Antibody Epitopes on G Protein-Coupled Receptors Mapped with Genetically Encoded Photoactivatable Cross-Linkers

We developed a strategy for creating epitope maps of monoclonal antibodies (mAbs) that bind to G protein-coupled receptors (GPCRs) containing photo-cross-linkers. Using human CXC chemokine receptor 4 (CXCR4) as a model system, we genetically incorporated the photolabile unnatural amino acid <i>p</i>-azido-l-phenylalanine (azF) at various positions within extracellular loop 2 (EC2). We then mapped the interactions of the azF-CXCR4 variants with mAb 12G5 using targeted loss-of-function studies and photo-cross-linking in whole cells in a microplate-based format. We used a novel variation of a whole cell enzyme-linked immunosorbent assay to quantitate cross-linking efficiency. 12G5 cross-linked primarily to residues 184, 178, and 189 in EC2 of CXCR4. Mapping of the data to the crystal structure of CXCR4 showed a distinct mAb epitope footprint with the photo-cross-linked residues clustered around the loss-of-function sites. We also used the targeted photo-cross-linking approach to study the interaction of human CC chemokine receptor 5 (CCR5) with PRO 140, a humanized mAb that inhibits human immunodeficiency virus-1 cellular entry, and 2D7. The mAbs produced distinct cross-linking patterns on EC2 of CCR5. PRO 140 cross-linked primarily to residues 174 and 175 at the amino-terminal end of EC2, and 2D7 cross-linked mainly to residues 170, 176, and 184. These results were mapped to the recent crystal structure of CCR5 in complex with maraviroc, showing cross-linked residues at the tip of the maraviroc binding crevice formed by EC2. As a strategy for mapping mAb epitopes on GPCRs, our targeted photo-cross-linking method is complementary to loss-of-function mutagenesis results and should be especially useful for studying mAbs with discontinuous epitopes

Site-Specific Epitope Tagging of G Protein-Coupled Receptors by Bioorthogonal Modification of a Genetically Encoded Unnatural Amino Acid

We developed a general strategy for labeling expressed membrane proteins with a peptide epitope tag and detecting the tagged proteins in native cellular membranes. First, we genetically encoded the unnatural amino acid <i>p</i>-azido-l-phenylalanine (azF) at various specific sites in a G protein-coupled receptor (GPCR), C-C chemokine receptor 5 (CCR5). The reactive azido moiety facilitates Staudinger ligation to a triarylphosphine-conjugated FLAG peptide. We then developed a whole-cell-based enzyme-linked immunosorbent assay approach to detect the modified azF-CCR5 using anti-FLAG mAb. We optimized conditions to achieve labeling and detection of low-abundance GPCRs in live cells. We also performed an accessibility screen to identify azF positions on CCR5 amenable to labeling. Finally, we demonstrate a preparative strategy for obtaining pure bioorthogonally modified GPCRs suitable for single-molecule detection fluorescence experiments. This peptide epitope tagging strategy, which employs genetic encoding and bioorthogonal labeling of azF in live cells, should be useful for studying biogenesis of polytopic membrane proteins and GPCR signaling mechanisms

Prediction of /isomerization in proteins using PSI-BLAST profiles and secondary structure information-0

<p><b>Copyright information:</b></p><p>Taken from "Prediction of /isomerization in proteins using PSI-BLAST profiles and secondary structure information"</p><p>BMC Bioinformatics 2006;7():124-124.</p><p>Published online 9 Mar 2006</p><p>PMCID:PMC1450308.</p><p>Copyright © 2006 Song et al; licensee BioMed Central Ltd.</p

Prediction of /isomerization in proteins using PSI-BLAST profiles and secondary structure information-3

<p><b>Copyright information:</b></p><p>Taken from "Prediction of /isomerization in proteins using PSI-BLAST profiles and secondary structure information"</p><p>BMC Bioinformatics 2006;7():124-124.</p><p>Published online 9 Mar 2006</p><p>PMCID:PMC1450308.</p><p>Copyright © 2006 Song et al; licensee BioMed Central Ltd.</p>d on proline

Prediction of /isomerization in proteins using PSI-BLAST profiles and secondary structure information-1

<p><b>Copyright information:</b></p><p>Taken from "Prediction of /isomerization in proteins using PSI-BLAST profiles and secondary structure information"</p><p>BMC Bioinformatics 2006;7():124-124.</p><p>Published online 9 Mar 2006</p><p>PMCID:PMC1450308.</p><p>Copyright © 2006 Song et al; licensee BioMed Central Ltd.</p

Characterization of Low-Frequency Modes in Aqueous Peptides Using Far-Infrared Spectroscopy and Molecular Dynamics Simulation

Far-infrared spectroscopy was used to study the dynamics of three aqueous peptides having varied helicity. Experimental data were compared to the molecular dynamics simulated far-infrared absorbance spectrum derived from the dipole time correlation function. Vibrational density of state (VDOS) simulation was then used to analyze the contribution of different structural elements to the bands. Frozen aqueous peptide samples were studied in the frequency range between 325 and 540 cm^–1 where the ice absorbance is low. Three resonances were identified; band I centered at approximately 333 cm^–1, band II centered at approximately 380 cm^–1, and band III comprising two constituent bands at approximately 519 and 528 cm^–1. The peak height and frequency of the maximum absorbance of bands I and II varied depending on the helicity of the peptide. VDOS of the far-infrared absorbance spectrum confirmed that bands I and II were associated with the peptide backbone and that band III had both potential backbone and side chain components

Open state conformation of NS2B generated using GPS-Rosetta.

