Anticooperativity in a Glu−Lys−Glu Salt Bridge Triplet in an Isolated α-Helical Peptide

Abstract

Phosphorylation is ubiquitous in control of protein activity, yet its effects on protein structure are poorly understood. Here we investigate the effect of serine phosphorylation in the interior of an α-helix when a salt bridge is present between the phosphate group and a positively charged side chain (in this case lysine) at i,i + 4 spacing. The stabilization of the helix is considerable and can overcome the intrinsically low preference of phosphoserine for the interior of the helix. The effect is pH dependent, as both the lysine and phosphate groups are titratable, and so calculations are given for several charge combinations. These results, with our previous work, highlight the different, context-dependent effects of phosphorylation in the α-helix. The interaction between the phosphate 2− group and the lysine side chain is the strongest yet recorded in helix-coil studies. The results are of interest both in de novo design of peptides and in understanding the structural modes of control by phosphorylation.Phosphorylation and dephosphorylation have long been known as control mechanisms for various processes in biochemistry, and roughly one-third of all proteins in eukaryotes are estimated to undergo reversible phosphorylation (1). Since the middle of the last century it has been known that protein activity can be controlled by the addition or removal of a phosphate group (2-5). The control of gene expression, macromolecule production, and cellular proliferation have since been shown to be orchestrated through multiple intracellular signal transduction pathways. These pathways, or protein kinase cascades, propagate signals received at the plasma membrane to the interior of the cell through a series of phosphorylation-controlled events.The mechanisms of action of phosphorylation are, however, still relatively poorly understood at the structural level, though many studies of individual systems now show some conformational differences between proteins and synthetic peptides before and after phosphorylation (see, e.g., refs 6-13). Previous work from our own group (14) and others (15-17) has shown that phosphorylation at the N-terminus stabilizes α-helices but also that the effect is position dependent. When interior serine residues are phosphorylated, the effect is greatly destabilizing, but at the three N-terminal positions it is greatly stabilizing (phosphoserine at the N2 position in a helix is the most stabilizing interaction yet found at this position). More recent work has suggested that protein phosphorylation sites, especially those for serine and threonine, are predominantly disordered prior to phosphorylation and that phosphorylation sites resemble natively unstructured proteins in terms of their charge, hydrophobicity, and amino acid composition. Some of the exceptions to these sites may be crystallization artifacts, and in others it may be that the substrate undergoes an order- † This work was funded by The Wellcome Trust (award reference 065106). Europe PMC Funders Author ManuscriptsEurope PMC ...

Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

“…7). Other simultaneous salt bridges (X → E(−4) & E(+4) and X → E(−3) & E(+3), for X = K or R) were not significantly populated, in agreement with the experimental finding of non-cooperativity in an alanine-based peptide with a K → E(−4) & E(+4) triplet pattern18. Glu residues are also able to form simultaneous salt bridges with two Lys or Arg partners (Supplementary Table S4).…”

“…Single E–K or E–R pairs of this type are known to promote the folding of monomeric α-helices in short (<20 residue) alanine-based peptides1516, and thus are assumed to stabilize long SAHs. Studies on short peptides have additionally suggested that K → E(+4) pairs are more helix-stabilizing than the reversed E → K(+4) orientation17, resolving previous conflicting reports161819. However, the equivalent study for E–R pairs is lacking.…”

Amino acid pair- and triplet-wise groupings in the interior of α-helical segments in proteins

“…It has been known for some time that particular sequences of amino acids may have significant roles in protein folding and stability (Nagano, 1973;Nagano, 1974) and, more recent studies have shown how these patterns in pairs in β-sheets, (Fooks et al, 2006) loop sequences, (Crasto and Feng, 2001) particular (i, i + 4) pairs with helix-stabilizing effects (Andrew et al, 2001;Andrew et al, 2002;Butterfield et al, 2002) and dipole-affecting triplets (Iqbalsyah and Doig, 2005a;Olson et al, 2001) may have a stabilizing or destabilizing influence.…”

“…In particular for α-helices, these effects are consequence of several types of interactions between amino acid side chains -including aromatic interactions, (Butterfield et al, 2002;Meurisse et al, 2004;Thomas et al, 2002a;Thomas et al, 2002b) nonpolar/polar interactions, (Andrew et al, 2001) surface salt bridges (Iqbalsyah and Doig, 2005a;Olson et al, 2001) -and may be dependent on the relative positioning of the intervening side-chains. There is also the possibility of interactions between amino acid residue side chains and the α-helix backbone.…”

“…(Iqbalsyah and Doig, 2005a) (FIGURE 1) Figure 1. Amino acid Triplets considered at the interior of α-helices:…”