Homophilic adhesion of E-cadherin occurs by a co-operative two-step interaction of N-terminal domains.

Cluster formation of E-cadherin on the cell surface is believed to be of major importance for cell-cell adhesion. To mimic this process the extracellular part of mouse E-cadherin (ECAD) was recombinantly fused to the assembly domain of rat cartilage oligomeric matrix protein (COMP), resulting in the chimeric protein ECAD-COMP. The COMP domain formed a five-stranded alpha-helical coiled-coil. This enabled the formation of a pentameric ECAD with bundled C-termini and free N-termini. The pentameric protein construct ECAD-COMP and the monomeric ECAD were expressed in human embryonal kidney 293 cells. Electron microscopy, analytical ultracentrifugation, solid phase binding and cell attachment assays revealed that pentamers showed strong self-association and cell attachment, whereas monomers exhibited no activity. At the high internal concentration in the pentamer the N-terminal EC1 domains of two E-cadherin arms interact to form a ring-like structure. Then the paired domains interact with a corresponding pair from another pentamer. None of the four other extracellular domains of E-cadherin is involved in this interaction. Based on these results, an in vivo mechanism is proposed whereby two N-terminal domains of neighbouring E-cadherins at the cell surface first form a pair, which binds with high affinity to a similar complex on another cell. The strong dependence of homophilic interactions on C-terminal clustering points towards a regulation of E-cadherin mediated cell-cell adhesion via lateral association.     

Effect of linker segments on the stability of epithelial cadherin Domain 2

Epithelial cadherin is a transmembrane protein that is essential in calcium-dependent cell-cell recognition and adhesion. It contains five independently folded globular domains in its extracellular region. Each domain has a seven-strand beta-sheet immunoglobulin fold. Short seven-residue peptide segments connect the globular domains and provide oxygens to chelate calcium ions at the interface between the domains (Nagar et al., Nature 1995;380:360-364). Recently, stability studies of ECAD2 (Prasad et al., Biochemistry 2004;43:8055-8066) were undertaken with the motivation that Domain 2 is a representative domain for this family of proteins. The definition of a domain boundary is somewhat arbitrary; hence, it was important to examine the effect of the adjoining linker regions that connect Domain 2 to the adjacent domains. Present studies employ temperature-denaturation and proteolytic susceptibility to provide insight into the impact of these linkers on Domain 2. The significant findings of our present study are threefold. First, the linker segments destabilize the core domain in the absence of calcium. Second, the destabilization due to addition of the linker segments can be partially reversed by the addition of calcium. Third, sodium chloride stabilizes all constructs. This result implies that electrostatic repulsion is a contributor to destabilization of the core domain by addition of the linkers. Thus, the context of Domain 2 within the whole molecule affects its thermodynamic characteristics.     

Light-scattering studies of the alpha-ketoglutarate dehydrogenase complex from escherichia coli. I. Characterization of the self-association of the complex

The self-association of Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) purified by a column Chromatographic technique, was characterized by light-scattering photometry. The complex adopts a solution conformation somewhat larger than that observed in the electron microscope. The evidence suggests a nonideal indefinite self-association model for KGDC in KCl, phosphate buffer. The KGDC monomer has a molecular charge of about -3 x 10(2) at neutral pH. The self-association is promoted by increasing KCl concentrations, pH (in the range from 6.3 to 7.4) and temperature (from 20 to 30 degrees C). The effects of pH changes suggest a release of protons during the self-association and a minor 'preferential' interaction of phosphate ions. For the association of one monomer to the aggregate at neutral pH and 25 degrees C. DeltaG degrees = -7.8 kcal mol(-1). DeltaH degrees = 24 kcal mol(-1) and DeltaS degrees = 1.1 x 10(2) cal mol(-1) K(-1). These data indicate that hydrophobic interactions drive the association. Thermodynamically, the self-association of KGDC is a complex phenomenon and may serve to stabilize the enzyme complex in solution.     

