Type I and II classical cadherins help to determine the adhesive specificities of animal cells. Crystal-structure determination of ectodomain regions from three type II cadherins reveals adhesive dimers formed by exchange of N-terminal beta strands between partner extracellular cadherin-1 (EC1) domains. These interfaces have two conserved tryptophan side chains that anchor each swapped strand, compared with one in type I cadherins, and include large hydrophobic regions unique to type II interfaces. The EC1 domains of type I and type II cadherins appear to encode cell adhesive specificity in vitro. Moreover, perturbation of motor neuron segregation with chimeric cadherins depends on EC1 domain identity, suggesting that this region, which includes the structurally defined adhesive interface, encodes type II cadherin functional specificity in vivo.

Vertebrate genomes encode nineteen "classical" cadherins and about a hundred non-classical cadherins. Adhesion by classical cadherins depends on binding interactions in their amino terminal EC1 domains, which swap N-terminal β-strands between partner molecules from apposing cells. However, strand swapping sequence signatures are absent from non-classical cadherins, raising the question of how these proteins function in adhesion. Here we show that T-cadherin, a GPI-anchored cadherin, forms dimers through an alternative non-swapped interface near the EC1-EC2 calcium binding sites. Mutations within this interface ablate the adhesive capacity of T-cadherin. These nonadhesive T-cadherin mutants also lose the ability to regulate neurite outgrowth from T-cadherin expressing neurons. Our findings reveal the likely molecular architecture of the T-cadherin homophilic interface, and reveal its requirement for axon outgrowth regulation. The adhesive binding mode employed by T-cadherin may also be used by other non-classical cadherins.

Summary Cadherin-mediated cell adhesion is achieved through dimerization of cadherin N-terminal extracellular (EC1) domains presented from apposed cells. The dimer state is formed by exchange of N-terminal β-strands and insertion of conserved tryptophan indole side chains from one monomer into hydrophobic acceptor pockets of the partner molecule. The present work characterizes individual monomer and dimer states and the monomer-dimer equilibrium of the mouse Type II cadherin-8 EC1 domain using NMR spectroscopy. Limited picosecond-to-nanosecond timescale dynamics of the tryptophan indole moieties for both monomer and dimer states are consistent with well-ordered packing of the N-terminal β-strands intramolecularly and intermolecularly, respectively. However, pronounced microsecond-to-millisecond timescale dynamics of the side chains are observed for the monomer, but not the dimer, state, suggesting that monomers transiently sample configurations in which the indole moieties are exposed. Dimer formation is favored at low pH and by the presence of calcium, indicating a role for calcium in the strand swapping mechanism. The results are discussed in terms of possible kinetic mechanisms for EC1 dimerization.

Bullous Pemphigoid Antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the socalled plakin domain, which is a common feature of the plakins. In this study, we report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction (SAD) phasing from a selenomethionine-substituted crystal at 3.0 Å resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.

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The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.

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Traditional metabolic engineering approaches, including homologous recombination, zinc finger nucleases, and short hairpin RNA (shRNA), have previously been employed to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors, This approach has the following potential advantages : no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 monoclonal antibody. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an ADCC assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods.