Structural insight into distinct mechanisms of protease inhibition by antibodies

Wu et al. 10.1073/pnas.0708251104.

Supporting Information

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SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Table 1
SI Figure 10
SI Table 2
SI Figure 11
SI Figure 12
SI Figure 13
SI Table 3

Fig. 6. CDR sequences of anti-HGFA antibodies Ab58 and Ab75. The residues are numbered according to the Kabat numbering system (1). The residues within the CDR sequences are indicated by black lines, and the residues chosen for diversification in the V_H libraries are shaded.

1. Wu TT, Kabat EA (1970) J. Exp. Med., 132: 211-250.

Fig. 7. Antibody specificity. In a direct binding ELISA, 96-well plates were coated with 2 mg/ml of HGFA, matriptase (1), urokinase (American Diagnostica), or factor XIIa (American Diagnostica) and incubated with 10 mg/ml of anti-HGFA antibodies in PTB buffer. After washing, bound antibodies were detected by addition of anti-human antibody HRP conjugate (diluted 1:2,500 in PTB buffer) and TMB substrate. Absorbance at 450 nm was measured on a microplate reader.

1. Kirchhofer D, Peek M, Li W, Stamos J, Eigenbrot C, Kadkhodayan S, Elliott JM, Corpuz RT, Lazarus RA, Moran P (2003) J Biol Chem 278:36341-36349.

Fig. 8. Scheme for equilibria of a partial (hyperbolic) competitive inhibition system. E, enzyme (HGFA); S, substrate (Spectrozyme FVIIa); I, inhibitor (Ab75); P, product (para-nitroanilide). K_s is the dissociation constant of the enzyme-substrate complex ([E] [S]/[ES]). K_i is the dissociation constant of the enzyme-inhibitor complex ([E] [I]/[EI]). k_p is the rate constant of the breakdown of ES to E + P. a is the factor by which K_s changes when I is bound to E. For Ab75, the determined value for a was 2.6 ± 0.4 (n = 3).

Fig. 9. The Fab58:HGFA complex from the 3.5-Å crystal structure. (A) Fab58 (heavy chain, teal; light chain, light blue) and HGFA protease domain (beige). The side chains of the catalytic triad residues His-57, Asp-102, and Ser-195 ("triad") are shown to indicate the position of the active site. Orange parts of HGFA are within 4 Å of the Fab58 heavy chain, and the blue part of HGFA is within 4 Å of the Fab58 light chain. Fab58 is orange (heavy chain) or yellow (light chain) when it is within 4 Å of HGFA. Residues that contact HGFA are restricted to CDRs H1, H2, H3, and L3 with the addition of a residue from a proximal non-CDR loop, Ser74H. The Fab58 epitope on HGFA is centered on Phe-97 at the distal edge of one side of the substrate-binding cleft, with significant contacts caused by CDRs H2 and H1 close approach to subsites S2 and S3, respectively. Nonrandomized CDR H1 residues Phe27H and Thr28H, plus randomized residues Thr30H, Ser32H, and Ala33H are within 4 Å of HGFA. (B) Paratope of Fab58 and epitope on HGFA as open-book representation colored the same as for A. The spots on the Fab58 surface indicate CDR residues and are colored as L1 (orange), L2 (beige), L3 (gray), H1 (yellow), H2 (green), and H3 (violet). Fab58 buried ≈890 Ų of solvent-accessible surface on each side of the interaction, 85% of which is attributable to the Fab58 heavy chain. Representative electron density appears in SI Fig. 13. Molecular figures were prepared by using PyMOL (Delano WL (2002) The PyMol Molecular Graphics System. Available at www.pymol.org.).

