Staple W100 (H) H3. Electron density maps of region C of the CDR-H3 loop in the 4E10 Fab SAH-MPER (671

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1 4E10/MPER ( KKK) 4E10/SAH-MPER ( KKK) (q) 4E10/SAH-MPER ( KKK) (q)pser W100B (H) Da-Gly-pSer Tether C-term L100C (H) P4 G100A (H) W100 (H) H3 G99 (H) T98 (H) N-term Supplementary Figure 1 Electron density maps of region C of the CDR-H3 loop in the 4E10 Fa SAH-MPER ( KKK)(q) structure and comparison of the 4E10 Fa SAH-MPER ( KKK) (q), 4E10 Fa SAH- MPER ( KKK) (q)pser and 4E10 Fa MPER ( KKK) (PDB 2FX7) complexes. (a) 2Fo-Fc electron density map (1α level) for residues C of the flexile CDR-H3 region of 4E10. () View of the of the variale domain of the superposed 4E10 Fa/SAH-MPER ( KKK) (q), 4E10 Fa/SAH-MPER ( KKK) (q)pser and 4E10 Fa/MPER ( KKK) (PDB 2FX7) complexes. 1Cardoso, R.M., Brunel, F.M., Ferguson, S., Zwick, M., Burton, D.R., Dawson, P.E. & Wilson, I.A. Structural asis of enhanced inding of extended and helically constrained peptide epitopes of the roadly neutralizing HIV-1 antiody 4E10. J Mol Biol 365, (2007).

2 * H N NWFNITNZLWXI*KKK SAH-MPER ( KKK) (q)pser (Da683-Gly-pSer) NWFNITNZLWXIKK*K SAH-MPER ( KKK) (q)pser (Da685-Gly-pSer) Da683 Gly K683 HN K685 L-pSer Da685 NH NH 2 H P H K685 Da683 Gly L-pSer Gly Da685 L-pSer K683 Relative asorance Compound Da683-Gly-pSer Da685-Gly-pSer IC 50, nm % CI 22 to to 83 Supplementary Figure Competitor peptide concentration (M) Design and 4E10 inding activity of SAH-MPER ( KKK) (q) containing a phosphate tether. (a) To engage the identified phosphate inding site at the 4E10 interface, a phosphate tether comprised of Da-Gly-pSer (10-atom linker) was attached at position 683 of SAH-MPER ( KKK) (q). As a control, the Da-Gly-pSer tether was alternatively attached at position 685 to orient the phosphate group in the opposite direction from the inding interface. Z and X represent R3 and S5, respectively, in the staple (red ar aove sequences). () SAH-MPER ( KKK) (q) earing the phosphate tether at position 683 manifested a 1.5-fold improvement in competitive 4E10 inding compared to the corresponding construct lacking the phosphate tether (Fig. 2c). Locating the tether at position 685, away from the inding interface, resulted in an approximate 1.5- and 2.5-fold decrease in inding compared to the parent SAH-MPER ( KKK) (q) construct and that earing the phosphate tether at position 683, respectively. Error ars, s.e.m. (n = 8 inding assay replicates). CI, confidence interval.

3 c K683 Da-Gly-pSer Tether Supplementary Figure 3 Interpretation of the electron density maps of the staple region of SAH-MPER ( KKK) (q)pser and comparison of the SAH-MPER ( KKK) (q) and phosphate-derivatized SAH-MPER ( KKK) (q)pser peptides. (a) Positive density contoured at 3σ level (green) for the staple region in the initial difference Fourier Fo-Fc map. The staple of the final refined model is shown in the picture as red sticks. () 2Fo-Fc electron density (1σ level) for the staple region of the SAH-MPER ( KKK) (q)pser in the refined model. (c) Superposition of the SAH-MPER ( KKK) (q)pser (rown) with SAH-MPER ( KKK) (q) (gray) otained y superposing all Cα atoms of the respective Fa/peptide complexes.

