Supporting Information

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1 Supporting Information Horne et al /pnas SI Materials and Methods Peptide Synthesis. Protected 3 -amino acids were purchased from PepTech. Cyclically constrained -residues, Fmoc-ACPC and Fmoc-APC(Boc), were prepared as previously described (1, 2). Peptides 1 7 were prepared on NovaSyn TGR resin by standard automated Fmoc solid phase peptide synthesis methods. / - Peptides 8 11 were prepared manually by microwave-assisted Fmoc solid phase peptide synthesis as previously described (3). Each peptide was cleaved from resin by treatment with 94:2.5:2.5:1 TFA/H 2 O/ethanedithiol/triisopropylsilane, precipitated by addition of cold ethyl ether, and purified by reversephase HPLC on a prep-c 18 column using gradients between 0.1% TFA in water and 0.1% TFA in acetonitrile. The identity and purity of the final products were confirmed by MALDI- TOF-MS and analytical HPLC, respectively. Stock solution concentrations were determined by UV absorbance (4). MALDI-TOF-MS (monoisotopic [M H], m/z): 1: Obsd , calc ; 2: Obsd , calc ; 3: Obsd , calc ; 4: Obsd , calc ; 5: Obsd , calc ; 6: Obsd , calc ; 7: Obsd , calc ; 8: Obsd , calc ; 9: Obsd , calc ; 10: Obsd , calc ; 11: Obsd , calc Synthesis of Fluorescein-Labeled CHR Peptide (Flu-C38). NovaSyn TGR resin (Novabiochem) bearing C38 peptide (WMEWDRE- INNYTSLIHSLIEESQNQQEKNEQELLELDK) was prepared by standard Fmoc solid phase peptide synthesis methods. 5-Carboxyfluorescein (28 mg, mmol) and HOBT H 2 O (11 mg, mmol) were dissolved in N-methyl-2-pyrrolidinone (0.75 ml). Diisopropylcarbodiimide (12 L, mmol) was added, and the resulting solution was transferred to the peptide-bearing resin. The reaction vessel was covered in foil and placed on a shaker overnight. The resin was washed with DMF (3 ), and the coupling reaction was repeated with fresh reagents. The resin was then washed with DMF (3 ), 20% piperidine (2 ) (5), DMF (3 ), CH 2 Cl 2 (3 ), and MeOH (3 ). The crude peptide was cleaved and purified as described above. Stock solutions, prepared in water, were quantified by visible absorbance ( ,000 M 1 cm 1 at ph 8.0). MALDI-TOF-MS (monoisotopic [M H], m/z): Obsd , calc Crystallization. Hanging drops were prepared by mixing 1 L of crystallization stock and 1 L of reservoir buffer followed by room temperature equilibration over a well containing 0.7 ml of buffer. Stock solutions of the 1 3 and 1 8 complexes were prepared by mixing concentrated stocks of the individual peptides in a 1:1 ratio to a final concentration of 2.2 mm total peptide in water. Crystals of 1 3 were obtained from a reservoir buffer consisting of 0.1 M Tris, ph 8.5, 1 M (NH 4 )H 2 PO 4. Crystals of the 1 8 complex were grown a reservoir buffer consisting of 0.4 M Li 2 SO 4 H 2 O, 12% vol/vol PEG 8000, 20% vol/vol glycerol. In our initial attempts to crystallize the 1 10 complex, a stock solution was prepared by mixing concentrated stocks of the individual peptides in a 1:1 ratio to a final concentration of 0.76 mm total peptide in water. Stocks of 1 10 prepared in this way were not fully soluble; however, the resulting viscous suspension yielded crystals of / -peptide 10 alone from a well buffer composed of 0.5 M ammonium sulfate, 0.1 M HEPES-Na, ph 7.5, 30% vol/vol 2-methyl-2,4-pentanediol. For subsequent crystallization trials of 1 10, the stock solution of the complex was prepared by refolding the 1:1 peptide mixture at 130 M total peptide in water followed by concentration to 1.1 mm by centrifugation at 4 C through a 10-kDa molecular weight cutoff membrane. Crystals of 1 10 were obtained from a stock prepared in this way and a reservoir buffer composed of 0.2 M NaCl, 0.1 M Tris, ph 8.5, 25% wt/vol PEG X-Ray Data Collection and Structure Determination. All crystals were flash-frozen in liquid nitrogen. Crystals of the 1 3 complex were briefly soaked in 0.08 M Tris, ph 8.5, 1.6 M (NH 4 )H 2 PO 4, 20% vol/vol glycerol before freezing. Crystals of 10 and 1 8 were frozen directly from the crystallization drop. Crystals of the 1 10 complex were soaked briefly in 0.2 M NaCl, 0.1 M Tris, ph 8.5, 25% wt/vol PEG 3350, 20% vol/vol glycerol before freezing. Diffraction data for the 1 3 and 1 8 complexes were collected on a Bruker X8 Proteum Diffractometer using Cu K radiation and were processed with the Bruker Proteum2 software package. Diffraction data for the crystals of 10 and the 1 10 complex were collected at the Life Sciences Collaborative Access Team beamline 21-ID-G at the Advanced Photon Source, Argonne National Laboratory, and were processed with HKL2000. Structure determination was carried out using the CCP4 software suite (6). Molecular replacement was carried out with Phaser (7) or Molrep (8). Refinement was accomplished by a combination of Refmac (9) for automated refinement, Coot (10) for manual model building, and ARP/wARP (11) for automated water building and free atom density modification. The structure of the 1 3 complex was solved using a search model derived from a published gp41 hexamer structure (PDB ID: 1AIK) (12). The structure of / -peptide 10 was solved using a CHR helix from the 1 3 complex as a search model. The structure of the 1 10 complex was solved using two search models, an NHR helix from the 1 3 complex and a CHR helix from the structure of / -peptide 10 alone. The structure of the 1 8 complex was solved using two search models, an NHR helix from the 1 3 complex, and a chimeric CHR helix prepared from the structures of 1 3 and Protease Stability. Stock solutions of peptides were prepared at a concentration of 25 M (based on UV absorbance) in TBS. A solution of proteinase K was prepared at a concentration of 50 g/ml (based on weight to volume) in TBS. For each proteolysis reaction, 40 L peptide stock was mixed with 10 L proteinase K stock. The reaction was allowed to proceed at room temperature and quenched at the desired time point by addition of 100 L 1% TFA in water. A portion of the resulting quenched reaction (125 L) was injected onto an analytical reverse-phase HPLC and run on a gradient between 0.1% TFA in water and 0.1% TFA in acetonitrile. The amount of starting peptide present quantified by integration of the peak at 220 nm. Duplicate reactions were run for each time point. Half-lives were determined by fitting time-dependent peptide concentration to an exponential decay using GraphPad Prism. Crude samples for some time points were analyzed by MALDI-MS, and the products observed were used to identify amide bonds cleaved in the course of the reaction. 1. Lee HS, LePlae PR, Porter EA, Gellman SH (2001) An efficient route to either enantiomer of orthogonally protected trans-3-aminopyrrolidine-4-carboxylic acid. J Org Chem 66: LePlae PR, Umezawa N, Lee HS, Gellman SH (2001) An efficient route to either enantiomer of trans-2-aminocyclopentanecarboxylic acid. J Org Chem 66: of8

