Design of a Protein Kinase-Inducible Domain

Abstract

Protein phosphorylation is a critical regulatory strategy. New tools are necessary which may be used to interrogate and are responsive to the activities of protein kinases and phosphatases. We have used protein design to develop a protein motif, termed a protein kinase-inducible domain, whose structure is dependent on its phosphorylation state. Based on an EF hand calcium-binding loop, the key design element is the replacement of a structurally critical Glu residue, which binds metal in a bidentate manner, with a serine residue, which is expected to bind metal tightly when phosphorylated but poorly when not phosphorylated. The design comprises an EF hand consensus sequence, a tryptophan at residue 7 to sensitize lanthanide luminescence, and the recognition sequence of a serine/threonine kinase. Designed peptides, which contain minimal substrate recognition motifs of the protein kinases PKA, PKC, or the MAP kinase Erk, form complexes with Tb3+ when phosphorylated, showing strong Tb3+ luminescence emission at 544 nm, but show weak luminescence when not phosphorylated. The change in fluorescence on phosphorylation is comparable to or greater than that observed in described kinase sensors. Site-specific lanthanide binding was confirmed by NMR with diamagnetic and paramagnetic metals. The kinase-inducible domain peptides comprise an expressible sequence, potentially enabling their use as genetically encoded tags of protein kinase activity. The motif is general and potentially applicable to the majority of serine/threonine kinases.

Application of Metal Coordination Chemistry To Explore and Manipulate Cell Biology

“…Our lab has shown that the phosphorylation state of a peptide can dramatically increase its affinity for metal ions 221,222. As a consequence, Tb 3+ can be an effective reporter of phosphorylation status 221,223. In these cases, the peptides are short sequences resembling calcium-binding loops where the phosphorylated amino acid is either embedded within the peptide, as in DPDNEA(pY)EMPSEEG,221 or as the anchoring metal-ligating residue near the end of the peptide, as in DKNADGWIDRA(pS)LA,223 where pY and pS are phosphotyrosine and phosphoserine, respectively.…”

Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

“…[3][4][5][6] The incorporation of lanthanides within designed proteins provides a functional handle to potentially introduce long-lifetime fluorescence, luminescence resonance energy transfer over large distances, orientation in magnetic fields, and hydrolytic activity into proteins. [7][8][9][10][11] Although lanthanides are not known to exist natively within proteins, lanthanides have long been used as spectroscopic probes of structure and function in calcium-binding proteins; lanthanides bind to calcium-binding sites in proteins due to similarities in metallic ionic radii and hardness. [7,8,12,13] EF-hand proteins represent the smallest and most broadly utilized natural calcium-binding proteins, comprising a 70 amino-acid motif that binds two calcium ions.…”

Controlling Peptide Folding with Repulsive Interactions between Phosphorylated Amino Acids and Tryptophan

“…Phopshorylated residues can also induce structural changes via metal binding 15–18. However, to our knowledge, no one has investigated whether phosphorylation can result in a repulsive interaction with a hydrophobic or aromatic group that could affect peptide structure.…”