Sedimentation Equilibrium Experiments Research Articles

Domain-swapped dimerization of ZO-1 PDZ2 generates specific and regulatory connexin43-binding sites

PDZ domain scaffold proteins are capable of assembling macromolecular protein complexes in diverse cellular processes through PDZ-mediated binding to a short peptide fragment at the carboxyl tail of target proteins. How each PDZ domain specifically recognizes its target protein(s) remains a major conceptual question, as at least a few out of the several hundred PDZ domains in each eukaryotic genome share overlapping binding properties with any given target protein. Here, we show that the domain-swapped dimerization of zonula occludens-1 PDZ2 generates a distinct interface that functions together with the well-separated canonical carboxyl tail-binding pocket in each PDZ unit in binding to connexin43 (Cx43). We further demonstrate that the charge-charge interaction network formed by residues in the PDZ dimer interface and upstream residues of the Cx43 peptide not only provides the unprecedented interaction specificity for the complex but may also function as a phosphorylation-mediated regulatory switch for the dynamics of the Cx43 gap junctions. Finally, we provide evidence that such domain-swapped dimer assembly also occurs in other PDZ domain scaffold proteins. Therefore, our findings present a new paradigm for understanding how some PDZ domain proteins specifically bind to and regulate the functions of their target proteins.

Cooperative Complexation of α-Cyclodextrin with Alternating Copolymers of Sodium Maleate and Dodecyl Vinyl Ether with Varying Molecular Weights

In this study, we investigated the effect of the polymer molecular weight on the cooperative complexation of α-cyclodextrin (α-CD) with alternating copolymers of sodium maleate and dodecyl vinyl ether (pC12M) by 1H NMR measurements. It was confirmed that the pC12M prepared formed micelle-like aggregates in aqueous media by steady state fluorescence and sedimentation equilibrium experiments. Using the NMR data, the concentrations of free and complexed α-CD (CCD,f and CCD,c, respectively) were calculated and the CCD,c values were plotted as a function of CCD,f to prepare binding isotherms. The binding isotherms exhibited sigmoidal curves for all the polymers examined, indicative of cooperative complexation. The binding isotherms also indicated that the complexation was more cooperative for pC12M of a higher molecular weight. Analysis of the binding isotherms using a model proposed based on the one-dimensional lattice theory indicated that the molecular weight dependency of the cooperative complexation was d...

Osmotic Pressure and Phase Boundary Determination of Multiphase Systems by Analytical Ultracentrifugation

We show that analytical ultracentrifugation can be applied to derive full equations of state of colloids in a single sedimentation equilibrium experiment, by determination of single-phase boundaries as well as of osmotic pressure versus concentration at fixed temperatures. A continuous dependence of the osmotic pressure, over orders of magnitude between at least approximately 10(1) and 10(4) Pa, and a wide concentration range, are determined in agreement with standard theoretical considerations. Two model experimental colloidal systems are investigated: For a well-known synthetic clay system (laponite), it is shown that two regimes-counter-ion ideal gas and interacting double layers-can easily be identified in the equation of state, whereas metastable glass- or microphase-separated gel states previously encountered in osmotic stress measurements of laponite are circumvented. For the case of rigid, crystallized catanionic bilayers, single phase domains can be identified. Osmotic pressure results in this case disagree with results obtained using the classical osmotic stress technique, as a result of sample adhesion to the ultracentrifuge cell windows and uncertainty due to possible micromolar ion contamination.

Structure of the two-subsite β-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

The three-dimensional structure of the catalytically efficient β-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3Å resolution). Most H bonds between inhibitor and protein occur within subsite −1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pKa 7.2) serves as catalytic acid. The pH (6–10) profile for 1/Ki(BTP) is bell-shaped with pKa’s 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pKa 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14−E186−. A sedimentation equilibrium experiment estimates a Kd ([dimer]2/[tetramer]) of 7×10−9M. Similar kcat and kcat/Km values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.

