Phenotypic and genotypic characteristics of O157:non-H7 and O157:H7 strains used in this study.

a<p>Sor, Sorbitol fermentation.</p>b<p>GUD, β-glucuronidase activity.</p>c<p>O-serogroup detected by the <i>E. coli</i> O157-specific antibody.</p>d<p>H-serogroup detected by the <i>E. coli</i> H-specific antibodies. NM; non-motile, UT; untypeable.</p>e<p>genotype detected by the PCR-RFLP assay of the <i>fliC</i> gene. UT; untypeable.</p>f<p>genotype detected by the PCR assay of the <i>eae</i> gene.</p

Comparisons of the O157-antigen biosynthesis gene clusters and their flanking regions in six O157 strains.

<p>(A) Genetic organization of the O157-antigen gene clusters and their flanking regions. Red arrows indicate orthologs associated with the O157-antigen biosynthesis, and white arrows indicate ORFs that are not conserved in all six strains. Arrowheads indicate insertion sites of REP sequences. (B–D) Pairwise sequence comparisons. (B) Comparisons between O157 strains carrying “Sakai-type <i>rfbE</i>”. (C) Comparisons between O157 strains carrying “PV01-185-type <i>rfbE</i>”. (D) Comparisons between “Sakai-type <i>rfbE</i>” and “PV01-185-type <i>rfbE</i>” strains. Sakai is compared with PV01-185, and EC95-42 is compared with PV276. The genetic organization of the O157-antigen gene clusters and their flanking regions are shown in upper panels, and levels of % DNA sequence identity calculated with a 100 bp sliding window and a 10 bp step size are shown in lower panels. The vertical lines indicate regions showing insertion and/or deletion of fragments, and of them, lines with circular heads indi cate indels containing REP sequences.</p

Schematic drawing of REP sequence-containing regions of O157-antigen biosynthesis gene cluster flanking regions.

<p>(A) Sequence alignment of the REP sequences located in the O157-antigen gene cluster flanking regions. The consensus sequence is derived from previously published data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023250#pone.0023250-Stern2" target="_blank">[40]</a>. The palindromic motif is underlined. The non-consensus sequences were highlighted. (B) Four regions showing insertion and/or deletion of fragments including REP sequence(s); <i>yeeZ-hisG</i>, <i>hisI-wzz wcaK-wzxC</i> and <i>cpsG-cpsB</i> are compared between strains. REP sequences are indicated by arrowheads and gray boxes indicate missing regions on each of the compared strains. (C) The nucleotide sequences from <i>wcaK</i> to <i>wzxC</i> and from <i>wcaA</i> to <i>wzc</i> on EC95-42 are compared with those of PV57, and the sequences from <i>hisI</i> to <i>wzz</i> on PV01-185 are compared with those of Sakai. Locations of SNPs by pairwise sequence comparison are indicated by vertical lines (lower panel).</p

Affinity Improvement of a Therapeutic Antibody by Structure-Based Computational Design: Generation of Electrostatic Interactions in the Transition State Stabilizes the Antibody-Antigen Complex

<div><p>The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen <i>in vitro</i>, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of <i>in silico</i> calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.</p></div

Thermodynamic analysis.

<p>(<b>A</b>) Regression analysis of the temperature dependence of the dissociation constant <i>K_D</i> yields the van’t Hoff enthalpy (Δ<i>H°</i>), entropy (-<i>T</i>Δ<i>S°</i>) and free energy (Δ<i>G°</i>). Empty squares and filled circles correspond to wild-type and L-N31R antibodies, respectively. (<b>B</b>) Thermodynamic parameters corresponding to the binding of wild-type antibody to antigen. (<b>C</b>) Same parameters obtained for L-N31R.</p

Effect of the ionic strength.

<p>(<b>A</b>) Association rate constant (<i>k_on</i>), (<b>B</b>) dissociation rate constant (<i>k_off</i>), and (<b>C</b>) dissociation constant (<i>K_D</i>). The kinetic parameters of the binding of wild-type 11K2 (or mutein L-N31R) to the antigen MCP-1 were determined in running buffer containing three different concentrations of NaCl (137, 300, or 500 mM) at 25°C.</p

Analysis of the binding enthalpy.

<p>(<b>A</b>) Favorable changes of binding enthalpy with respect to wild-type antibody at the transition state (empty bar, ΔΔ<i>H^‡</i>) and at equilibrium (filled bar, ΔΔ<i>H°</i>). (<b>B</b>) Suggested model of the new interactions formed at the antibody/antigen contact surface upon mutation. Residues depicted in yellow and dark green correspond to 11K2 and MCP-1, respectively. The conformation of the side-chain of the mutated residues was modeled from the Dunbrak library of rotamers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087099#pone.0087099-Dunbrack1" target="_blank">[44]</a> as implemented in the program Chimera <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087099#pone.0087099-Pettersen1" target="_blank">[45]</a> (the most probably rotamer was always selected, except in L-Arg31, where the second most probable rotamer was chosen). Because Lys35 of MCP-1 is not interacting with a neighboring residue in the crystal structure, the conformation of this residue was also modeled as above. The dotted lines and distances represent putative interactions between the mutated residues and the antigen.</p

Mutagenesis <i>in silico</i>.

<p>Average energy values (as the sum of electrostatic and van der Waals energies) of all possible mutations of two different residues (L-Asn31 and L-Ser53) of antibody 11K2 with respect to wild-type. Negative values suggest higher affinity between the mutated protein and the antigen. The mutations indicated by the asterisks were selected for further examination by SPR.</p

Thermodynamic parameters of scFv 11K2.

<p>Values of –<i>T</i>Δ<i>S°</i> and Δ<i>G°</i> are given at 25°C.</p

Binding sensorgrams.

<p>(<b>A</b>) Binding of wild-type 11K2 to its antigen MCP-1. (<b>B</b>) Binding of mutein L-N31R to MCP-1. The arrows pointing downward indicate injection of running buffer with 11K2 antibody. The arrows pointing upward correspond to the injection of buffer with no antibody. The response signal is proportional to the amount of scFv 11K2 binding to a chip decorated with antigen MCP-1. The straight dotted line at the top curve in each panel is drawn to appreciate the slower dissociation rate of the mutein with respect to the wild-type protein. The concentration of antibody is given in each panel.</p