Stability studies of extracellular domain two of neural-cadherin

Neural- (NCAD) and epithelial- (ECAD) cadherin are calcium-dependent cell-adhesive molecules, and are localized at excitatory and inhibitory synapses respectively. They play an important role in synaptogenesis, synapse maintenance and plasticity. The extracellular region plays a critical role in cadherin-mediated cell adhesion, and has five tandemly repeated ectodomains (EC1-EC5). Calcium binding is required for dimer formation between first two N-terminal domains (EC1-EC2). Despite similarity in the primary structure, the extracellular domains of NCAD and ECAD have different intrinsic stability, dimerization affinity and kinetics of disassembly. To investigate the origin of these differences, we are characterizing the modular domains individually. Here, we report studies of NCAD2, EC2 of NCAD. This domain is important for calcium binding and is the physical linkage between the dimerization interface in EC1 and the membrane proximal modular domains. Thermal-denaturation studies show that NCAD2 is less stable than ECAD2 and less influenced by the adjoining 7-residue, N- and C-terminal linker segments. In addition the NCAD2 constructs are less influenced by added salt. This difference is likely due to variation in the overall number and distribution of charges on these anionic proteins. Our studies indicate that despite their sequence similarity and apparently passive role in adhesive dimer formation, EC2 of E- and N-cadherins are distinctly different and may contribute to the differences in energetics and kinetics of dimerization.     

Calcium-dependent stability studies of domains 1 and 2 of epithelial cadherin

Epithelial cadherin is important in establishing and maintaining cell to cell interactions in epithelial cells, thereby playing an important role during morphogenesis. The epithelial cadherin molecules have three main regions: the N-terminal extracellular region, the transmembrane region that spans the cell membrane once, and the C-terminal cytoplasmic region that communicates with the cytoskeletal actin filaments through catenins. We report studies of the calcium-dependent stability of extracellular domains 1 and 2 of epithelial cadherin as a two-domain construct (MECAD12). Circular dichroism (CD) spectra of MECAD12 indicated a typical beta-sheet conformation in all solution conditions. Thermal- and denaturant-induced unfolding was monitored by CD. Distinct calcium stabilization was observed as a shift in T(m) from 40 (apo) to 65 degrees C (10 mM Ca2+). Spectroscopic experiments agreed well with calorimetric (DSC). In the absence of calcium, the unfolding transition was shallow (deltaH(m) = 40 kcal/mol) but not obviously three state. Model-dependent analysis indicated that a second transition could be assigned to the unfolding of domain 2. A calcium-binding constant was derived from the calcium-dependent shift in temperature denaturation profiles. The Kd that was obtained (55 microM) was consistent with literature values. Thus, the modular domains of epithelial cadherin exhibit context-dependent behavior in both the apo and calcium-bound states. This cooperativity between the modules is consistent with the physiological role of epithelial cadherin in signal transduction through cell-adhesive contacts.     

Dimeric states of neural- and epithelial-cadherins are distinguished by the rate of disassembly

Epithelial- and neural-cadherins are specifically localized at synapses in neurons which can change the shape and contact surface on a time scale of seconds to months. We have focused our studies on the role of the extracellular domains of cadherins in the dynamics of synapses. The kinetics of dimer disassembly of the first two extracellular domains of E- and N-cadherin, ECAD12 and NCAD12, were studied with analytical size exclusion chromatography and sedimentation velocity. NCAD12 forms three different dimers that are distinguished by assembly conditions and kinetics of dissociation. ECAD12 dimer disassembles rapidly regardless of the calcium concentration, whereas the disassembly of NCAD12 dimers was strongly dependent on calcium concentration. In addition to the apo- and saturated-dimeric forms of NCAD12, there is a third dimeric form that is a slow exchange dimer. This third dimeric form for NCAD12, formed by decalcification of the calcium-saturated dimer, was kinetically trapped in apo-conditions and did not disassemble over a period of months. Sedimentation velocity experiments showed that this dimer, upon addition of calcium, had similar weighted averages as a calcium-saturated dimer. These studies provide evidence that the kinetics of dimer disassembly of the extracellular domains may be a major contributor to the morphological dynamics of synapses in vivo.     

Glycan Dependence of Galectin-3 Self-Association Properties

Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions.     

