Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display

Protein Engineering, Design & Selection vol. 26 no. 4 pp. 307– 315, 2013 Published online February 1, 2013 doi:10.1093/protein/gzt003 Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display Seema Patel, Pascale Mathonet, Agnes M.Jaulent and Christopher G.Ullman1 Isogenica Ltd, Little Chesterford, Essex CB10 1XL, UK 1 Received August 29, 2012; revised January 4, 2013; accepted January 6, 2013 Edited by Dan Tawfik WW domains are small b-sheet motifs that are involved in intracellular signalling through the recognition of proline-rich or phosphorylated linear peptide sequences. Here, we describe modification of this motif to provide a framework for engineering the side chains exposed on its concave surface. This non-natural scaffold incorporates an additional tryptophan, has a shorter loop 1 and supports modification of 25% of the natural protein to form a novel affinity reagent. We demonstrate the utility of this structure by selecting a high-affinity binder to the extracellular region of human vascular endothelial growth factor receptor isoform 2 (VEGFR-2) from a library of modifications, using a cell-free molecular display platform, CIS display. The isolate has low nanomolar affinity to VEGFR-2 and inhibits binding of human VEGF to its receptor with nanomolar activity. The structure is amenable to cyclisation to improve its proteolytic stability and has advantages over larger protein scaffolds in that it can be synthesised chemically to high yields offering potential for therapeutic and non-therapeutic applications. Keywords: CIS display/in vitro display/WW domain/Pin1/ scaffold Introduction Protein domains are natural modulators of biological function through ligand binding. These domains may be engineered in order to produce altered activities and novel affinity reagents. Typically, engineering efforts focus upon modification or randomisation of the loops in globular structures. Antibody variable domains are the most widely studied affinity ligands, but other structures have been developed to obtain new and useful properties (Hoogenboom, 2005; Hosse et al., 2006). WW domains are small peptide domains, 40 amino acids in length, which bind linear peptide sequences and mediate intracellular protein – protein interactions (Staub and Rotin, 1996). The name ‘WW domain’ refers to a pair of conserved tryptophan (W) residues known to be essential for the formation of the structure. However, there are rare # The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: 307 To whom correspondence should be addressed. E-mail: exceptions to this rule, where a phenylalanine or a tyrosine residue substitutes one of the tryptophan residues. Since WW domains are relatively small structures, they have served as good models for understanding the folding energetics of small b-sheet proteins. Several structures of single or paired WW domains have been determined, with and without a bound ligand, and have been shown to adopt an anti-parallel three-stranded b-sheet (or ‘b-meander’) with a cup-shaped binding surface. Generally, these peptide ligands adopt an extended conformation in the binding groove of the WW domain in a manner similar to a rope being loosely held by the grip of a hand although, in one known structure, the peptide ligand is looped across the palm (Huang et al., 2000; Kanelis et al., 2001; Wintjens et al., 2001; Wiesner et al., 2002; Meiyappan et al., 2007). The binding specificity of the WW domain has led to the classification of these domains into five groups dependent upon the ligand sequence. group I domains recognise PPXY motifs; group II domains recognise PPLP motifs; group III domains bind proline-rich stretches containing methionine and glycine residues (PGM motif ); group IV domains bind phosphoserine and phosphothreonine residues; and group V domains bind stretches of proline that are rich in arginine (Macias et al., 2002). Recently, 42 WW domains have been studied by nuclear magnetic resonance spectroscopy and peptide library screens, leading to further classification of WW peptides into six groups based upon their recognition of proline, phosphoserine and threonine peptide motifs (Otte et al., 2003). Two WW domains from group I (Nedd4.3, hYAP) have been modified for altered ligand specificity (Dalby et al., 2000; Kasanov et al., 2001). hYAP has been displayed on the surface of phage particles to select improved binding to a known peptide ligand containing the consensus motif PPXY from a combinatorial library. In silico approaches have identified new group I WW domains for binding to peptide libraries derived from consensus motifs and identified the coevolving contact residues as L3P4G6E8I/V21D/N22H23T28. No group II or IV domains were identified that bound these peptide libraries (Russ et al., 2005; Socolich et al., 2005). The group IV WW domain, Pin1, has previously been mutated in order to modulate its binding and stability, and was shown to bind exclusively to phosphorylated serine/ threonine targets ( poS/poT), and not to phosphotyrosine peptides or non-phosphorylated target sequences. In fact, the tightest binding was recorded for poT peptides from the sequence wwpoTPP (where w is a hydrophobic residue and P is proline) (Jiang et al., 2001; Otte et al., 2003). Introduction of a single or a pair of tryptophan residues into the wild-type Pin1 sequence as well as substitution of its native loop with the sequence of loop1 from the FBP WW domain resulted in an improved thermostability. However, the binding affinities S.Patel et al. of these engineered Pin1 WW domains for their natural peptide ligands were reduced below detectable limits (Jäger et al., 2006, 2007, 2009). Despite the wealth of mutagenesis data available for WW domains, it has not been shown that the WW domain can be engineered to bind extracellular ligands. Here, we demonstrate that the Pin1 sequence can be used as a framework for engineering and can be displayed on an in vitro molecular display system, CIS display. Using this system, high-affinity, folded, stable, active peptides have been identified via selection against an extracellular protein. In addition, the selected WW domain has been chemically synthesised and modified to add further stability. This approach has potential for applications in therapy or as diagnostic tools or affinity reagents. DNA library construction CIS display library construction was performed as previously described (Odegrip et al., 2004; Eldridge et al., 2009). Library primers were designed to alter the appropriate sequence of the Pin1 WW domain. The tac-PinLib-RepACIS-ori polymerase chain reaction (PCR) construct was prepared as follows; the RepA-CIS-ori region was amplified by PCR from the R1 plasmid (GenBank accession no. V00351) using primers 1StepRepFor and M13rev. A second PCR was then performed on RepA-CIS (...truncated)