1. Volume 25, Issue 3, Pages 395-406 (March 2017)
Mapping Protein Binding Sites and Conformational Epitopes Using Cysteine Labeling and Yeast Surface Display 
Tariq Ahmad Najar, Shruti Khare, Rajesh Pandey, Satish K. Gupta, Raghavan Varadarajan 
Structure 
Volume 25, Issue 3, Pages (March 2017)
DOI: /j.str
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2. Structure 2017 25, 395-406DOI: (10.1016/j.str.2016.12.016)
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3. Figure 1 Schematic Outline of the Epitope Mapping Approach
The gene encoding the protein antigen (P) having a cysteine mutation (shown as a red star) in the antibody/ligand binding site (shown in purple) is displayed as a fusion protein on the surface of a yeast cell (A). The antibody binding to the displayed protein is monitored by flow cytometry (B). The cysteine on the displayed protein is labeled with biotin-PEG2-maleimide (shown as a green crescent) (C). If cysteine is a part of the antibody/ligand binding site, then the label will prevent the binding of antibody/ligand to the displayed protein and no binding signal will be observed (D). The c-myc tag was removed from the expression cassette as it interfered with binding of GyrA-14 fragment to the displayed CcdB protein. Surface expression of CcdB WT and cysteine mutants was monitored by binding of fluorescently labeled anti-HA antibody and GyrA having a C-terminal FLAG tag as described in Supplemental Experimental Procedures and Figure S3.
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4. Figure 2
Heat Map Summarizing the Binding Data for GyrA-14 and 26 Mouse mAbs to a Panel of 21 Chemically Masked Single Cysteine Mutants and WT Protein by Flow Cytometry
The yeast cells displaying single cysteine mutants were first labeled with biotin-PEG2-maleimide. Subsequently, loss in binding of the corresponding mAb or GyrA-14 to each labeled cysteine mutant was monitored individually by flow cytometry. Binding was quantitated in terms of mean fluorescence intensity (MFI) and was normalized with respect to the corresponding MFI for surface expression probed with anti-HA antibody. Dark blue indicates complete loss in mAb or GyrA-14 binding to the respective labeled cysteine mutant while green to red indicates little to no effect on mAb binding. The binding of mAbs and GyrA-14 to the displayed WT antigen is shown in the last column. Most mAbs (except MA-2003, -2007, -2024, and -2025) bound to the wild-type antigen at very dilute concentration (50 nM). MA-2008, -2019, -2020, and showed very weak binding to the displayed wild-type antigen. MA-2001 binding was completely lost when residues at position 49, 92, 96, and 100 were mutated to cysteine and subsequently masked by cysteine-specific label, while intermediate effects were observed with residues at 23, 26, and 88. Similar results were observed with MA-2002 (residues 23, 88, 92, and 100), MA-2026, MA-2027 (residues 23, 49, 92, 96, and 100), and GyrA-14 (23, 26, 88, 92, 96, and 100). mAb concentration in culture supernatants (by ProteOn XPR36) and binding to the purified, immobilized antigen (CcdB; by ELISA) were determined as described in Supplemental Experimental Procedures and Figures S1 and S2.
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5. Figure 3
Chemical Labeling of Exposed and Buried Residues with Biotin-PEG2-Maleimide
Labeling was carried out in the absence (A) and in the presence (B) of denaturant, and labeling was monitored as a function of GyrA-14 binding by flow cytometry. No effect was observed for binding of GyrA-14 to either WT or R15C mutant following labeling under either native or denaturing conditions. However, the buried residue V18C is labeled only under denaturing conditions and fails to refold back to its native conformation. Hence, upon labeling, V18C loses binding to GyrA-14. Error bars represents SEs of two replicate experiments.
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6. Figure 4
Design of a Panel of Single Cysteine Mutants and Conformational Epitope Mapping
The amino acid residues whose side chains were exposed (≥20% accessibility and ≤5 Å depth) were individually mutated to Cys so as to probe the entire surface of the antigen (CcdB protein) for conformational epitope mapping studies (Table S1).
(A) Surface and ribbon diagram of antigen (CcdB protein); light and dark gray colors show the two monomers of CcdB protein, and the green highlighted regions show the positions of residues, along with the amino acid sequence, that were mutated to cysteine for epitope mapping. Twenty-one residues, distributed all over the surface of the folded protein, were selected and individually mutated to cysteine, displayed on the yeast cell surface, chemically masked by biotin-PEG2-maleimide, and subsequently probed with mAbs or GyrA-14 for epitope mapping. The highlighted colored regions represent the location of residues that form the corresponding antibody or GyrA-14 binding site (epitope). The dotted circles represent the residues that were found important for GyrA-14 binding by our method highlighted on the CcdB structure (B) and compared with the crystal structure data of the CcdB:GyrA14 complex (C) and saturation mutagenesis data (Adkar et al., 2012) (D). Similarly encircled are the residues that form a conformational epitope for MA-2001 (E), MA-2002 (F), and linear epitope for other mAbs (MA-2005, -2009, -2010, -2011, -2012, -2014, -2015, -2016, -2017, -2018, -2022, and -2023) (G) (see Figure S4).