2 3. Horne WS, Price JL, Gellman SH (2008) Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly. Proc Natl Acad Sci USA 105: Gill SC, Von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182: Fischer R, Mader O, Jung G, Brock R (2003) Extending the applicability of carboxyfluorescein in solid-phase synthesis. Bioconjugate Chem 14: Collaborative Computational Project, Number 4 (1994) The CCP4 suite: Programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ (2005) Likelihood-enhanced fast translation functions. Acta Crystallogr D Biol Crystallogr 61: Vagin A, Teplyakov A (1997) MOLREP: An automated program for molecular replacement. J Appl Crystallogr 30: Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: Lamzin VS, Wilson KS (1993) Automated refinement of protein models. Acta Crystallogr D Biol Crystallogr 49: Chan DC, Fass D, Berger JM, Kim PS (1997) Core structure of gp41 from the HIV envelope glycoprotein. Cell 89(2): of8

3 Fig. S1. (A D) Stereo views of electron density from the refined structures of 1 3 (A), 10 (B), 1 10 (C), and 1 8 (D). The maps depict a weighted 2Fo Fc electron density contoured at 1. 3 of 8

4 Fig. S2. Crystal structure of / -peptide 10 alone. (A) Cartoon representation of the helix bundle formed by 10 ( -residues are colored yellow, -residues are colored cyan). The three helices in the bundle are parallel but out of register relative to one another. (B) Overlay of the three helices from the asymmetric unit. Two of the three helices are straight (orange and white), whereas the third (purple) has two kinks, one near each terminus. (C and D) Packing of the kinked helix (shown as sticks) against the two straight helices (shown as surface). The two kinks appear to result from structural rearrangements necessary for a well-packed hydrophobic core. 4of8

5 Fig. S3. Inhibition of infection of TZM-bl cells by the indicated virus strain as a function of the concentration of gp41-derived fusion-blocking peptides. Each data point is the mean SEM from three independent experiments. 5of8

6 Fig. S4. Proteolytic stability of 3, 4, and 10. Solutions of 20 M peptide in TBS were incubated at room temperature with 10 g/ml proteinase K. (A) Time-dependent degradation data with curves resulting from fits to a simple exponential decay. (B) Proteolysis products observed by mass spectrometry at the indicated time point. Vertical lines indicate observation by MALDI-MS of one or both products consistent with hydrolysis of the backbone amide bond between the indicated residues. 6of8

7 Fig. S5. / -Peptides with different / 3 backbone patterns based on gp41 that showed little or no activity in the competition fluorescence polarization assay. 7of8

8 Table S1. X-ray data collection and refinement statistics 1 3 complex complex 1 8 complex Data collection Space group P C2 P H32 Cell dimensions a, b, c (Å) 37.6, 179.0, , 44.0, , 84.9, , 57.0, a,, ( ) 90, 90, 90 90, 105.4, 90 90, 90, 90 90, 90, 120 Resolution (Å) ( )* ( )* ( )* ( )* R sym (%) 5.0 (26.2) 6.7 (35.6) 6.1 (51.2) 5.8 (38.8) I/ I 28.0 (4.7) 16.1 (3.5) 31.6 (2.8) 16.8 (3.7) Completeness (%) 99.9 (100) 99.8 (99.8) 99.8 (98.2) 99.5 (100) Redundancy 8.6 (3.6) 3.5 (3.4) 7.8 (6.2) 5.9 (6.2) Refinement Resolution (Å) No. reflections 15,123 9,769 2,730 2,947 R work /R free (%) 20.9 / / / / 31.1 Avg. B factor (Å 2 ) RMSD Bond lengths (Å) Bond angles ( ) *Highest resolution shell is shown in parentheses. 8of8