structures of wild-type and mutant mnemiopsin 2 occur at optimum concentrations 6 and 2 mM of calcium chloride, respectively. The addition of calcium to both proteins increases the proportions of their secondary structures in the amide I and II regions. The major amide I bands in the IR spectra of both mnemiopsin‑calcium complexes shift towards smaller wavenumbers, whereas their main amide II bands are identified at larger wavenumbers.

Constructing a structural model of troponin using site-directed spin labeling: EPR and PRE-NMR.

The relative ease of introducing a paramagnetic species onto a protein, and advances in electron paramagnetic resonance (EPR) over the past two decades, have established spin labeling as a vital structural biology technique for revealing the functional workings of the troponin muscle regulatory complex-an ~80kDa heterotrimeric protein switch for turning on striated muscle contraction. Through the site-directed spin labeling (SDSL) of cysteine residues at key sites in troponin, a molecular-level understanding of the troponin muscle regulatory system across all levels of structural hierarchy has been achieved. Through the application of EPR, mobility and accessibility trends in the EPR signals of the spin labels attached to consecutive residues can reveal the secondary structure of troponin elements and also help map the interaction between subunits. Distance restraints calculated from the interspin interactions between spin label pairs have helped with building a structural model of the troponin complex. Further, when SDSL is paired with NMR, paramagnetic relaxation enhancement (PRE)-NMR has been used to obtain high-resolution structural detail for both intra- and interdomain interactions in troponin and revealed details of protein conformational changes and dynamics accompanying troponin function. In this review, we provide an overview of the SDSL labeling methodology and its application towards building a dynamic structural model of the multi-subunit troponin complex which details the calcium-induced conformational changes intimately linked to muscle regulation. We also describe how the SDSL method, in conjunction with EPR or NMR, can be used to obtain insights into structural perturbations to troponin caused by disease-causing mutations.

Monitoring Interactions Between S100B and the Dopamine D2 Receptor Using NMR Spectroscopy.

S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and interacts with a wide range of proteins to modulate their functions. The dopamine D2 receptor is one potential S100B binding partner that may play a key role in neurological processing. In this chapter, we describe the use of NMR spectroscopy to examine the interaction between calcium-bound S100B and the third intracellular loop (IC3) from the dopamine D2 receptor. We provide details that allow the strength of the interaction (K d) between the two proteins to be determined and the IC3 site of interaction on the structure of S100B to be identified. Both these characteristics can be identified from a single series of nondestructive experiments.

The Role of Calmodulin Methionine Oxidation in Regulating Conformational Change

We have used molecular dynamics simulations to understand the role of calmodulin (CaM) methionine (Met) oxidation in disrupting calcium-induced conformational changes associated with CaM's regulation of its target proteins. Previous work suggests that C-terminal CaM methionines, particularly M109 and M124, are critical for functional interaction with the major calcium release channel in muscle, the ryanodine receptor (RyR). Methionine to glutamine (Gln) substitutions at M109 or M124 designed to mimic oxidation of Met to Met sulfoxide block CaM activation of RyR at low calcium levels and inhibition of RyR at high calcium levels (Journal of Biological Chemistry 278(18) 2003). Spectroscopic distance measurements across CaM's lobes suggest that these same Met to Gln substitutions altered the equilibrium between key CaM conformations (Biochemical and biophysical research communications 456(2) 2015). We carried out microsecond molecular dynamics simulations of the open and closed CaM structural states with Met to Gln substitutions at M109 or M124 to gain an atomic-level understanding of (1) the role of methionines in stabilizing CaM structure, and (2) how Met oxidation might influence conformational equilibria and regulatory interactions. We found that every CaM C-terminal Met participates in a Met-aromatic interaction with nearby Phe residues with interaction energies of ∼4-10 kcal/mol. M109 stably interacts with F92, but only in the open structure, and M124 stably interacts with F141, but only in the closed structure. Met to Gln substitution at M109 or M124 alters interaction energies with these nearby Phe and alters the stability of the open and closed structures. We expect that these results will be applicable to understanding the general mechanism through which Met oxidation orchestrates redox cell signaling.

Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin.

Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α-helical content to CaMWt, and only modest differences in α-helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C-terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N-terminal CaM and CyaA-ACD.

