Cyril M. Kay

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1–EC5). Studies on cadherin specificity have implicated the NH2-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer–dimer equilibrium with an affinity constant of ∼64 μM. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment–based adhesion assay. A protein with only the first two NH2-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. Proc. Natl. Acad. Sci. USA. 96:11820–11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758–68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.

Dissociation of Antimicrobial and Hemolytic Activities in Cyclic Peptide Diastereomers by Systematic Alterations in Amphipathicity

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic β-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The β-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000- and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.

Ca2+ regulation of interactions between endoplasmic reticulum chaperones.

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 μm), whereas the protein complex dissociates at >400 μmCa2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ “sensor” for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.

Structural and biochemical characterization of the type III secretion chaperones CesT and SigE.

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir–CesT and enzymatic activity profiles of SigD–SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.

New ice‐binding face for type I antifreeze protein

Type I antifreeze protein (AFP) from winter flounder is an alanine‐rich, 37 amino acid, single α‐helix that contains three 11 amino acid repeats (Thr‐X2‐Asx‐X7), where X is generally Ala. The regularly spaced Thr, Asx and Leu residues lie on one face of the helix and have traditionally been thought to form hydrogen bonds and van der Waals interactions with the ice surface. Recently, substitution experiments have called into question the importance of Leu and Asn for ice‐binding. Sequence alignments of five type I AFP isoforms show that Leu and Asn are not well conserved, whereas Ala residues adjacent to the Thr, at right angles to the Leu/Asn‐rich face, are completely conserved. To investigate the role of these Ala residues, a series of Ala to Leu steric mutations was made at various points around the helix. All the substituted peptides were fully α‐helical and remained as monomers in solution. Wild‐type activity was retained in A19L and A20L. A17L, where the substitution lies adjacent to the Thr‐rich face, had no detectable antifreeze activity. The nearby A21L substitution had 10% wild‐type activity and demonstrated weak interactions with the ice surface. We propose a new ice‐binding face for type I AFP that encompasses the conserved Ala‐rich surface and adjacent Thr.

Modulation of Structure and Antibacterial and Hemolytic Activity by Ring Size in Cyclic Gramicidin S Analogs

We have evaluated the effect of ring size of gramicidin S analogs on secondary structure, lipid binding, lipid disruption, antibacterial and hemolytic activity. Cyclic analogs with ring sizes ranging from 4 to 14 residues were designed to maintain the amphipathic character as found in gramicidin S and synthesized by solid phase peptide synthesis. The secondary structure of these peptides showed a definite periodicity in β-sheet content, with rings containing 6, 10, and 14 residues exhibiting β-sheet structure, and rings containing 8 or 12 residues being largely disordered. Peptides containing 4 or 6 residues did not bind lipopolysaccharide, whereas longer peptides showed a trend of increasing binding affinity for lipopolysaccharide with increasing length. Destabilization of Escherichia coli outer membranes was only observed in peptides containing 10 or more residues. Peptides containing fewer than 10 residues were completely inactive and exhibited no hemolytic activity. The 10-residue peptide showed an activity profile similar to that of gramicidin S itself, with activity against Gram-positive and Gram-negative microorganisms as well as yeast, but also showed high hemolytic activity. Differential activities were obtained by increasing the size of the ring to either 12 or 14 residues. The 14-residue peptide showed no antibiotic activity but exhibited increased hemolytic activity. The 12-residue peptide lost activity against Gram-positive bacteria, retained activity against Gram-negative microorganisms and yeast, but displayed decreased hemolytic activity. Biological activities in the 12-residue peptide were optimized by a series of substitutions in residues comprising both hydrophobic and basic sites resulting in a peptide that exhibited activities comparable with gramicidin S against Gram-negative microorganisms and yeast but with substantially lower hemolytic activity. Compared with gramicidin S, the best analog showed a 10-fold improvement in antibiotic specificity for Gram-negative microorganisms and a 7-fold improvement in specificity for yeast over human erythrocytes as determined by a therapeutic index. These results indicate that it is possible to modulate structure and activities of cyclic gramicidin S analogs by varying ring sizes and further show the potential for developing clinically useful antibiotics based on gramicidin S.