<p>(A) Scatter plot of 5,000 all-atom structures showing their combined score of weighted PCS + Rosetta energy versus the Cα RMSD of NS2B in the homology model built on the crystal structure 2FOM of the open conformation. The final selected structure (red point) has the lowest combined energy score and is referred to as the “open GPS-Rosetta structure”. The four next-lowest combined score structures are represented by blue points. (B) Superimposition of the best GPS-Rosetta structures onto the homology model. The open GPS-Rosetta structure is shown in red (NS2B) and grey (NS3pro) and the homology model in green (NS2B) and grey (NS3pro). The Cα RMSD of NS2B in the open GPS-Rosetta model is 2.7 Å relative to the homology model. The NS2B segments of the next four lowest-energy structures have RMSDs ranging from 2.7 to 3.1 Å and are displayed in different shades of blue. The superimposition shown in the figure used the Cα atoms of NS2B only. (C) Same as (A), except that scoring used PCSs only. (D) Same as (B), except using the structures with the lowest PCS scores identified in (C). In all five models, NS2B has a Cα RMSD between 2.7 and 3.6 Å relative to the NS2B part in the homology model.</p

Correlations between experimental and back-calculated PCSs fitted to the closed and open GPS-Rosetta conformations.

<p>To illustrate the information content associated with the PCSs, experimental PCSs measured for the closed state and PCSs generated for the open state (both reported as PCS_exp) were used to fit Δχ-tensors to either the closed or open GPS-Rosetta structures. Subsequently, the Δχ-tensors were used to back-calculate PCS_calc values. Data from Mutant B_C1, Mutant B_C2, Mutant C_C1, and Mutant C_C2 are represented in black, red, cyan, and blue, respectively. Q-factor calculations used the PCSs of backbone amide protons of NS2B only. The correlation plots were produced for all four possible combinations: (A) experimental PCSs for the closed state and closed GPS-Rosetta structure; (B) experimental PCSs of the closed state and open GPS-Rosetta structure; (C) PCSs generated for the open state and closed GPS-Rosetta structure; (D) PCSs generated for the open state and open GPS-Rosetta structure.</p

Crystal structures of DENV NS2B-NS3pro and overview of PCSs measured versus the amino acid sequence.

<p>(A) Open state as observed in the ligand-free conformation in the crystal structure 2FOM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref026" target="_blank">26</a>]. NS2B is shown in green and NS3pro is shown in grey. The orange balls identify the locations of residues 34 and 68. We refer to the mutants S34C and S68C as mutants B and C, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref023" target="_blank">23</a>]. To induce PCSs in the protein, the mutants B and C were reacted with a single lanthanide binding tag to form a disulfide bond with either of the cysteine residues at these sites. (B) Closed state as observed in the crystal structure 3U1I [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref022" target="_blank">22</a>] with the same color coding as in (A). The closed conformation is presumed to represent the enzymatically active state. (C) Summary of the experimental PCSs. C1 and C2 denote two different lanthanide binding tags used. They differ only in chirality [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref028" target="_blank">28</a>]. Open circles, filled circles, and boxes identify the residues for which PCSs were observed with Tb³⁺, Tm³⁺, or both Tb³⁺ and Tm³⁺, respectively. The residue numbering used is shown at the top of the amino acid sequence. In this numbering scheme, the mutants B and C are at residues 94 and 129 (highlighted in orange). The green and grey characters identify, respectively, the NS2B and NS3pro segments for which coordinates are reported in the crystal structures (PDB ID: 2FOM, 3U1I). Sequence segments for which no electron density was observed, are highlighted in blue. These parts are probably flexible [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref025" target="_blank">25</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127053#pone.0127053.ref027" target="_blank">27</a>].</p

Closed state conformation of NS2B determined by GPS-Rosetta.

<p>(A) Scatter plot of 5,000 all-atom structures showing their combined score of weighted PCS + Rosetta energy versus the Cα RMSD of NS2B relative to the crystal structure in the closed conformation (PDB ID 3U1I). The RMSD was calculated for NS2B only (residues 50–87 of chain A in 3U1I). The conformation selected as the best structure (red point) has the lowest combined energy score and is referred to as the “closed GPS-Rosetta structure”. The four next-lowest combined score structures are represented by blue points. (B) Comparison between GPS-Rosetta structures and the crystal structure. The closed GPS-Rosetta structure is shown in red (NS2B) and grey (NS3pro) and the crystal structure 3U1I is shown in green (NS2B) and grey (NS3pro). The Cα RMSD of NS2B in the closed GPS-Rosetta model is 1.0 Å relative to the crystal structure. The NS2B segments of the next four lowest-energy structures have RMSDs ranging from 0.7 to 2.2 Å and are displayed in different shades of blue. The superimposition shown in the figure used the Cα atoms of NS2B only. (C) Same as (A), except that scoring used PCSs only. (D) Same as (B), except using the structures with the lowest PCS scores identified in (C). In all five models, NS2B has a Cα RMSD between 0.8 and 1.6 Å relative to the NS2B part in the crystal structure.</p