α-Helix stabilization by co-operative side chain charge-reinforced interactions to phosphoserine in a basic kinase-substrate motif

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 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.

Exploring the dynamics and interactions of the N-myc transactivation domain through solution NMR

“…Ac-PGEDIWKKFELLPTPPL-NH 2 (N-myc 45–61, MBI); Ac-EPPSWVTEMLLENELWG-NH 2 (N-myc 73–89, AIH); Ac-GFSAREKLERAVSEKLQ-NH 2 (N-myc 119–135, MBII+); Ac-GFSAAAKLVSEKLASYQ-NH 2 (c-myc 137–153, MBII+). Peptides were initially dissolved to give 0.5–1.0 mM stock solutions in buffer composed of 1 mM sodium borate, 1 mM sodium citrate, 1 mM (Na/H) 3 PO 4 and 10 mM NaCl, pH 7 [64, 65]. For measurements, samples were further diluted in buffer to concentrations of 10–100 μM.…”

“…Background spectra were recorded using buffer alone, buffer/15% TFE or buffer/30% TFE, and background ellipticity values were subtracted from raw sample ellipticity values ( θ ( λ )) to calculate MREs using: where M w is the peptide molecular weight, and n is the number of total peptide bonds, in each case taken to be 17 accounting for the acetyl capping group. Helicity values were estimated using the following equation: with MRE 222 in units of deg cm 2 dmol −1 res −1 [64, 66].…”

Mechanisms and influencing factors of peptide hydrogel formation and biomedicine applications of hydrogels

“…Batchelor et al 40 and Raza et al 41 studied the effects of pH on hydrogels. The results showed that different pH values resulted in the hydrogel formation of a-helix peptide Ac-AAQAAARQApSAAQKAY-NH 2 and the b-sheet peptide FER-8 (FEFEFRFK), respectively.…”

How phosphorylation impacts intrinsically disordered proteins and their function

“…The effects on helix populations from phosphorylation of an IDP are typically dual. Either the helix structure is stabilized when phosphorylation occurs in N-terminus facilitated by favorable electrostatic interactions and destabilized when positioned in the C-terminus, likely due to interference with the helix dipole (Figure 2A) [55,[60][61][62]. Although the underlying structural mechanism is not clear, the presence of a phosphoryl group close to a proline can impact the relative populations of cis and trans isomers [24,63], leading to switched binding capacities and therefore function.…”