Abstract
How cellular functions are regulated through protein phosphorylation events that promote or inhibit protein–protein interactions (PPIs) is key to understanding regulatory molecular mechanisms. Whilst phosphorylation can orthosterically or allosterically influence protein recognition, phospho-driven changes in the conformation of recognition motifs are less well explored. We recently discovered that clathrin heavy chain recognizes phosphorylated TACC3 through a helical motif that, in the unphosphorylated protein, is disordered. However, it was unclear whether and how phosphorylation could stabilize a helix in a broader context. In the current manuscript, we address this challenge using poly-Ala-based model peptides and a suite of circular dichroism and nuclear magnetic resonance spectroscopies. We show that phosphorylation of a Ser residue stabilizes the α-helix in the context of an Arg(i−3)pSeri Lys(i+4) triad through charge-reinforced side chain interactions with positive co-operativity, whilst phosphorylation of Thr induces an opposing response. This is significant as it may represent a general method for control of PPIs by phosphorylation; basic kinase-substrate motifs are common with 55 human protein kinases recognizing an Arg at a position −3 from the phosphorylated Ser, whilst the Arg(i−3)Seri Lys(i+4) is a motif found in over 2000 human proteins.
Highlights
We describe the effects of phosphorylation on helix stability using model poly-Ala based peptides, into which we grafted a motif inspired by studies on the Aurora A/TACC3/clathrin heavy chain (CHC) pathway, an unusual example of a protein–protein interaction centred on a phosphorylated helix.[22]
An interesting feature of the TACC3–CHC binding interface in the crystal structure is that it is does not exhibit the canonical recognition of phosphorylation, in which a cluster of basic residues on the binding partner clamps down on the phosphate group
The phosphorylated serine is positioned between two basic residues along one side of a helix in TACC3, and hydrophobic residues on another side of the helix line up to complement a groove on CHC
Summary
The determinants of α-helix stability are key in understanding the formation and strength of α-helix mediated protein-protein interactions (PPIs),[1] and in enabling peptide drugdiscovery.[2, 3] Prior studies established helix propensities of individual amino acids,[4, 5] the role of helix capping[6, 7] and effects of interaction between side chains,[8,9,10,11] the role of phosphorylation is less well explored. The phosphorylation state was altered, Arg and Lys side chains were systematically moved in and out of pSer registry, and the effect of substitution of Ser for Thr was studied (Scheme 1)
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