Fig. 10. The Fab75:HGFA complex from the 2.2-Å crystal structure. (A) Fab75 (heavy chain, dark blue; light chain, light blue) and HGFA serine protease domain (beige). Orange is used where the Fab75 heavy chain and HGFA are within 4 Å of each other, and yellow is used where the Fab75 light chain and HGFA are within 4 Å of each other. The side chains of the catalytic triad residues His-57, Asp-102, and Ser-195 ("triad") are shown to indicate the position of the active site. The Fab75 epitope on HGFA is centered on residues 87-101, comprising the 99-loop and the b-strand leading into it. There are additional distances less than 4 Å to residues in the 60-loop following the catalytic His-57 (Phe-59, Ser60b, Pro60c, and Arg-61), the last part of the 170-loop (Ser-177, Pro-178, and Asn-179), and the protease domain's C-terminal a-helix (Asn-233 and Arg-241). (B) Paratope of Fab75 and epitope on HGFA as open-book representation. The interacting surfaces, which are colored the same as in A, are shown after a 90° rotation. One HGFA residue, Thr-92, is within 4 Å of both the Fab75 chains and is shown in blue. The spots on the Fab75 surface indicate CDR residues and are colored as L1, orange; L2, beige; L3, gray; H1,yellow; H2, green; and H3, violet. All Fab75 CDRs except L2 are within 4 Å of HGFA and combine to bury ≈1,020 Ų of solvent-accessible surface on each side of the interaction, 75% of which is attributable to the Fab75 heavy chain. Representative electron density appears in SI Fig. 13.

Fig. 11. Active site region in Fab75:HGFA complex. The semitransparent surface of HGFA (beige) reveals small pockets used by substrate side chains during normal substrate processing. The yellow worm and side chains are from a neighboring molecule in the crystal, a crystal packing interaction. For comparison, a small section of KD1 from the KD1:HGFA X-ray structure (gray) is included. Note the close correspondence between P1 and P2 side chains (gray) of KD1 and the side chains of Pro-388' and Arg-390' (yellow) from the neighboring HGFA. The loops that help form the P2- and P3-binding pockets are labeled. It is on the opposite side of these loops that the Fab75 epitope begins. If our Fab75:HGFA structure includes Fab75-induced structural changes in HGFA suggestive of an allosteric influence on the enzyme active site, they are small ones. The rmsd of Ca atoms within b-strands from Fab75:HGFA and KD1:HGFA after their least-squares superposition is 0.21 Å for 71 pairs of atoms, and a similar superposition with the apo-HGFA structure yields an rmsd of 0.31 Å. Inspection of these superimposed structures for informative differences, after discounting regions subject to intermolecular contacts, leaves only a difference centered at the Ca of His60a of ≈1 Å. This distance is only 2-3 times the estimated error of atomic positions for the individual crystal structures. The 60-loop is part of the Fab75 epitope, is contacted by KD1 in the KD1:HGFA, and is not subject to any intermolecular contact in the apo-HGFA structure. The KD1 and apo structures align well here, whereas the main chain around His60a from the Fab75 complex is shifted toward the active site.

Fig. 12. Size exclusion chromatography of Fab58:HGFA and Fab75:HGFA complexes. The complexes were formed by mixing HGFA and Fabs at a 1:1.5 molar ratio in 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.01% Triton X-100 buffer and then analyzed on a Superdex-200 column (A₂₈₀ vs. elution volume in milliliters). (A) Reference proteins of known molecular weight. (B and C) Fab58:HGFA (B) and Fab75:HGFA (C) complexes. Peak fractions were analyzed by SDS/PAGE and are depicted below the elution profile. The peak elution volumes forFab58:HGFA and Fab75:HGFA complexes were 13.7 and 13.8 ml, respectively, indicating ≈80-kDa heterodimeric Fab:HGFA complexes at a 1:1 molar ratio. No higher order complexes were detected.

Fig. 13. Final 2Fo-Fc electron density maps contoured at 1 s. Backbone atoms are shown as worms, and side chains are colored as in SI Figs. 9 and 10. (A) 3.5-Å map from Fab58:HGFA where HGFA Phe-97 contacts Fab58. (B) 2.2-Å map from Fab75:HGFA where HGFA Leu-93 contacts Fab75, with red spheres for water atoms.

Table 1. Fab58-HGFA contacts

Table 2. Fab75-HGFA contacts

Table 3. Data collection and refinement

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