Calcium-induced conformational changes of Thrombospondin-1 signature domain: implications for vascular disease

Context: Thrombospondin1 (TSP1) participates in numerous signaling pathways critical for vascular physiology and disease. The conserved signature domain of thrombospondin 1 (TSP1-Sig1) comprises three epidermal growth factor (EGF), 13 calcium-binding type 3 thrombospondin (T3) repeats, and one lectin-like module arranged in a stalk-wire-globe topology. TSP1 is known to be present in both calcium-replete (Holo-) and calcium-depleted (Apo-) state, each with distinct downstream signaling effects.Objective: To prepare a homology model of TSP1-Sig1 and investigate the effect of calcium on its dynamic structure and interactions.Methods: A homology model of Holo-TSP1-Sig1 was prepared with TSP2 as template in Swissmodel workspace. The Apo-form of the model was obtained by omitting the bound calcium ions from the homology model. Molecular dynamics (MD) simulation studies (100 ns) were performed on the Holo- and Apo- forms of TSP1 using Gromacs4.6.5.Results and discussion: After simulation, Holo-TSP1-Sig1 showed significant reorientation at the interface of the EGF1-2 and EGF2-3 modules. The T3 wire is predicted to show the maximum mobility and deviation from the initial model. In Apo-TSP1-Sig1 model, the T3 repeats unfolded and formed coils with predicted increase in flexibility. Apo-TSP1-Sig1model also predicted the exposure of the binding sites for neutrophil elastase, integrin and fibroblast growth factor 2. We present a structural model and hypothesis for the role of TSP1-Sig1 interactions in the development of vascular disorders.Conclusion: The simulated model of the fully calcium-loaded and calcium-depleted TSP1-Sig1 may enable the development of its interactions as a novel therapeutic target for the treatment of vascular diseases.

Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier

The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane.

Calcium-induced conformational changes of the regulatory domain of human mitochondrial aspartate/glutamate carriers.

The transport activity of human mitochondrial aspartate/glutamate carriers is central to the malate–aspartate shuttle, urea cycle, gluconeogenesis and myelin synthesis. They have a unique three-domain structure, comprising a calcium-regulated N-terminal domain with eight EF-hands, a mitochondrial carrier domain, and a C-terminal domain. Here we present the calcium-bound and calcium-free structures of the N- and C-terminal domains, elucidating the mechanism of calcium regulation. Unexpectedly, EF-hands 4–8 are involved in dimerization of the carrier and form a static unit, whereas EF-hands 1–3 form a calcium-responsive mobile unit. On calcium binding, an amphipathic helix of the C-terminal domain binds to the N-terminal domain, opening a vestibule. In the absence of calcium, the mobile unit closes the vestibule. Opening and closing of the vestibule might regulate access of substrates to the carrier domain, which is involved in their transport. These structures provide a framework for understanding cases of the mitochondrial disease citrin deficiency.

Biosynthetic alginate–polyester hydrogels with inherent free radical scavenging activity promote cellular response

The prevention of deleterious effects of reactive oxygen species on the cell growth by biosynthetic hydrogels based on alginate–polyester copolymer was studied using H2O2as the model ROS molecule. Chemically cross-linked biosynthetic hydrogels of alginate- co-poly(propylene fumarate)–n-butyl methacrylate, alginate- co-poly(propylene fumarate)–methyl methacrylate, alginate- co-poly(propylene fumarate)–2-hydroxyethyl methacrylate, and alginate- co-poly(propylene fumarate)– N,N′-methylene bisacrylamide with different biostabilities were prepared. We found that they were able to resist reactive oxygen species penetration into the cell to a greater extent which was evident from the live/dead assay, and increased intracellular glutathione levels compared to the H2O2-treated control. The hydrogels maintained the genomic integrity which was confirmed by comet assay. The inherent protective effects of these hydrogels without any antioxidant moiety may be mediated by dual mechanism: (a) prevention of migration of H2O2into the cells by calcium-induced conformational changes and rigidity in phospholipids present in the surface membrane of cells by the calcium generated from degradation of hydrogel and (b) by the dilution of H2O2by the free water in the hydrogel. These hydrogels have potential as injectable hydrogels to manage myocardial infarction and ischemia.

