Photon Factory Activity Report 2014 #32 (2015) B

AR-NW12A/2012G116

Structure of the artificial protein AF.p17 developed by adaptive assembly
Hideki Watanabe and Shinya Honda*
National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba 305-8566, Japan
1 Introduction
Protein engineering that exploits known functional
peptides holds great promise for generating novel
functional proteins. We have proposed a combinatorial
approach termed adaptive assembly that provides a tailormade protein scaffold for a given functional peptide [1]. A
combinatorial library was designed to create a tailor-madescaffold which was generated from β-hairpins derived
from a 10-residue minimal protein “chignolin” andrandomized amino acid segments extended at the N- andC-terminus of a functional peptide. We applied adaptive
assembly to a peptide with low-affinity for the Fc regionof human immunoglobulin G, generating a 54-residue
protein AFFinger p17 (AF.p17) with a 40,600-foldenhanced affinity. Here, we report on the crystal structureof AF.p17 complexed with the Fc region, and discuss theenhancement mechanism at the atomic level.
2 Experiment
The complex of AF.p17 and the Fc region was dialyzed
against 20 mM Tris-HCl (pH 7.4) containing 10%
dimethyl sulfoxide (DMSO), and then concentrated to 10mg/ml. A crystal of the complex was obtained by using
sitting drop vapor diffusion under 40% polyethylene
glycol 4000, 0.1 M sodium citrate (pH 5.6), 0.2 M
ammonium acetate. The diffraction data were collectedunder cryogenic conditions (100 K) at Photon FactoryNW-12 and processed by HKL-2000. The crystal
structures were determined by molecular replacement(Molrep) in the CCP4 suite by using a search model(PDB code, 1DN2), and refined by using CNS,REFMAC5 and Coot. The atomic coordinates andstructural factors have been deposited in the Protein Data
Bank (code 3WKN).
3 Results and Discussion
The crystal structure of AF.p17 complexed with the Fcregion was determined at 2.9 Å resolution [1]. The
asymmetric unit contained the four complexes. The
overall structure of AF.p17 consisted of four β-strands,
three β-hairpins, a loop-like region, and a short α-helix(Figure 1). The C- and N-terminal extended segments
adopted different conformations. The C-terminal segment
formed a short α-helix with protruding side chains that
directly contacted with the binding region (Figure 1a). We
term this α-helix a “tail-helix-latch” that worked as alatch to support the active conformation of the bindingregion. Particularly, the hydrophobic side-chain of Met48
in the tail-helix-latch stabilized the conformation of the
Trp30 aromatic ring. The side-chain of Trp45 contactedwith Met48, resulting in a small hydrophobic cluster of
Trp30, Met43, and Trp45. Compared to the C-terminal
segment, the N-terminal segment formed a loop-likestructure that contacted with non-functional residues inthe two β-hairpin corresponding to the base of the binding
region (Figure 1b). This indicates that the N-terminal
segment supported the overall structure of the binding
region, in contrast to the C-terminal segment that directly
fixed the active conformation of the binding region. To
evaluate the effects of these residues on binding function,
we performed alanine scanning mutagenesis. All of thealanine-substituted mutants showed decreases in affinity.
In particular, Trp45Ala and Met48Ala resulted in morethan 100-fold decreases in affinity, indicating that theseresidues critically impacted the binding function by
supporting the functional segment.
In conclusion, adaptive assembly provided a functional
peptide with a tailor-made protein scaffold. We propose
adaptive assembly as a useful methodology for functional
enhancement of a given functional peptide.

Fig. 1: The crystal structure of AF.p17. (a) The Cterminal segment formed the tail-helix-latch (red) tosupport the conformation of the binding region (green).
(b) The N-terminal segment formed a loop-like structure(blue) to support the base of the binding region.
References[1] H. Watanabe et al., Chemistry & Biology, in press(2015).
* s.honda@aist.go.jp