Amino Acids Important for DNA Recognition by the Response Regulator OmpR

Response regulators undergo regulated phosphorylation and dephosphorylation at conserved aspartic acid residues in bacterial signal transduction systems. OmpR is a winged helix-turnhelix DNA-binding protein that functions as a global regulator in bacteria and is also important in pathogenesis. A detailed mechanistic picture of how OmpR binds to DNA and activates transcription is lacking. We used NMR spectroscopy to solve the solution structure of the C-terminal domain of OmpR (OmpR(C)) and to analyze the chemical shift changes that occur upon DNA binding. There is little overlap in the interaction surface with residues of PhoB that were reportedly involved in protein/protein interactions in its head-to-tail dimer. Multiple factors account for the lack of overlap. One is that the spacing between the OmpR half-sites is shorter than observed with PhoB, requiring the arrangement of the two OmpR molecules to be different from that of the PhoB dimer on DNA. A second is the demonstration herein that OmpR can bind to its high affinity site as a monomer. As a result, OmpR(C) appears to be capable of adopting alternative orientations depending on the precise base composition of the binding site, which also contributes to the lack of overlap. In the presence of DNA, chemical shift changes occur in OmpR in the recognition alpha-helix 3, the loop between beta-strand 4 and alpha-helix 1, and the loop between beta-strands 5 and 6. DNA contact residues are Val(203) (T), Arg(207) (G), and Arg(209) (phosphate backbone). Our results suggest that OmpR binds to DNA as a monomer and then forms a symmetric or asymmetric dimer, depending on the binding site. We propose that during activation OmpR binds to DNA and undergoes a conformational change that promotes phosphorylation of the N-terminal receiver domain, the receiver domains dimerize, and then the second monomer binds to DNA. The flexible linker of OmpR enables the second monomer to bind in multiple orientations (head-to-tail and head-to-head), depending on the specific DNA contacts.

Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies.

IAP Antagonists Induce Autoubiquitination of c-IAPs, NF-κB Activation, and TNFα-Dependent Apoptosis

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.

The Partly Folded Back Solution Structure Arrangement of the 30 SCR Domains in Human Complement Receptor Type 1 (CR1) Permits Access to its C3b and C4b Ligands

Human complement receptor type 1 (CR1, CD35) is a type I membrane-bound glycoprotein that belongs to the regulators of complement activity (RCA) family. The extra-cellular component of CR1 is comprised of 30 short complement regulator (SCR) domains, whereas complement receptor type 2 (CR2) has 15 SCR domains and factor H (FH) has 20 SCR domains. The domain arrangement of a soluble form of CR1 (sCR1) was studied by X-ray scattering and analytical ultracentrifugation. The radius of gyration R G of sCR1 of 13.4(±1.1) nm is not much greater than those for CR2 and FH, and its R G /R 0 anisotropy ratio is 3.76, compared to ratios of 3.67 for FH and 4.1 for CR2. Unlike CR2, but similar to FH, two cross-sectional R G ranges were identified that gave R XS values of 4.7(±0.2) nm and 1.2(±0.7) nm, respectively, showing that the SCR domains adopt a range of conformations including folded-back ones. The distance distribution function P(r) showed that the most commonly occurring distance in sCR1 is at 11.5 nm. Its maximum length of 55 nm is less than double those for CR2 or FH, even though sCR1 has twice the number of SCR domains compared to CR2 Sedimentation equilibrium experiments gave a mean molecular weight of 235 kDa for sCR1. This is consistent with the value of 245 kDa calculated from its composition including 14 N-linked oligosaccharide sites, and confirmed that sCR1 is a monomer in solution. Sedimentation velocity experiments gave a sedimentation coefficient of 5.8 S. From this, the frictional ratio ( f/ f 0) of sCR1 was calculated to be 2.29, which is greater than those of 1.96 for CR2 and 1.77 for FH. The constrained scattering modelling of the sCR1 solution structure starting from homologous SCR domain structures generated 5000 trial conformationally randomised models, 43 of which gave good scattering fits to show that sCR1 has a partly folded-back structure. We conclude that the inter-SCR linkers show structural features in common with those in FH, but differ from those in CR2, and the SCR arrangement in CR1 will permit C3b or C4b to access all three ligand sites.

Characterization of the C-terminal Domain of a Potassium Channel from Streptomyces lividans (KcsA)

KcsA, a potassium channel from Streptomyces lividans, is a good model for probing the general working mechanism of potassium channels. To date, the physiological activator of KcsA is still unknown, but in vitro studies showed that it could be opened by lowering the pH of the cytoplasmic compartment to 4. The C-terminal domain (CTD, residues 112-160) was proposed to be the modulator for this pH-responsive event. Here, we support this proposal by examining the pH profiles of: (a) thermal stability of KcsA with and without its CTD and (b) aggregation properties of a recombinant fragment of CTD. We found that the presence of the CTD weakened and enhanced the stability of KcsA at acidic and basic pH values, respectively. In addition, the CTD fragment oligomerized at basic pH values with a transition profile close to that of channel opening. Our results are consistent with the CTD being a pH modulator. We propose herein a mechanism on how this domain may contribute to the pH-dependent opening of KcsA.