4 Alanine Da-Gly-pSer Tether c Da-Gly-pSer Tether d Da-Gly-pSer Tether P4 Supplementary Figure 4 Interpretation of the electron density maps of the tethered phosphate moiety and comparison of the phospho-inding-site region of the SAH-MPER ( KKK) (q) and SAH-MPER ( KKK) (q)pser peptides. (a) Initial 2Fo-Fc (1σ level) and positive difference Fourier Fo-Fc (3σ level) electron density maps for the region of the Da-Gly-pSer tether are shown in lack and green, respectively. An alanine residue was initially used at the site of the expected tether. () Final 2Fo-Fc (1σ level) and Fo-Fc (3σ level for positive, green; -3σ for negative, red) electron density maps for the refined Da-Gly-pSer moiety. As can e oserved, there is neither positive nor negative density in the difference Fourier map at the proximity of the tether. (c) Final 2Fo-Fc electron density map of the refined Da-Gly-pSer residue contoured at 0.8σ level shows almost complete coverage of the tether. (d) Comparison of the Da-GlypSer tether inding site (rown) and the phosphate inding site (gray) shows 4E10 residues in a very similar orientation. The hydrogen ond interactions are shown as dashed lines.

5 1.3 Chymotrypsin, ph 7 Fraction intact Compound Time (min) Half-life, min Fold enhancement MPER ( KKK) NWFNITNWLWYIKKKK 20 1 SAH-MPER ( KKK) (q) NWFNITNZLWXIKKKK 38 2 Supplementary Figure 5 Comparative chymotrypsin proteolysis of MPER ( KKK) and SAH-MPER ( KKK) (q) peptides. Exposure of SAH-MPER ( KKK) constructs to chymotrypsin followed y LC/MS-ased monitoring of intact full-length peptide revealed relatively rapid degradation profiles. Insertion of the single (i, i+3) q staple produced a modest 2-fold prolongation of half-life compared to the corresponding unmodified peptide. Cleavage at those sites within or immediately adjacent to the staple was eliminated y the allhydrocaron constraint, and the kinetics of proteolysis was slowed at the N-terminal sites as a result of induced structure. Error ars, s.e.m. (n = 3 proteolysis reaction replicates). Green residues, theoretical chymotrypsin sites; orange arrowheads, oserved chymotrypsin cleavage; Z and X represent R3 and S5, respectively, in the staple (red ar aove sequence).

6 SAH-MPER ( KKK) (q) ELDKWASLWNWFNITNZLWXIKKKK Half-life, min. 8 SAH-MPER ( KKK) (D,q) ELDXWASXWNWFNITNZLWXIKKKK x 10 6 NITNZLWXIKKKK 2.0 x 10 7 NWFNITNZLWXIKKKK Ion aundance 6.0 x x x 10 6 Ion aundance 1.5 x x x Time (min) Time (min) Supplementary Figure 6 Comparative proteolytic-cleavage kinetics for discrete chymotryptic sites within SAH-MPER ( KKK) (q) and SAH-MPER ( KKK) (D,q) peptides. LC/MS analysis revealed rapid detection of two proteolytic fragments of the SAH-MPER ( KKK) (q) construct, whereas the production of these chymotryptic fragments was either markedly slowed (left) or completely eliminated (right) y doule stapling. Green residues, theoretical chymotrypsin sites; orange arrowheads, oserved chymotrypsin cleavage; Z and X represent R3 and S5, respectively, in the staple (red ar aove sequences).

7 MPER(RRR RRR) RRRNEQELLELDKWASLWNWFDITNWLWYIRRRR SAH-MPER ( KKK) (B,q) EXDKWXSLWNWFNITNZLWXIKKKK Supplementary Figure 7 Electron density map of SAH-MPER ( KKK) (B,q) and comparison of the 10E8-ound structures of MPER (RRR RRR) and SAH-MPER ( KKK) (B,q) peptides. (a) 2Fo-Fc electron density map (1σ level) of the antiody-ound SAH-MPER ( KKK) (B,q) peptide. () Superimposition of the visualized regions of unmodified MPER (RRR RRR) (green) and SAH- MPER ( KKK)(B,q) (gray) peptides highlights the areas of similarity at the antiody-inding surface (ottom). Z and X represent R3 and S5, respectively, in the staple (red ar aove sequence).