The Effects of Interhelical Electrostatic Repulsions between Glutamic Acid Residues in Controlling the Dimerization and Stability of Two-stranded α-Helical Coiled-coils

The effects of interhelical electrostatic repulsions in controlling the dimerization and stability of two-stranded α-helical coiled-coils have been studied using de novo designed synthetic coiled-coils. A native coiled-coil was synthesized, which consisted of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain 2-2′ disulfide bridge. This peptide, designed to contain no intrachain or interchain electrostatic interactions, forms a stable coiled-coil structure at 20°C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Five mutant coiled-coils were designed in which Gln residues at the e and g positions of the heptad repeat were substituted with Glu systematically from the N terminus toward the C terminus, resulting in each polypeptide chain having 2, 4, 6, 8, or 10 Glu residues. These substituted Glu residues are able to form interchain i to i‘+5 electrostatic repulsions across the dimer interface. As the number of interchain repulsions increases, a steady loss of helical content is observed by circular dichroism spectroscopy. The effects of the interchain Glu-Glu repulsions on the coiled-coil structure are partly overcome by the presence of an interchain disulfide bridge; the peptide with six Glu substitutions is only 15% helical in the reduced form but 85% helical in the oxidized form. The stabilities of the coiled-coils were determined by urea and guanidine hydrochloride (GdnHCl) denaturation studies at 20°C. The stabilities of the coiled-coils determined by urea denaturation indicate a decrease in stability, which correlates with an increasing number of interchain repulsions ([urea]1/2 values ranging from 8.4 to 3.7 M in the presence of 3 M KCl). In contrast, all coiled-coils had similar stabilities when determined by GdnHCl denaturation (approximately 2.9 M). KCl could not effectively screen the effects of interchain repulsions on coiled-coil stability as compared to GdnHCl.

Lateral self-assembly of E-cadherin directed by cooperative calcium binding

We report the Ca2+ binding characteristics of recombinant Ecad12, a construct spanning the first two repeats of epithelial cadherin, and demonstrate the links between Ca2+ binding and dimer formation. Sedimentation equilibrium and dynamic light scattering experiments show that weak dimerization of Ecad12 occurs in the presence of 10 mM Ca2+ (K P d=0.17 mM), while no appreciable dimer formation was detected in the absence of Ca2+. Ca2+‐induced dimerization was also observed in electron microscopy images of Ecad12. We conclude from Ca2+ titration experiments monitored by tryptophan fluorescence and flow dialysis that dimerization does not affect the equilibrium binding constant for Ca2+. However, the value of the Hill coefficient for Ca2+ binding increases from 1.5 to 2.4 as the protein concentration increases, showing that dimer formation largely contributes to the cooperativity in Ca2+ binding. Based on these observations and previous crystallographic studies, we propose that calcium acts more likely as a geometrical aligner ensuring the proper assembly of cadherin molecules, rather than a simple adhesive.

The Grb2-mSos1 Complex Binds Phosphopeptides with Higher Affinity than Grb2

Epidermal growth factor (EGF) stimulation leads to autophosphorylation of the epidermal growth factor receptor (EGFR) and tyrosine phosphorylation of Shc. The Grb2 SH2 domain binds to Tyr1068 of EGFR and Tyr317 of Shc while its SH3 domains bind to mSos1. Therefore, EGF treatment potentially results in the formation of several multimeric signaling complexes, including EGFR-Grb2-mSos1, EGFR-Shc-Grb2-mSos1, and Shc-Grb2-mSos1, linking the receptor to activation of the Ras GTPase. We have purified Grb2, mSos1, and the Grb2-mSos1 complex to high homogeneity, and used these isolated proteins to obtain binding affinities of mSos1 for Grb2 and of either Grb2 or Grb2-mSos1 for phosphotyrosine-containing peptides. mSos1 bound Grb2 with a KD of 0.4 μM; the stoichiometry of the Grb2-mSos1 complex was 1:1. An EGFR-derived phosphopeptide bound Grb2 with a KD of 0.7 μM, whereas the Shc-derived phosphopeptide bound Grb2 with a KD of 0.2 μM. Since Grb2 exists in a stable complex with mSos1, and both proteins can exist in a constitutive complex in unstimulated cells, we performed phosphopeptide binding studies on the Grb2-mSos1 complex to gain a better understanding of binding events in the intact cell. Grb2-mSos1 bound to both EGFR- and Shc-derived phosphopeptides with higher affinities (KD of 0.3 μM and 31 nM, respectively) than Grb2 alone. These findings suggest that the proximity of mSos1 to Grb2 in the complex can influence the interactions of the Grb2 SH2 domain with phosphopeptides and raise the possibility that in the Grb2-mSos1 complex the SH2 and SH3 domains of Grb2 are not independent of each other but may be indirectly linked by mSos1.

Gonococcal MinD Affects Cell Division in Neisseria gonorrhoeae and Escherichia coli and Exhibits a Novel Self-Interaction

The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae (MinD(Ng)) in this mutant. Hence, MinD(Ng) is required for maintaining proper cell division and growth in N. gonorrhoeae. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinD(Ng) is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinC(Ng) and MinD(Ng) from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinC(Ng) or MinD(Ng) alone did not display noticeable morphological changes. These studies suggest that MinD(Ng) is involved in inhibiting gonococcal cell division, likely in conjunction with MinC(Ng). The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinD(Ng) in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinD(Ng) revealed a novel MinD(Ng) self-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinD(Ng). These results indicate that MinD(Ng) is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.