Fast dissociation kinetics between individual E-cadherin fragments revealed by flow chamber analysis

E-cadherin is the predominant adhesion molecule of epithelia. The interaction between extracellular segments of E-cadherin in the membrane of opposing cells is homophilic and calcium dependent. Whereas it is widely accepted that the specificity of the adhesive interaction is localized to the N-terminal domain, the kinetics of the recognition process are unknown. We report the first quantitative data describing the dissociation kinetics of individual E-cadherin interactions. Aggregation assays indicate that the two outermost domains of E-cadherin (E/EC1-2) retain biological activity when chemically immobilized on glass beads. Cadherin fragment trans-interaction was analysed using a flow chamber technique. Transient tethers had first-order kinetics, suggesting a unimolecular interaction. The unstressed lifetime of individual E-cadherin interactions was as brief as 2 s. A fast off rate and the low tensile strength of the E-cadherin bond may be necessary to support the high selectivity and plasticity of epithelial cell interactions.     

Contribution of cation-pi interactions to protein stability

Calculations predict that cation- interactions make an important contribution to protein stability. While there have been some attempts to experimentally measure strengths of cation-pi interactions using peptide model systems, much less experimental data are available for globular proteins. We have attempted to determine the magnitude of cation-pi interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP, and Trx). In each case, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-pi pair was replaced by Leu. Stabilities of wild-type (WT) and mutant proteins were characterized by both thermal and chemical denaturation. Gln and aromatic --> Leu mutants were consistently less stable than corresponding Met mutants, reflecting the nonisosteric nature of these substitutions. The strength of the cation-pi interaction was assessed by the value of the change in the free energy of unfolding [DeltaDeltaG(degrees) = DeltaG(degrees)(Met) - DeltaG(degrees)(WT)]. This ranged from +1.1 to -1.9 kcal/mol (average value -0.4 kcal/mol) at 298 K and +0.7 to -2.6 kcal/mol (average value -1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-pi interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than calculated values with an average difference /DeltaG(degrees)expt - DeltaG(degrees)calc/av of 2.9 kcal/mol. At room temperature, the data indicate that cation-pi interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However, at elevated temperatures, close to typical Tm's, cation-pi interactions are generally stabilizing.     

Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy (T delta S degrees = +10.32 kcal/mol) and no change in enthalpy. Binding to albumin is driven by enthalpy (delta H degrees = -8.34 kcal/mol) and is accompanied by a decrease in entropy (T delta S degrees = -2.88 kcal/mol). Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic (delta H degrees was -3.3 and -5.5 kcal/mol, respectively) and by entropic (T delta S degrees was +4.44 and +2.91 kcal/mol, respectively) components. The implications of these finding are discussed.     

Calcium-dependent homoassociation of E-cadherin by NMR spectroscopy: changes in mobility,conformation and mapping of contact regions

Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion. The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 microM-5 mM) and calcium (0-5 mM) concentrations. Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms. Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts. Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments. The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure. In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible. This mechanism provides a rational for the calcium-induced adhesiveness. Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2. These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations. Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2. According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.     

Effects of tryptophan microenvironment, soluble domain, and vesicle size on the thermodynamics of membrane protein folding: lessons from the transmembrane protein OmpA

Refolding curves of the integral membrane protein outer membrane protein A (OmpA) were measured to determine the conformational stabilities of this model system for membrane protein folding. Wild-type OmpA exhibits a free energy of unfolding (DeltaG degrees H2O) of 10.5 kcal/mol. Mutants, containing a single tryptophan residue at the native positions 7, 15, 57, 102, or 143, are less stable than wild-type OmpA, with DeltaG degrees H2O values of 6.7, 4.8, 2.4, 4.7, and 2.8 kcal/mol, respectively. The trend observed here is discussed in terms of noncovalent interactions, including aromatic interactions and hydrogen bonding. The effect of the soluble tail on the conformational stability of the transmembrane domain of OmpA was also investigated via truncated single-Trp mutants; DeltaG degrees H2O values for four of the five truncated mutants are greater by >2.7 kcal/mol relative to the full-length versions, suggesting that the absence of the soluble domain may destabilize the unfolded transmembrane domain. Finally, dynamic light scattering experiments were performed to measure the effects of urea and protein on vesicle size and stability. Urea concentrations greater than 1 M cause an increase in vesicle size, and these diameters are unaltered in the presence of protein. These dynamic light scattering results complement the fluorescence studies and illustrate the important effects of vesicle size on protein conformational stability.     

