Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

Abstract

We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state-specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.intrabodies | X-ray crystallography

Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing

“…In addition to the wide array of endogenous proteins that can be targeted with newly designed ubiquibodies, the existing R4-uAb construct described here could be used in conjunction with ␤-gal protein trapping (38) to silence virtually any ␤-galtagged protein expressed from its endogenous loci in cultured cells and whole organisms. Furthermore, because ubiquibodies operate at the post-translational level and their DBP domains can be created to bind specific protein conformations or posttranslational modifications such as phosphorylation (39), the ubiquibody technique has the potential for depleting certain protein isoforms while sparing others. This is significant because post-translational modification-specific silencing of proteins is not possible using current knockdown methods that function at the level of DNA or RNA.…”

Delivery of antibodies to the cytosol

“…[6][7][8] Various solutions for this problem have recently been provided by the use of various alternative scaffolds which fold correctly in the cytosol. [9][10][11][12] Correct folding of antibodies is assured only in the endoplasmic reticulum (ER). Consequently, there are many examples where ER-retained antibodies have been used to generate antigen knockdowns, [13][14][15] but this method is restricted to membrane proteins and secreted targets.…”

Knowledge-Based Design of a Biosensor to Quantify Localized ERK Activation in Living Cells

“…The reagentless fluorescent DARPin (RFD) biosensors described here are based on DARPin pE59, which has been selected by ribosome display to bind selectively to phosphorylated ERK2 (pERK2) (Kummer et al, 2012). pE59 reliably detects pERK, but does not bind to any other closely related member of the MAPK family in either phosphorylated or non-phosphorylated form, as shown both in vitro and in cellular assays (Kummer et al, 2012).…”

“…DARPins consist of 33 amino acid long, consecutive homologous structural modules with fixed framework and variable potential interaction residues, which stack together to form elongated protein domains (Binz et al, 2003). Specific high-affinity binders derived from DARPin libraries can be generated against virtually any protein antigen by in vitro selections (Binz et al, 2004; Boersma and Plückthun, 2011; Kawe et al, 2006; Zahnd et al, 2006) and can serve as basis for the design of biosensors using fluorescence readouts, such as BRET (Kummer et al, 2012) or via the attachment of environmentally sensitive dyes (Brient-Litzler et al, 2010). Importantly, the defined interaction surface and the uniformity of the DARPin scaffold simplify the sensor design through knowledge-guided attachment of fluorophores, thus minimizing previously required extensive optimization steps in order to yield functional biosensors (Brient-Litzler et al, 2010; Miranda et al, 2011; Nalbant et al, 2004).…”