Characterization of CBEL and <i>Tv</i>XynB binding to Avicel by solid state depletion assay.

<p>¹ Bmax is expressed in μmol of protein per g of substrate.</p><p>²<i>K</i>_D is expressed as μmol.L^-1 of proteins.</p><p>Characterization of CBEL and <i>Tv</i>XynB binding to Avicel by solid state depletion assay.</p

Schematic representation of CBEL protein domains.

<p>CBM1s and PAN/Apple are numbered from the N- to the C-terminus of the protein. CBM1s and PAN/Apple domains are symbolized by grey and white boxes respectively. The linker is represented by a black line.</p

Binding interaction between CBEL and cellohexaose as investigated by isothermal titration calorimetry (ITC).

<p>The upper panel shows the thermogram representing the heat of binding. The lower panel shows the titration curve. Fitting procedure was performed using a two sequential binding site model.</p

Characterization of CBEL binding to cellulose nanocrystals by fluorescence spectroscopy.

<p>N.D: no binding detected.</p><p>Characterization of CBEL binding to cellulose nanocrystals by fluorescence spectroscopy.</p

Wheat straw hydrolysis by <i>Tv</i>XynB xylanase and its derivatives.

<p>Black, grey and white bars represent <i>Tv</i>XynB-CBM1-1, <i>Tv</i>XynB and <i>Tv</i>XynBΔCBM respectively. Increasing amounts of <i>Tv</i>XynB, <i>Tv</i>XynBΔCBM and <i>Tv</i>XynB-CBM1-1 (0.1 to 0.5 mg enzyme/g substrate) were incubated with 0.02 g of wheat straw. The amount of solubilized reducing extremities was measured after 24h of hydrolysis at 40°C, pH 3.0 using the DNS assay. A control without enzyme was included and all experiments were conducted in triplicate. Asterisks indicate significant differences (p-value<0.05, <i>t</i>-test compared to <i>Tv</i>XynB, the wild type reference).</p

A Carrier Protein Strategy Yields the Structure of Dalbavancin

Many large natural product antibiotics act by specifically binding and sequestering target molecules found on bacterial cells. We have developed a new strategy to expedite the structural analysis of such antibiotic–target complexes, in which we covalently link the target molecules to carrier proteins, and then crystallize the entire carrier–target–antibiotic complex. Using native chemical ligation, we have linked the Lys-d-Ala-d-Ala binding epitope for glycopeptide antibiotics to three different carrier proteins. We show that recognition of this peptide by multiple antibiotics is not compromised by the presence of the carrier protein partner, and use this approach to determine the first-ever crystal structure for the new therapeutic dalbavancin. We also report the first crystal structure of an asymmetric ristocetin antibiotic dimer, as well as the structure of vancomycin bound to a carrier–target fusion. The dalbavancin structure reveals an antibiotic molecule that has closed around its binding partner; it also suggests mechanisms by which the drug can enhance its half-life by binding to serum proteins, and be targeted to bacterial membranes. Notably, the carrier protein approach is not limited to peptide ligands such as Lys-d-Ala-d-Ala, but is applicable to a diverse range of targets. This strategy is likely to yield structural insights that accelerate new therapeutic development