Divalent cation and ionic strength effects on Vinca alkaloid-induced tubulin self-association.

We present here a systematic study of ionic strength and divalent cation effects on Vinca alkaloid-induced tubulin spiral formation. We used sedimentation velocity experiments and quantitative fitting of weight-average sedimentation coefficients versus free drug concentrations to obtain thermodynamic parameters under various solution conditions. The addition of 50-150 mM NaCl to our standard buffer (10 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM Mg, 50 microM GDP or GTP, pH 6.9) enhances overall vinblastine- or vincristine-induced tubulin self-association. As demonstrated in previous studies, GDP enhances overall self-association more than GTP, although in the presence of salt, GDP enhancement is reduced. For example, in 150 mM NaCl, GDP enhancement is 0.24 kcal/mol for vinblastine and 0.36 kcal/mol for vincristine versus an average enhancement of 0.87 (+/- 0.34) kcal/mol for the same drugs in the absence of salt. Wyman linkage analysis of experiments with vinblastine or vincristine over a range of NaCl concentrations showed a twofold increase in the change in NaCl bound to drug-induced spirals in the presence of GTP compared to GDP. These data indicate that GDP enhancement of Vinca alkaloid-induced tubulin self-association is due in part to electrostatic inhibition in the GTP state. In the absence of NaCl, we found that vinblastine and 1 mM Mn2+ or Ca2+ causes immediate condensation of tubulin. The predominant aggregates observed by electron microscopy are large sheets. This effect was not found with 1 mM Mg2+. At 100 microM cation concentrations (Mn2+, Mg2+, or Ca2+), GDP enhances vinblastine-induced spiral formation by 0.55 (+/- 0.26) kcal/mol. This effect is found only in K2, the association of liganded heterodimers at the ends of growing spirals. There is no GDP enhancement of K1, the binding of drug to heterodimer, although K1 is dependent upon the divalent cation concentration. NaCl diminishes tubulin condensation, probably by inhibiting lateral association, and allows an investigation of higher divalent cation concentrations. In the presence of 150 mM NaCl plus 1 mM divalent cations (Mn2+, Mg2+, or Ca2+) GDP enhances vinblastine-induced spiral formation by 0.35 (+/- 0.21) kcal/mol. Relaxation times determined by stopped-flow light scattering experiments in the presence of 150 mM NaCl and vincristine are severalfold longer than those in the presence of vinblastine, consistent with a mechanism involving the redistribution of longer polymers. Unlike previous results in the absence of NaCl, relaxation times in the presence of NaCl are only weekly protein concentration dependent, suggesting the absence of annealing or an additional rate-limiting step in the mechanism.     

Self-association of lysozyme. Thermochemical measurements: effect of chemical modification of Trp-62, Trp-108, and Glu-35.

Heats of dilution and of saccharide binding for hen egg white lysozyme have been measured at 30 degrees, 0.1 ionic strength, and pH 7 over the range 3 to 95 mg of protein/ml. The concentration dependence of the apparent relative molar enthalpy of lysozyme derived from these results gives the thermodynamic parameters for the formation of an intermolecular contact in an indefinite (head-to-tail) self-association process as delta G 0 = -3.9 kcal/mol, delta H 0 = -6.4 kcal/mol, and delta S 0 = -8,3 e.u. Oxindolealanine-62-lysozyme does not undergo self-association reactions that can be detected calorimetrically. This derivative reacts with native lysozyme to form hybrid polymeric species with free energy and enthalpy of interaction similar to those for the polymers of native lysozyme. These results are consistent with the intermolecular contact in the self-assocaition of lysozyme being asymmetric (head-to-tail). The heat of dilution of the derivative of lysozyme in which Glu-35 is blocked as the ester with oxindolealanine-108 is like that observed for native lysozyme in acid solution and is independent of pH. The concentration difference spectrum that develops through self-association is of the shape expected for introduction of an indole chromophore into a charge-free region of the intermolecular contact. The foregoing results indicate that Glu-35 and Trp-62 are part of the contact, that perturbation of Trp-108 does not make a principle contribution to the concentration difference spectrum, and that no acid group other than Glu-35 is perturbed by self-association. There is a small change in the heat of (GlcNAc)3 binding over the range 0.005 to 0.034 M saccharide. These data give the value of -1 kcal/mol for the enthalpy change for formation of the 2:1 saccharide-enzyme complex (ES2) from ES and S.     

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme.The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.     

E-cadherin and cell adhesion: a role in architecture and function in the pancreatic islet.

BACKGROUND/AIMS: The efficient secretion of insulin from beta-cells requires extensive intra-islet communication. The cell surface adhesion protein epithelial (E)-cadherin (ECAD) establishes and maintains epithelial tissues such as the islets of Langerhans. In this study, the role of ECAD in regulating insulin secretion from pseudoislets was investigated. METHODS: The effect of an immuno-neutralising ECAD on gross morphology, cytosolic calcium signalling, direct cell-to-cell communication and insulin secretion was assessed by fura-2 microfluorimetry, Lucifer Yellow dye injection and insulin ELISA in an insulin-secreting model system. RESULTS: Antibody blockade of ECAD reduces glucose-evoked changes in [Ca(2+)](i) and insulin secretion. Neutralisation of ECAD causes a breakdown in the glucose-stimulated synchronicity of calcium oscillations between discrete regions within the pseudoislet, and the transfer of dye from an individual cell within a cell cluster is attenuated in the absence of ECAD ligation, demonstrating that gap junction communication is disrupted. The functional consequence of neutralising ECAD is a significant reduction in insulin secretion. CONCLUSION: Cell adhesion via ECAD has distinct roles in the regulation of intercellular communication between beta-cells within islets, with potential repercussions for insulin secretion.     

FGFR3 Transmembrane Domain Interactions Persist in the Presence of Its Extracellular Domain

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Förster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.     

X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.     

The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters

Cell adhesion molecules (CAMs) sense the extracellular microenvironment and transmit signals to the intracellular compartment. In this investigation, we addressed the mechanism of signal generation by ectodomains of single-pass transmembrane homophilic CAMs. We analyzed the structure and homophilic interactions of carcinoembryonic antigen (CEA)–related CAM 1 (CEACAM1), which regulates cell proliferation, apoptosis, motility, morphogenesis, and microbial responses. Soluble and membrane-attached CEACAM1 ectodomains were investigated by surface plasmon resonance–based biosensor analysis, molecular electron tomography, and chemical cross-linking. The CEACAM1 ectodomain, which is composed of four glycosylated immunoglobulin-like (Ig) domains, is highly flexible and participates in both antiparallel (trans) and parallel (cis) homophilic binding. Membrane-attached CEACAM1 ectodomains form microclusters in which all four Ig domains participate. Trans-binding between the N-terminal Ig domains increases formation of CEACAM1 cis-dimers and changes CEACAM1 interactions within the microclusters. These data suggest that CEACAM1 transmembrane signaling is initiated by adhesion-regulated changes of cis-interactions that are transmitted to the inner phase of the plasma membrane.     

Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: a kinetic and thermodynamic analysis

Structural studies have shown that electrostatic interactions play a major part in the binding of dihydrolipoyl dehydrogenase (E3) to the peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acyltransferase (E2) in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The binding is characterized by a small, unfavorable enthalpy change (deltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TdeltaS degrees = +14.8 kcal/mol). The contributions of individual surface residues of the PSBD of E2 to its interaction with E3 have been assessed by alanine-scanning mutagenesis, surface plasmon resonance detection, and isothermal titration calorimetry. The mutation R135A in the PSBD gave rise to a significant decrease (120-fold) in the binding affinity; two other mutations (R139A and R156A) were associated with smaller effects. The binding of the R135A mutant to E3 was accompanied by a favorable enthalpy (deltaH degrees = -2.6 kcal/mol) and a less positive entropy change (TdeltaS degrees = +7.2 kcal/mol). The midpoint melting temperature (T(m)) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 degrees C) in the T(m), compared with the wild-type complex. The results reveal the importance of Arg135 of the PSBD as a key residue in the molecular recognition of E3 by E2, and as a major participant in the overall entropy-driven binding process. Further, the effects of mutagenesis on the deltaCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.