Comparative Biophysical Characterization of p53 with the Pro-apoptotic BAK and the Anti-apoptotic BCL-xL

The p53 transcription-independent apoptosis in mitochondria, mediated by its interaction with the pro-apoptotic and the anti-apoptotic members of the Bcl2 family of proteins, has been described in vivo, especially in radiosensitive tissues. We have characterized the interaction of p53 with both the pro-apoptotic Bak and the anti-apoptotic Bcl-x(L) proteins, comparing their affinity and their interaction surfaces, using biophysical techniques such as fluorescence anisotropy, analytical ultracentrifugation, and NMR. We have shown that both proteins interact with only the p53 core domain and not with its N- and C-terminal regions. Further, p53 has a higher affinity for Bcl-x(L) than for Bak, which is consistent with the previously described sequential binding of Bcl-x(L) and Bak by p53. Interestingly, although the interaction with both proteins is electrostatic in character, they have different binding sites. Using NMR spectroscopy, we have determined that Bcl-x(L) interacts with the DNA binding site of p53, but Bak does not interact with this site. A new potential interaction surface for Bak is proposed.

Lumazine Synthase from Candida albicans as an Anti-fungal Target Enzyme: STRUCTURAL AND BIOCHEMICAL BASIS FOR DRUG DESIGN

Lumazine synthase is an enzyme involved in riboflavin biosynthesis in many plants and microorganisms, including numerous human pathogens. The fact that the enzymes of the riboflavin biosynthesis pathway are not present in the human or animal host makes them potential targets for anti-infective agents. The crystal structure of lumazine synthase from Candida albicans was solved by molecular replacement and refined at 2.5-Angstrom resolution. The results of crystallographic investigations and sedimentation equilibrium experiments clearly indicated the presence of pentameric assemblies of the enzyme either in crystals or in solution. Isothermal titration calorimetry measurements of the binding reactions of four different inhibitors revealed high affinity for all four compounds with binding constants in the micromolar range. Structural comparison with previously determined structures of the enzyme.ligand complexes of other orthologue allowed modeling of the binding of four different inhibitors into the active site of lumazine synthase from Candida albicans.

Structure Analysis of Activity-dependent Neurotrophic Factor 9 by Circular Dichroism and Sedimentation Equilibrium

Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and Alzheimer's disease-related and amyotrophic-lateral-sclerosis-related insults at femto-molar concentrations. However, the mechanism of the femto-molar neuroprotection by the peptide has not been elucidated. The characterization of the peptide structure in solution at molecular level should shed light in the mechanism of such extremely high biological activity. From that point of view, the secondary and quaternary structure analysis of ADNF9, an active core fragment peptide of ADNF, was performed by circular dichroism (CD) and sedimentation equilibrium. ADNF9 has also been shown to exhibit a neurotrophic activity in femto-molar concentrations; in this study it showed sub-pM neuroprotective activity against V642I-APP-induced cytotoxity in the mouse primary cortical neuron. CD analysis showed that the secondary structure of ADNF9 is identical in water and phosphate-buffered saline (PBS) and is independent of the peptide concentration. The CD spectra appear to be characterized most likely as disordered. The sedimentation equilibrium experiments demonstrated monomeric structure of the protein over the wide range of peptide concentration. There is a slight enhancement of CD intensity at 37 degrees C relative to 20 degrees C, suggesting a possible hydrophobic association of the peptide. There is no change in the secondary structure in PBS upon freeze-thaw treatment, which has previously been suggested to cause activity loss.

In Vivo and in Vitro Analyses of Single-amino Acid Variants of the Salmonella enterica Phosphotransacetylase Enzyme Provide Insights into the Function of Its N-terminal Domain

The function of the N-terminal domain ( approximately 350 residues) of the Pta (phosphotransacetylase) enzyme of Salmonella enterica is unclear. Results from in vivo genetic and in vitro studies suggest that the N-terminal domain of Pta is a sensor for NADH and pyruvate. We isolated 10 single-amino acid variants of Pta that, unlike the wild-type protein, supported growth of a strain of S. enterica devoid of Acs (acetyl-CoA synthetase; AMP-forming) activity on 10 mm acetate. All mutations were mapped within the N-terminal domain of the protein. Kinetic analyses of the wild type and three variant Pta proteins showed that two of the variant proteins were faster enzymes (k(cat) 2.5-3-fold > k(cat) Pta(WT). Results from sedimentation equilibrium experiments are consistent with Pta(WT) being a trimer. Pta variants formed more hexamer than the Pta(WT) protein. NADH inhibited Pta(WT) activity by inducing a conformational change detectable by limited trypsin proteolysis; NADH did not inhibit variant protein Pta(R252H). Pyruvate stimulated Pta(WT) activity, and its effect was potentiated in the variants, being most pronounced on Pta(R252H).

