Koide et al. 10.1073/pnas.0700149104. |
Fig. 6. Epitope mapping using NMR spectroscopy. HSQC spectra of [2H,13C,15N]-MBP in the absence (black) and presence (red) of the unlabeled MBP-74 monobody.
Fig. 7. The interaction network between the binding complex of MBP and the monobody MBP-74. Interface residues are defined as those with buried surface area ³5 Å2. The MBP residues are listed on the right side and the monobody residues on the left side. Intermolecular interactions are defined as distances £5.6 Å (the minimal distance cutoff that identifies at least one interaction partner for each interface residue) and are shown as lines. Only interactions involving the interface residues are shown. Interactions involving the BC, DE and FG loop residues are colored in blue, yellow and green, respectively, and those involving the monobody scaffold residues are in gray.
Fig. 8. Shotgun-scanning mutagenesis of the MBP-74 monobody. We mutated either six positions of the BC or nine positions of the FG loop of the shaved template using the KHT codon (K = G and T; H = A, C and T) and introduced the loop sequence of MBP-74. Note that the MBP-74 contains the template DE loop, and thus we did not diversify this loop. Resulting libraries were separated sorted using phage display either for binding to MBP ("binding selection"; A) or for binding to the V5 epitope tag ("display selection"; B). The latter selects for phages that display a monobody. The amino acid sequences of the two loop regions of recovered clones and their numbers of occurance are shown. Tyr is shaded yellow, Ser in red, and unchanged residue (V29) in gray. Because these libraries were constructed by introducing mutations in the "shaved" template, significant fractions of the starting libraries contain the template sequence (listed at the bottom of the sequence lists). No clones containing the template sequence in the FG loop were recovered from the binding selection.
Fig. 9. Paratopes of binding proteins with a Y/S-binary interface. (A) The paratope of the MBP-74 monobody. The solvent-accessible surfaces of atoms that are located within 5 Å of the binding partner (MBP) are shown. The surfaces for Tyr carbon atoms are shown in yellow. The surfaces for the other types of carbon atoms are in white, those for oxygen in red and those for nitrogen in blue. The monobody backbone is shown as a cartoon model. (B) The paratope of Fab-YSd1 (PDB ID code 1ZA3) (1). The paratope surfaces are shown in the same manner as in A except that the surfaces for the Tyr carbon atoms of CDR-H3 are shown in orange. (C) The amino acid compositions of the binding interfaces for the MBP-74 monobody/MBP complex (filled bars) and the Fab-YSd1/hDR5 complex. The upper panel show the buried surface areas for the paratopes plotted for different amino acid types, and the lower panel for the epitopes.
1. Fellouse FA, Li B, Compaan DM, Peden AA, Hymowitz SG, Sidhu SS (2005) J Mol Biol 348:1153-1162.
SI Methods
Phage Display Selection.
Phagemid particles were prepared by growing XL1-Blue cells transfected with the phagemid library in the presence of 0.2 mM IPTG and helper phage (1, 2). Three rounds of phagemid library selection were performed as follows. In the first round, 0.5 mM of a target protein that had been modified with EZ-Link Sulfo-NHS-SS-Biotin (Sulfosuccinimidyl 2(biotinamido)-ethyl-1,3-dithiopropionate; Pierce) was mixed with a sufficient amount of streptavidin-conjugated magnetic beads (Streptavidin MagneSphere Pramagnetic Particles; Promega, Z5481/2) in TBS (50 mM Tris HCl buffer pH 7.5 150 mM NaCl) containing 0.5% Tween20 (TBST). To this target solution, 1012-13 phagemids suspended in 1 ml TBST plus 0.5% BSA were added, and the solution was mixed and incubated for 15min at room temperature. After washing the beads twice with TBST, the beads suspension containing bound phagemids were added to fresh E. coli culture. Phagemids were amplified as described before (2). In the second round, phagemids were incubated with 0.1 mM target in TBST plus 0.5% BSA, and then captured by streptavidin-conjugated magnetic beads. Phagemids bound to the target protein were eluted from the beads by cleaving the linker within the biotinylation reagent with 100 mM DTT in TBST. The phagemids were washed and recovered as described above. After amplification, the third round of selection was performed using 0.02 mM target.Yeast Surface Display.
Yeast surface experiments were performed according to Boder and Wittrup with minor modifications (3). The Express-tag in the yeast display vector, pYD1, (Invitrogen) was removed because it cross-reacts with anti-FLAG antibodies (Sigma). The genes for monobodies in the phagemid library after three rounds of selection were amplified using PCR and mixed with the modified pYD1 cut with EcoRI and XhoI, and yeast EBY100 cells were transformed with this mixture. The transformed yeast cells were grown in the SD-CAA media at 30°C for two days, and then monobody expression was induced by growing the cells in the SG-CAA media at 30°C for 24 h.Sorting of monobody-displaying yeast cells were performed as follows. The yeast cells were incubated with a biotinylated target (50 nM) and mouse anti-V5 antibody (Sigma), then after washing incubated with anti-mouse antibody-FITC conjugate (Sigma) and neutravidin-PE conjugate (Invitrogen). The stained cells were sorted based on the FITC and PE intensities. Typically, cells exhibiting the top ~1% PE intensity and top 10% FITC intensity were recovered.
After FACS sorting, individual clones were analyzed. Approximate Kd values were determined from a titration curve by FACS analysis (3). Their amino acid sequences were deduced from DNA sequencing.
