Neighboring phosphoSer‐Pro motifs in the undefined domain of IRAK1 impart bivalent advantage for Pin1 binding

Abstract

The peptidyl prolyl isomerase Pin1 has two domains that are considered to be its binding (WW) and catalytic (PPIase) domains, both of which interact with phosphorylated Ser/Thr-Pro motifs. This shared specificity might influence substrate selection, since many known Pin1 substrates have multiple sequentially close phosphoSer/Thr-Pro motifs, including the protein IRAK1. The IRAK1 undefined domain (UD) contains two sets of such neighboring motifs (Ser131/Ser144 and Ser163/Ser173), suggesting possible bivalent interactions with Pin1. Using a series of NMR titrations with 15N-labeled full-length Pin1 (Pin1-FL), PPIase, or WW domain and phosphopeptides representing the Ser131/Ser144 and Ser163/Ser173 regions of IRAK1-UD, bivalent interactions were investigated. Binding studies using singly-phosphorylated peptides showed that individual motifs displayed weak affinities (>100 μM) for Pin1-FL and each isolated domain. Analysis of dually phosphorylated peptides binding to Pin1-FL showed that inclusion of bivalent states was necessary to fit the data. The resulting complex model and fitted parameters were applied to predict the impact of bivalent states at low micromolar concentrations, demonstrating significant affinity enhancement for both dually phosphorylated peptides (3.5 μM and 24 μM for peptides based on the Ser131/Ser144 and Ser163/Ser173 regions, respectively). The complementary technique biolayer interferometry confirmed the predicted affinity enhancement for a representative set of singly and dually phosphorylated Ser131/Ser144 peptides at low micromolar concentrations, validating model predictions. These studies provide novel insights regarding the complexity of interactions between Pin1 and activated IRAK1, and more broadly suggest that phosphorylation of neighboring Ser/Thr-Pro motifs in proteins might provide competitive advantage at cellular concentrations for engaging with Pin1.

A Noncanonical Binding Site in the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein Regulates Its Interactions with the Proline Rich Region of Zyxin

“…Therefore, the binding affinity was determined using a four-state model (free [ 15 N]EVH1, only primary site bound, only secondary site bound, and both primary and secondary sites bound) as previously described. 36 For all four peptides, this analysis yielded relatively weak affinities for the primary binding site and considerably weaker affinities for the secondary site (Table 1). These affinities are mostly weaker than the previously reported values for the same M1 P –M4 P peptides (74 μ M, 61 μ M, 197 μ M, and no binding observed, respectively) determined by Trp fluorescence spectroscopy and fitted to a two-state model (free and 1:1 bound).…”

“…Fitting of the binding data for six residues in the primary and six residues in the secondary binding surface to this model yields an apparent K d value of 32.2 ± 1.7 μ M (Figure 2E). The goodness of fit, evaluated by a χ 2 analysis as described in detail elsewhere, 36 yielded a reduced χ 2 value of 4.19. This χ 2 value is consistent with the two-state bivalent model being an oversimplification, as expected because the population distribution determined above indicates that multiple bound species are sampled.…”

“…Additionally, TITAN offers the flexibility of including personalized binding models. For the [ 15 N]EVH1 wild type titrated with individual motif peptides, we used the four-state binding model described in detail elsewhere 36 and fit 10 peak profiles in the primary binding site and six in the secondary binding site. Peaks included in these analyses were selected on the basis of trajectories that displayed maximum Δ δ values that exceeded the corresponding error in Δ δ by a factor of at least 10.…”

Pin1 facilitates isoproterenol‑induced cardiac fibrosis and collagen deposition by promoting oxidative stress and activating the MEK1/2‑ERK1/2 signal transduction pathway in rats

“…As MAPK family members, activation of the MAPK kinase (MEK)1/2-extracellular signal-regulated kinase (ERK)1/2 signaling pathway involves significant modifications in the emergence and development of the myocardial fibrotic process, by interacting with and phosphorylating protein kinases and transcription factors. It is known that protein phosphorylation is a critical process consisting of complex signaling cascades, which mediate protein structure and folding ( 8 , 9 ) and exert a profound effect on pathophysiological cardiac conditions.…”

An Alternative Pin1 Binding and Isomerization Site in the N-Terminus Domain of PSD-95

“…Thus, indicating that these sites are not sensitive to the configuration of this assay or that they do not undergo much cis-trans isomerization. Although singly phosphorylated S290 could be isomerized by Pin1, it is highly unlikely due to bivalency requirements for Pin1 binding/isomerization (Daum et al, 2007;Zhang et al, 2012;Eichner et al, 2016;Rogals et al, 2016). Furthermore, a mutant containing both sets of mutations (N3A/C2A) behaved just like the N3A-PSD-95 mutant ( Figures 4K,M, left bar graph).…”