This review summarizes the influence of inserting constraints on biophysical, conformational, structural and cellular behaviour for peptides targeting α-helix mediated protein–protein interactions.

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 recognises 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)pSeriLys(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 recognising 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.

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 recognises 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 recognising an Arg at a position –3 from the phosphorylated Ser, whilst the Arg(i – 3)pSeri Lys(i + 4) is a motif found in over 2000 human proteins.