Blueprint Assignment Examples.

<p>Blueprint Assignment Examples.</p

Examples of backbone manipulation using RosettaRemodel.

<p>In the center is the crystal structure of protein G (PDB ID: 1PGA), which was used as the starting point for all the different cases. The colored regions highlights changed made with RosettaRemodel.</p

RosettaAntibodyDesign (RAbD): A general framework for computational antibody design

<div><p>A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., <i>J</i>. <i>Mol</i>. <i>Biol</i>., 406:228–256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody–antigen complexes, using two design strategies—optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native amino acid types for residues that contact the antigen in the native structures was 72% in simulations performed in the presence of the antigen and 48% in simulations performed without the antigen, for an ARR of 1.5. For the non-contacting residues, the ARR was 1.08. This shows that the sequence profiles are able to maintain the amino acid types of these conserved, buried sites, while recovery of the exposed, contacting residues requires the presence of the antigen-antibody interface. We tested RAbD experimentally on both a lambda and kappa antibody–antigen complex, successfully improving their affinities 10 to 50 fold by replacing individual CDRs of the native antibody with new CDR lengths and clusters.</p></div

Binding of designed antibodies to HIV gp120.

<p>(A) Apparent binding affinity (KD) of WT CH103 antibody and designed antibodies to a panel of gp120 antigens. Here, 30 designs were expressed and tested, where 7 had detectable binding to these gp120s. (B) Binding affinity (KD) of the designed antibody, H2-6, versus the wild-type antibody CH103. (C) Kinetic sensorgrams of CH103 WT and design H2-6 to two select GP120s, Core Bal and PVO as determined through a Biacore 4000. (D) Model of the interface changes in design H2-6, with designed H2 cluster H2-10-1 (cyan), superimposed onto the WT antibody from PDB ID 4JAN (gray) <b>(E)</b> Alignment of H2-6 and the WT antibody CH103 from PDB ID 4JAN.</p

An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells

<div><p>Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env_IL-21 molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env_IL-21 may prove useful in improving antibody responses against HIV-1.</p></div

Computational benchmarking of the opt-E protocol.

<p>Recovery metrics on 60 antibodies for the opt-E protocol (optimization of total Rosetta energy) for each CDR that underwent GraftDesign in the RAbD design protocol. (A) %Recovered and %Sampled for each CDR length and cluster for the opt-E simulations. (B) Design risk ratios (DRR) for recovery of CDR length and cluster for the opt-E simulations. 95% confidence intervals for the Risk Ratio statistics are calculated as described in Methods.</p

Packing shell setup.

<p>During the inner optimization cycle, a packing shell is created (cyan) around the chosen CDR (in this case, L1 in yellow), and its neighbors (in this case, L3 and the DE loop (L4) in blue). By default, 6 Å is used as the packing shell distance. During the inner loop, all side chains are optimized and amino acid changes are made to any CDRs or regions set to sequence. The chosen CDR and its neighbors additionally undergo backbone optimization during this stage according to the minimization type chosen.</p

Schematic diagrams of RosettaAntibodyDesign.

<p>A. The outer loop: The protocol starts by (1) Choosing a CDR from those that are set to design [L1, L2, etc.] randomly according to set weights (default is equal weighting) and (2) grafting a random structure for that CDR from the CDRSet, a set of CDR structures from the PDB that satisfy user-defined input rules. (3) Regional Sequence Design is then setup for all designable regions and (4) structural constraints on the CDRs and SiteConstraints on the antibody-antigen orientation, if any, are set. (5) N Inner cycles are then completed, followed by (6) the application of the Monte Carlo criterion to either accept or reject the preliminary designs. (7) Finally, the lowest energy designs are output. B. The inner loop: (1) The antigen-antibody interface is first optionally optimized by running N cycles of <i>RosettaDock</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006112#pcbi.1006112.ref045" target="_blank">45</a>]. Interface residues set to undergo sequence design will be designed. (2) The inner Monte Carlo criterion is then applied. The conformations of the CDR, its stem, and surrounding residues, and CDRs are then optimized according to the instruction file. (3) Residues from neighboring regions are designed if enabled (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006112#pcbi.1006112.g002" target="_blank">Fig 2</a> shows this packing/design shell). (4) The inner Monte Carlo criterion is then applied again and (5) the lowest energy decoy found in the inner loop is returned to the outer loop.</p

Benchmark antibody complexes.

<p>Benchmark antibody complexes.</p

Antigenic characterization of Env_IL-4 and Env_IL-21 molecules.

<p>ELISA reactivity of Env_IL-4 and Env_IL-21 with 2G12 and HIV-Ig (A); b12 and CD4-IgG2 (B); and 48d (CD4i) in the absence and presence of sCD4 at 1 µg/ml (C). All ELISA results are representative for at least three independent experiments using proteins derived from three independent transfections.</p