Structure-Function Relationship in Human Phospholipid Scramblase 1: Role of C-Terminal α-Helix

Phospholipid scramblases (PLSCRs) constitute a group of homologous bidirectional lipid translocators that are conserved in all eukaryotic organisms. In humans, four related PLSCR genes have been identified, named hPLSCR1- hPLSCR4. The first described member and prototype of this family is hPLSCR1, a 37 kDa type II endofacial membrane protein, that is multipalmitoylated and widely expressed in most human tissues. hPLSCR1 is involved in the rapid calcium dependent translocation of plasma membrane phospholipids, although neither the detailed calcium-induced conformational change nor the mechanism of phospholipid scrambling are known yet. In addition to this role hPLSCR1 may also function in regulating processes including signaling, cell differentiation, apoptosis, injury, cell proliferation and transcription.In the present contribution we have studied the role of the C-terminal α-helix (30 aa residues) in the structure-function relationship of hPLSCR1. With that aim a truncated mutant was constructed lacking the C-terminal α-helix (hPLSCR1αC290). A combination of structural and functional studies (fluorescence and infrared spectroscopies, partial trypsin digestion and functional characterization using liposomes) reveal that the α-helix is crucial for the scramblase activity. Furthermore in the presence of calcium the truncated mutant displays a much lower affinity for this ion and, although it still undergoes conformational changes, it requires higher Ca2+ concentrations than the wild type. Calcium binding increases the truncated mutant stability, inducing protection against trypsin digestion and thermal denaturation.

Thermodynamic and kinetic analysis of peptides derived from CapZ, NDR, p53, HDM2, and HDM4 binding to human S100B.

S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2, and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, we screened these peptides against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone (1)H and (15)N resonances were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic K(on) rate constants observed in this study (≥1 × 10(7) M(-1) s(-1)) suggest that S100B utilizes a "fly casting mechanism" in the recognition of these peptide targets.

Calcium-induced Conformational Changes in C-terminal Tail of Polycystin-2 Are Necessary for Channel Gating

Polycystin-2 (PC2) is a Ca(2+)-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca(2+). PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca(2+)-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca(2+) remains unknown. We propose that the PC2 EF-hand is a Ca(2+) sensor required for channel gating. Consistent with this, Ca(2+) binding causes a dramatic decrease in the radius of gyration (R(g)) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca(2+) concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D(max)) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca(2+) via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca(2+) release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca(2+) levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca(2+)-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.

Probing the Ca2+ Switch of the Neuronal Ca2+ Sensor GCAP2 by Time-Resolved Fluorescence Spectroscopy

We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.

The Role of the Transmembrane Alpha-Helix of Human Phospholipid Scramblase 1 in Protein Structure and Function

Human Phospholipid Scramblase 1 (hPLSCR1) is an endofacial plasma membrane protein that is multipalmitoylated and is widely expressed in most human tissues. hPLSCR1 is involved in the rapid calcium dependent translocation of plasma membrane phospholipids, although neither the detailed calcium-induced conformational change nor the mechanism of phospholipid scrambling are known yet. In addition to this role in phospholipid scrambling, hPLSCR1 may also function to regulate processes including signaling, cell differentiation, apoptosis, injury, cell proliferation and transcription.We have constructed a truncated mutant lacking the transmembrane alpha helix at C-terminal end (hPLSCR1ΔC290) in order to make the protein more stable and soluble. Preliminary structural studies(fluorescence and infrared spectroscopies) in the presence of calcium suggest that the truncated protein binds the ion and undergoes conformational changes similarly to the wild type.Functional studies using the Langmuir balance show that the ion increases the mutant adsorption to lipid monolayers, and this increase is enhanced by the presence of PS. Nevertheless the protein reconstituted in liposomes does not have any scramblase activity. We conclude that the mutant lacking the transmembrane domain retains two important properties of the native protein, the calcium-dependent conformational change and the capacity of membrane interaction.

Designed Red Fluorescent Calcium Sensor with Ultrafast Kinetic Properties

Transient change of cytosolic calcium level leads to physiological actions, which are modulated by the intracellular calcium store, as well as membrane calcium channels and vesicles/endosomes. To probe calcium responses in such high calcium environments simultaneously with the calcium transients in intracellular plasma, there is a pressing need to develop a calcium sensor featured with 1) fast kinetic properties, 2) a different spectral window from other calcium indicators, and 3) pH-independent fluorescence change. In this study, we first report our development of fast red calcium sensors using our protein design instead of using endogenous calcium binding proteins or natural calcium binding motifs. Calcium binding results in fluorescence signal increase with a Kd in the submilimolar level. The red calcium sensors with fluorescence emission in the near Infrared region allow us to monitor calcium signaling together with GFP-based calcium indicators. The pH stability of the red sensors was dramatically enhanced, with pKa below 5, compared to the GFP-derived calcium sensors. High resolution NMR has been applied to probe the mechanism of calcium-induced conformational change of designed calcium binding proteins. Our results show the developed calcium sensors are able to monitor calcium dynamics at several cellular environments responding to perturbations of extracellular calcium signaling.

