File SI - Binding of HIV-1 gp41-Directed Neutralizing and Non-Neutralizing Fragment Antibody Binding Domain (Fab) and Single Chain Variable Fragment (ScFv) Antibodies to the Ectodomain of gp41 in the Pre-Hairpin and Six-Helix Bundle Conformations

Five supporting figures. Figure S1. ITC analysis of Fab and ScFv binding to two pre-hairpin intermediate mimetics. Binding of (A) Fab8066 and (B) Fab8062 to the disulfide-linked trimer N35CCG-N13 in 50 mM sodium acetate pH 5.2 buffer (required to maintain the solubility of N35CCG-N13). 10 µM Fab was in the cell and N35CCG-N13 (∼100 µM in monomer) was the titrant. (C to F) Binding of ScFvs to 5-helix in 10 mM Tris-HCl, pH 7.6, 150 mM NaCl. 5-helix (5–7 µM) in the cell was titrated with the designated ScFv at a concentration of 50–70 µM. HIV-1 neutralizing antibodies are shown in the left-hand panels and non-neutralizing on the right-hand panels. Values of the equilibrium association constant (KA) are shown in each panel. Figure S2. Expression, purification and folding of functional ScFvs. Sc66 and Sc62 are derived from the broadly neutralizing Fab8066 and the non-neutralizing Fab8062, respectively [S1]. (A) Amino acid sequence of Sc66. Sc62 differs from Sc66 at 4 positions (displayed in green) within the CDR-H2 loop. In the ScFvs, the light (blue) and heavy (orange) variable regions are separated by the 15 residue spacer sequence 3×(GGGGS) that connects the C-terminus of the light chain variable domain to the N-terminus of the heavy chain variable domain. (B) 20% homogeneous SDS-PAGE of Sc66 under oxidizing (lanes O) and reducing (lanes R) conditions shows that complete disulfide bridge formation can be visualized by the faster migration of oxidized Sc66 (lanes O) relative to the reduced sample (lanes R) and can therefore be monitored during ScFv folding. (C) 20% homogeneous SDS-PAGE of Sc66, Sc62 and four Sc66 mutants containing point mutations within the CDR-H2. Numbering of the mutations corresponds to their position in the heavy chain variable sequence of Fab8066 [S2]. Numbers beside the marker lane (M) in both gel panels denote molecular weights in kDa. (D) The mass of Sc66 determined by SEC-MALS corresponds to a monomer (calculated mass, 25,540). Figure S3. Native- and SDS-PAGE of peak fractions from SEC-MALS analysis of 1∶1 mixtures of coreS-Fab8066 and coreS-Sc66 complexes. Gels shown in (A) and (B) correspond to elution profiles and masses shown for coreS-Fab8066 and coreS-Sc66 in Figs. 3A and B, respectively, in the main text. Numbering of lanes corresponds to elution volume. Figure S4. Native-PAGE of coreS-Sc66 single mutant complexes. No binding is observed with Sc66T56F, and relatively weak (1∶1) binding is seen with Sc66N58V. Shifts in the presence of mutants Sc66I53L and Sc66T57A closely resemble those for Sc66. CoreS and ScFv are color coded orange and blue, respectively. Calculated molecular weights of coreS, ScFv and their corresponding complexes are indicated in kDa. Figure S5. Raw CD data for Fab8066 and Sc66 complexed to the six-helix bundles coreSP and 6-helix. (A) coreSP+Fab8066; (B) coreSP+Sc66; (C) the C-HR peptide C34; (D) 6 helix+Sc66. (PDF)