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7. Figure 5
Binding of Conformation-Specific Antibody to Cysteine Mutants Labeled under Native or Denaturing Conditions
Exposed (R15C) and buried (V18C) single cysteine mutants and wild-type (WT) protein displayed on the yeast cells were labeled under native (A) and denaturing (B) conditions; subsequently, antibody binding under native conditions was examined by flow cytometry. Labeling of the buried V18C residue is expected to disrupt the protein structure. When labeling is carried out under native conditions, both mutants retain binding to all three mAbs, suggesting that no labeling of the buried V18C has occurred. When labeling is carried out under denaturing conditions, WT and R15C, which is at an exposed non-epitopic site, retain mAb binding. However, labeled V18C selectively loses binding to the conformation-specific mAbs MA-2001 and MA-2002 but retains binding to MA-2010, which recognizes a linear epitope. Error bars represents SEs of two replicate experiments.
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8. Figure 6
FACS Sort of Labeled Mutant Pool Followed by Deep Sequencing to Determine the Epitopes of Four mAbs, MA-2001, -2002, -2010, and -2018, and Ligand GyrA-14
(A–F) A pooled library of 21 single cysteine mutants of CcdB expressed on the yeast cell surface was labeled with cysteine-specific label (biotin-PEG2-maleimide) and subsequently probed with (A) the mAb MA-2001 plus anti-HA, (B) MA-2002 plus anti-HA, (C) GyrA-14 plus anti-HA, (D) MA-2010 plus anti-HA, and (E) MA-2018 plus anti-HA. After probing with appropriate fluorescent secondary antibodies, positive (orange) and negative (magenta) cells were sorted using FACS. In the case of GyrA-14, we also sorted cells that showed partial loss of binding (green) (C). The mutant library labeled only with anti-HA (F) was used to probe relative surface expression of all the mutants and was used for normalization of surface expression. Residues important for binding of MA-2001, -2002, -2010, and GyrA are determined by DNA sequencing (Sanger sequencing; Table S2) of about 30 clones from each sorted population. Deep sequencing results are plotted as mutant frequency (y axis) of a particular cysteine mutant (x axis) that lost binding (“–” values) or retained binding (“+” values), after labeling, to GyrA-14 (G) or mAb (H) (z axis).
(G) Partial (GyrA+/−) to complete (GyrA−) loss of binding was observed when residues at positions 23, 26, 88, 92, 96, and 100 were mutated to Cys and subsequently labeled. However, no change in binding to GyrA-14 (GyrA+) was observed with other Cys mutants.
(H) Similarly, loss of binding of MA-2001 (pink bars) and MA-2002 (blue bars) was observed when residues at positions 23, 49, 92, 96, 100), and (23, 88, 92, 100) respectively, were mutated to cysteine and subsequently labeled. For MA-2010 (green bars) and MA-2018 (red bars), binding was not affected for most mutants (except 55 and 58; plus value). Both MA-2001 and target the gyrase binding site on CcdB and thus compete with GyrA as described in the Supplemental Experimental Procedure and Figure S5.
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9. Figure 7
Rabbit Polyclonal Sera Binding to Displayed, Chemically Masked Single Cysteine Mutants by Flow Cytometry and to Overlapping Peptides by ELISA
(A) Individual Cys mutants were displayed on the yeast surface and labeled with biotin-PEG2-maleimide. Binding of polyclonal serum was quantitated in terms of mean fluorescence intensity (MFI) and was normalized with respect to the corresponding MFI for surface expression probed with anti-HA antibody. Asterisks on color bars indicate the decrease in polyclonal sera binding to the corresponding cysteine mutant after labeling compared with wild-type protein (green bar).
(B) The immuno-dominant regions targeted by rabbit polyclonal sera are also highlighted in the crystal structure of CcdB protein.
(C and D) Sixteen biotinylated overlapping peptides of CcdB were captured onto a streptavidin-coated plate, and 50 μL of rabbit polyclonal sera was added at 1:1,000 dilutions to each well. The wells were probed with alkaline-phosphatase-conjugated goat anti-rabbit antibody at 1:10,000 dilutions and subsequently developed by adding 100 μL of chromogenic substrate, p-nitrophenyl phosphate. The optical density was measured at 405 nm. Peptides -04, -10, and -16 showed strong binding to the polyclonal sera (blue, red, and purple bars) while other peptides (black bars) showed very weak binding. Full-length protein (WT) was used as a positive control (green bar). Rabbit polyclonal sera target the gyrase binding site on CcdB and thus compete with GyrA as well as with MA-2001 and as described in the Supplemental Experimental Procedure and Figure S6. Error bars represents SEs of two replicate experiments.
Structure  , DOI: ( /j.str )
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