The S100A10-Annexin A2 Complex Provides a Novel Asymmetric Platform for Membrane Repair

Membrane repair is mediated by multiprotein complexes, such as that formed between the dimeric EF-hand protein S100A10, the calcium- and phospholipid-binding protein annexin A2, the enlargeosome protein AHNAK, and members of the transmembrane ferlin family. Although interactions between these proteins have been shown, little is known about their structural arrangement and mechanisms of formation. In this work, we used a non-covalent complex between S100A10 and the N terminus of annexin A2 (residues 1-15) and a designed hybrid protein (A10A2), where S100A10 is linked in tandem to the N-terminal region of annexin A2, to explore the binding region, stoichiometry, and affinity with a synthetic peptide from the C terminus of AHNAK. Using multiple biophysical methods, we identified a novel asymmetric arrangement between a single AHNAK peptide and the A10A2 dimer. The AHNAK peptide was shown to require the annexin A2 N terminus, indicating that the AHNAK binding site comprises regions on both S100A10 and annexin proteins. NMR spectroscopy was used to show that the AHNAK binding surface comprised residues from helix IV in S100A10 and the C-terminal portion from the annexin A2 peptide. This novel surface maps to the exposed side of helices IV and IV' of the S100 dimeric structure, a region not identified in any previous S100 target protein structures. The results provide the first structural details of the ternary S100A10 protein complex required for membrane repair.

Identification of Calcium-Independent and Calcium-Enhanced Binding between S100B and the Dopamine D2 Receptor

S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and exposes a hydrophobic protein-binding surface. Recently S100B was identified as a binding partner of the dopamine D2 receptor in a bacterial two-hybrid screen involving the third intracellular loop (IC3). The low in vivo calcium concentration in bacteria (100–300 nM) suggests this interaction may occur in the absence of calcium. In this work the calcium-sensitive ability for S100B to recruit the IC3 of the dopamine D2 receptor was examined, and regions in both proteins required for complex formation were identified. Peptide array experiments identified the C-terminal 58 residues of the IC3 (IC3-C58) as the major interacting site for S100B. These experiments along with pull-down assays showed the IC3 interacts with S100B in the absence and presence of calcium. 1H–15N HSQC experiments were used to identify residues, primarily in helices III and IV, utilized in the IC3-C58 interaction. NMR titration data indicated that although an interaction between apo-S100B and IC3-C58 occurs without calcium, the binding was enhanced more than 100-fold upon calcium binding. Further, it was established that shorter regions within IC3-C58 comprising its N- and C-terminal halves had diminished binding to Ca2+-S100B and did not display any observable affinity in the absence of calcium. This indicates that residue or structural components within both regions are required for optimal interaction with Ca2+-S100B. This work represents the first example of an S100B target that interacts with both the apo- and calcium-saturated forms of S100B.

Interactions between factor XIII and the αC region of fibrinogen

AbstractFibrinogen αC residues 242-424 have been shown to have a major regulatory role in the activation of factor XIII-A2B2 (FXIII-A2B2); however, the interactions underpinning this enhancing effect have not been determined. Here, we have characterized the binding of recombinant (r)FXIII-A subunit and FXIII-A2B2 with fibrin(ogen) and fibrin αC residues 233-425. Using recombinant truncations of the fibrin αC region 233-425 and surface plasmon resonance, we found that activated rFXIII-A bound αC 233-425 (Kd of 2.35 ± 0.09μM) which was further localized to αC 389-403. Site-directed mutagenesis of this region highlighted Glu396 as a key residue for binding of activated rFXIII-A. The interaction was specific for activated rFXIII-A and depended on the calcium-induced conformational change known to occur in rFXIII-A during activation. Furthermore, nonactivated FXIII-A2B2, thrombin-cleaved FXIII-A2B2, and activated FXIII-A2B2 each bound fibrin(ogen) and specifically αC region 371-425 with high affinity (Kd < 35nM and Kd < 31nM, respectively), showing for the first time the potential involvement of the αC region in binding to FXIII-A2B2. These results suggest that in addition to fibrinogen γ′ chain binding, the fibrin αC region also provides a platform for the binding of FXIII-A2B2 and FXIII-A subunit.