Engineering a monomeric miniature protein.

The stability, size, and structure of avian pancreatic polypeptide (aPP) makes it a useful starting point for the design of miniature proteins that bind with high affinity and specificity to DNA1-4 and proteins5-14 and inhibit their interactions, both with high affinity and specificity, both in vitro1-6,8,9,11-14 and in mammalian cells and extracts.7,10 The utility of these molecules in a cellular context could be diminished, however, by self-association: aPP forms a dimer at 10-6 M concentration,15,16 a property shared by some, but not all, aPP-based miniature proteins.8,9,17-19 Interestingly, two miniature proteins, p007, which binds DNA,2,4 and pGolemi, which binds EVH1 domains,7 remain monomeric and well-folded at 10-4 M concentration despite primary sequence differences that exceed 50% (Figure 1). This observation suggests that it should be possible to identify aPP variants that are both monomeric and well-folded. Here we systematically isolate, characterize, and remove two structural elements responsible for aPP dimerization and install a new elementsa proline switchsthat single-handedly repacks aPP’s signature fold. The result is a monomeric and well-folded miniature proteins that can serve as a starting point for the in Vitro and in ViVo applications of these molecules. The structures of pancreatic fold proteins (PP) are composed of an N-terminal type II polyproline helix that folds upon a C-terminal R-helix to generate a stable, well-packed hydrophobic core.21-24 The first evidence of aPP self-association was the X-ray structure, which revealed an antiparallel dimer (Figure 1A).20 Molecular sieve chromatography indicated that formation of the aPP dimer was pHand temperature-dependent, with an equilibrium dissociation constant (Kd) between 400 pM and 5 μM.15,16 However, not all PPfold proteins self-associate; the NMR structure of peptide YY (PYY), an aPP ortholog, shows an aPP-like hydrophobic core but no evidence of dimerization.22,23,25 Subsequent sedimentation equilibrium experiments confirmed that PYY remains monomeric in the micromolar concentration range, forming a dimer only at a very high concentration (Kd ) 21 mM).21 Visual inspection of the aPP structure identifies three potentially stabilizing interactions at the dimer interface (Figure 1A). An intermolecular π-stacking interaction between Y7 side chains is evident (Figure 1B), with the orientation of each Y7 side chain defined by an intramolecular edge-to-face interaction with F20 (Figure 1B); an intermolecular π-stacking interaction between Y21 side chains is also observed (Figure 1C). Comparison of the sequences of natural and designed PP-fold miniature proteins reveals that all self-associating molecules contain tyrosine at position 7, whereas the nonassociating pGolemi, p007, and PYY do not (Figure 1D). The identities of the side chains at positions 20 and 21, however, do not correlate with self-association; most miniature proteins (including pGolemi) contain phenylalanine at position 20, and the residue at position 21 varies widely across miniature proteins in the PP family. To determine the extent to which these residues contribute to aPP dimer stability, we prepared aPP variants containing alanine in place of each of the residues present at the dimer interface (aPPY7A, aPPF20A, aPPY21A) and characterized them using circular dichroism (CD) and analytical ultracentrifugation (AU). aPPY7A and aPPF20A both assemble into tetramers, with Kd values of 3.9 × 10-12 and 7.6 × 10-11 M3, respectively. Because of the stoichiometry of self-association, however, aPPY7A and aPPF20A are tetrameric only at very high concentration: at 30 μM more than 97% of the molecules remain monomeric. By contrast, aPP, with Kd ) 4.1 × 10-6 M, exists predominantly (>94%) in the dimer state at 30 μM. Although less prone to self-association than aPP, as monomers neither aPPY7A nor aPPF20A assembled into the hairpin fold that characterizes PP-fold proteins, as judged by minimal negative ellipticity at 222 nm and a shift in the 208 nm minima to 205 nm (Figure 2A). By contrast, aPPY21A formed a modestly more stable dimer than did aPP, as judged by analytical ultracentrifugation (Kd ) 1.3 × 10-6 M) and CD (16 500 and 14 700 deg‚cm2‚dmol-1 at 208 and 222 nm, respectively). These data suggest that Y7 and F20 of aPP contribute to both dimer stability and maintenance of the characteristic aPP fold.26 Y21, although positioned at the dimer interface in the X-ray structure, contributes modestly to dimer stability. Figure 1. (A) Ribbon diagram of the aPP dimer20 highlighting pairs of Y7 (red, pink), F20 (teal, light teal), and Y21 (blue, light blue) side chains. (B) Close-up of intermolecular network comprising Y7 and F20 from both monomers. (C) Close-up of intermolecular π-stacking interaction between Y21 side chains. In panels B and C, equivalent residues on each monomer of the aPP dimer are distinguished by black and white residue labels. (D) Alignment of natural and designed PP-fold proteins and variants prepared as part of this work. Residues that differ from wt aPP are shaded; those that differ between p007 and pGolemi are starred. Published on Web 08/18/2007