Structural Basis for the Conformational Integrity of the Arabidopsis thaliana HY5 Leucine Zipper Homodimer

The leucine zipper (LZ) domain of the HY5 transcription factor from Arabidopsis thaliana has unique primary structural properties, including major occupation by the Leu residues as well as two buried polar residues in the a positions and a localized distribution of charged and polar residues in the first three heptad repeats. In this study, we solved the crystal structure of the HY5 LZ domain and show that the peculiarities in the primary sequence yield unusual structural characteristics. For example, the HY5 LZ domain exhibits a bipartite charge distribution characterized by a highly negative electrostatic surface potential in its N-terminal half and a nearly neutral potential in its C-terminal half. The LZ N-terminal region also contains two consecutive putative trigger sites for dimerization of the coiled coils. In addition, two buried asparagines at a positions 19 and 33 in the HY5 LZ domain display distinct modes of polar interaction. Whereas Asn(19) shows a conformational flip-flop, Asn(33) is engaged in a permanent hydrogen bond network. CD spectropolarimetry and analytical ultracentrifugation experiments performed with versions of the HY5 LZ domain containing mutations in the a positions yielded further evidence that position a amino acid residues are crucial for achieving an oligomeric state and maintaining stability. However, a low correlation between position a amino acid preference, core packing geometry, and rotamer conformations suggests that the oligomeric state of the LZ domain is not governed entirely by known structural properties. Taken together, our results suggest structural factors conferring conformational integrity of the HY5 LZ homodimer that are more complicated than proposed previously.

The c-Myc Target Gene Rcl (C6orf108) Encodes a Novel Enzyme, Deoxynucleoside 5′-monophosphate N-Glycosidase

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.

Biochemical Characterization of a Regulatory Cascade Controlling Transcription of the Pseudomonas aeruginosa Type III Secretion System

Many Gram-negative pathogens utilize type III secretion systems (T3SS) to translocate effector proteins into eukaryotic host cells. Expression of T3SS genes is highly regulated and is often coupled to type III secretory activity. Transcription of the Pseudomonas aeruginosa T3SS genes is coupled to secretion by a cascade of interacting regulatory proteins (ExsA, ExsD, ExsC, and ExsE). ExsA is an activator of type III gene transcription, ExsD binds ExsA to inhibit transcription, ExsC inhibits ExsD activity, and ExsE inhibits ExsC activity. The entire process is coupled to secretion by virtue of the fact that ExsE is a secreted substrate of the T3SS. Changes in the intracellular concentration of ExsE are thought to govern formation of the ExsC-ExsE, ExsC-ExsD, and ExsD-ExsA complexes. Whereas formation of the ExsC-ExsE complex allows ExsD to bind ExsA and transcription of the T3SS is repressed, formation of the ExsC-ExsD complex sequesters ExsD from ExsA and transcription of the T3SS is induced. In this study, we characterized the self-association states of ExsC, ExsD, and ExsE and the binding interactions of ExsC with ExsE and ExsD. ExsC exists as a homodimer and binds one molecule of ExsE substrate. Dimeric ExsC also interacts directly with ExsD to form a heterotetrameric complex. The difference in binding affinities between the ExsC-ExsE (K(d) 1 nm) and ExsC-ExsD (K(d) 18 nm) complexes supports a model in which ExsC preferentially binds cytoplasmic ExsE, resulting in the inhibition of T3SS gene transcription.