Effects of E. coli lysate on monobody-target interaction were tested by comparing the MBP binding in the presence and absence of E. coli lysate prepared from cell suspension with OD600 of 50.
Protein Expression and Purification.
The genes for ySUMO and hSUMO4 were respectively cloned in pHFT1. The hSUMO4 gene was a gift of Drs. Irina Dementieva and Steve Goldstein (University of Chicago). The genes for monobodies were cloned in the expression vector, pHFT2, which is a derivative of pHFT1 (4) in which the His-6 tag had been replaced with a His-10 tag. The MBP gene was also cloned in pHFT2. Protein expression and purification were performed as described previously (4).Uniformly 2H/13C/15N-enriched MBP was prepared by culturing E. coli BL21(DE3) cells harboring a pHFT2 derivative containing the MBP gene in the M9 media prepared in 2H2O containing 13C-glucose and 15N NH4Cl as sole carbon and nitrogen sources, respectively. Protein expression was induced by an addition of 1 mM IPTG. The protein was purified using a Ni-Sepharose column (Amersham Pharmacia). After cleaving the N-terminal tag sequence with TEV protease, the protein was concentrated and dissolved in 20 mM Na phosphate buffer containing 50 mM EDTA, pH 7.2.
Surface Plasmon Resonance Measurements.
A monobody containing a His-10 tag (50 nM) was immobilized on a NTA-chip (Biacore) in a BIAcore 2000 instrument, and binding of MBP (10-100 nM) was monitored. Sensorgrams were analyzed with the BIAEvaluation program.X-Ray Crystallography.
The MBP-monobody fusion protein was crystallized in 20% polyethyleneglycol-1000, 0.1 M Na/K phosphate buffer and 0.2 M NaCl, pH 6.5 using the sitting drop vapor diffusion method. Crystals were frozen in an 80% mixture of this solution combined with 20% glycerol as cryoprotectant.The x-ray diffraction data were collected at APS beamline 24-ID. (Advanced Photon Source at the Argonne National Laboratory). Crystal data and data collection statistics are summarized in Table 2, which is also published on the PNAS web site. x-ray diffraction data were processed and scaled with HKL2000 (5). The structure was determined by molecular replacement using multicopy search with two different models with the program MOLREP in CCP4 (6). The MBP structure (PDB ID code 1DBM) was used as a search model, along with the FN3 structure (PDB ID code 1FNA) (7). The rigid body refinement was carried out with CNS1.1 (8). The SigmaA-weighted 2Fobs-Fcalc and Fobs-Fcalc Fourier maps were calculated and examined. The engineered loops and linker were built at this stage. The model building was carried out using the Turbo-Frodo program (9). The simulated annealing and the search for water molecules were performed in CNS1.1. The TLS (Translation/Libration/Screw) and bulk solvent parameters, restrained temperature factor, and final positional refinement were completed with REFMAC5 (10). Molecular graphics were generated using PyMOL (www.pymol.org).
NMR Spectroscopy.
1H, 15N-HSQC and HNCO spectra of the free 2H/13C/15N-enriched MBP (0.3 mM) and those of a mixture of 2H/13C/15N-enriched MBP (92 mM) and the unlabeled MBP74 monobody (165 mM) were collected on a Varian (Palo Alto, CA) INOVA 600 NMR spectrometer using pulse sequences provided by the manufacturer. The HNCO resonances of the free MBP were in a good agreement with previously established assignments generously provided by Drs. Lewis Kay and Vitali Tugarinov (University of Toronto). Residues affected by monobody binding were identified by comparing the two HNCO spectra. We classified amide cross peaks into four categories: strongly affected, a peak that migrates more than two linewidths; weakly affected, a peak exhibiting a significantly reduced intensity at the same position as in the free spectrum, or a peak that has a corresponding peak in the complex spectrum to the vicinity (within two linewidths) of its original position in the free spectrum; not affected; and excluded from analysis, a peak that overlaps with another in the spectra (11, 12).1. Koide A, Koide S (2007) Methods Mol Biol 352:95-109.
2. Sidhu SS, Lowman HB, Cunningham BC, Wells JA (2000) Methods Enzymol 328:333-363.
3. Boder ET, Wittrup KD (2000) Methods Enzymol 328:430-444.
4. Huang J, Koide A, Nettle KW, Greene GL, Koide S (2006) Protein Expr Purif 47:348-354.
5. Otwinowski Z, Minor W (1997) Methods Enzymol 276:307-326.
6. Collaborative Computational Project. (1994) Acta Crystallogr D 50:760-763.
7. Dickinson CD, Veerapandian B, Dai X-P, Hamlin RC, Xuong N-H, Ruoslahti E, Ely KR (1994) J Mol Biol 236:1079-1092.
8. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, et al. (1998) Acta Crystallogr D 54:905-921.
9. Emsley P, Cowtan K (2004) Acta Crystallogr D 60:2126-2132.
10. Murshudov GN, Vagin AA, Dodson EJ (1997) Acta Crystallogr D 53:240-255.
11. Farmer BT, Constantine KL, Goldfarb V, Friedrichs MS, Wittekind M, Yanchunas J, Robertson JG, Mueller L (1996) Nat Struct Biol 3:995-997.
12. Huang X, Yang X, Luft B, Koide S (1998) J Mol Biol 281:61-67.