Characterization of the pH-dependent Interaction between the Gap Junction Protein Connexin43 Carboxyl Terminus and Cytoplasmic Loop Domains

A prevailing view regarding the regulation of connexin43 (Cx43) gap junction channels is that, upon intracellular acidification, the carboxyl-terminal domain (Cx43CT) moves toward the channel opening to interact with specific residues acting as a receptor site. Previous studies have demonstrated a direct, pH-dependent interaction between the Cx43CT and a Cx43 cytoplasmic loop (Cx43CL) peptide. This interaction was dependent on alpha-helical formation for the peptide in response to acidification; more recent studies have shown that acidification also induces Cx43CT dimerization. Whether Cx43CT dimerization is an important structural component in Cx43 regulation remains to be determined. Here we used an assortment of complimentary biophysical techniques to characterize the binding of Cx43CT or its mutants to itself and/or to a more native-like Cx43CL construct (Cx43CL(100-155), residues 100-155). Our studies expand the observation that specific Cx43CT domains are important for dimerization. We further show that properties of the Cx43CL(100-155) are different from those of the Cx43CL peptide; solvent acidification leads to Cx43CL(100-155) oligomerization and a change in the stoichiometry and binding affinity for the Cx43CT. Homo-Cx43CT and Cx43CL(100-155) oligomerization as well as the Cx43CT/Cx43CL(100-155) interaction can occur under in vivo conditions; moreover, we show that Cx43CL(100-155) strongly affects resonance peaks corresponding to Cx43CT residues Arg-376-Asp-379 and Asn-343-Lys-346. Overall, our data indicate that many of the sites involved in Cx43CT dimerization are also involved in the Cx43CT/Cx43CL interaction; we further propose that chemically induced Cx43CT and Cx43CL oligomerization is important for the interaction between these cytoplasmic domains, which leads to chemically induced gating of Cx43 channels.

Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation

This study explores the capability of modern analytical ultracentrifugation (AUC) to characterize the homogeneity, under product formulation conditions, of preparations of adenovirus vectors used in gene therapy and to assess the lot-to-lot consistency of this unique drug product. We demonstrate that a single sedimentation velocity run on an adenovirus sample can detect and accurately quantify a number of different forms of virus particles and subvirus particles. These forms include (a) intact virus monomer particles, (b) virus aggregates, (c) empty capsids (ECs), and (d) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly (or as a result of damage to the intact adenovirus or EC material during all phases of virus production). This information, which is collected on adenovirus samples under the exact formulation conditions that exist in the adenovirus vial, is obtained by direct boundary modeling of the AUC data generated from refractometric and/or UV detection systems using the computer program SEDFIT developed by Peter Schuck. Although both detectors are useful, refractometric detection using the Rayleigh interferometer offers a key advantage for providing accurate concentration information due to the similar response factors for both protein and DNA and its insensitivity to light scattering effects. Additional AUC data obtained from analytical band sedimentation velocity and density gradient sedimentation equilibrium experiments in CsCl with UV detection were also generated. These results further support conclusions concerning the solution properties of adenovirus, the identity of the different virus species, and the overall capability of boundary sedimentation velocity analysis.

Src Tail Phosphorylation Is Limited by Structural Changes in the Regulatory Tyrosine Kinase Csk

Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.

Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region

Stromal interaction molecule 1 (STIM1) has recently been identified as a key player in store-operated Ca2+ entry. Endoplasmic reticulum (ER) luminal Ca2+ depletion results in STIM1 redistribution from ER membrane homogeneity to distinctly localized aggregates near the plasma membrane; these changes precede and are linked to cytoplasmic Ca2+ influx via Ca2+ release-activated channels (CRACs). The molecular mechanisms initiating ER STIM1 redistribution and plasma membrane CRAC activity are not well understood. We recombinantly expressed the Ca2+-sensing region of STIM1 consisting of the EF-hand together with the sterile alpha-motif (SAM) domain (EF-SAM) to investigate its Ca2+-related conformational and biochemical features. We demonstrate that Ca2+-loaded EF-SAM (holo) contains high alpha-helicity, whereas EF-SAM in the absence of Ca2+ (apo) is much less compact. Accordingly, the melting temperature (Tm) of the holoform is approximately 25 degrees C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca2+-induced stabilization of 3.2 kcal mol(-1). We show that holoEF-SAM exists as a monomer, whereas apoEF-SAM readily forms a dimer and/or oligomer, and that oligomer to monomer transitions and vice versa are at least in part mediated by changes in surface hydrophobicity. Additionally, we find that the Ca2+ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (Kd) of approximately 0.2-0.6 mM and a binding stoichiometry of 1. Our results suggest that EF-SAM actively participates in and is the likely the molecular trigger initiating STIM1 punctae formation via large conformational changes. The low Ca2+ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca2+ sensor.