Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient a-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.Keywords: intrinsically disordered protein (IDP); transient secondary structure; temperature dependence; polyproline II; circular dichroism (CD); nuclear magnetic resonance spectroscopy (NMR); sodium-proton (Na1/H1) exchanger 1 (NHE1); S-phase delayed 1 (Spd1); activator for thyroid hormone and retinoid receptors (ACTR) Abbreviations: ACTR, activator for thyroid hormone and retinoid receptors; CBP, CREB-binding protein; CD, circular dichroism; IDP, intrinsically disordered protein; NCBD, nuclear coactivator binding domain; NHE1cdt, sodium-proton (Na þ /H þ ) exchanger 1 C-terminal distal tail; NMR, nuclear magnetic resonance; PPII, polyproline II; RDC, residual dipolar coupling; SAXS, small-angle X-ray scattering; Spd1, S-phase delayed 1.

Galectins are defined by a conserved β-galactoside binding site that has been linked to many of their important functions in e.g. cell adhesion, signaling, and intracellular trafficking. Weak adjacent sites may enhance or decrease affinity for natural β-galactoside-containing glycoconjugates, but little is known about the biological role of this modulation of affinity (fine specificity). We have now produced 10 mutants of human galectin-3, with changes in these adjacent sites that have altered carbohydrate-binding fine specificity but that retain the basic β-galactoside binding activity as shown by glycan-array binding and a solution-based fluorescence anisotropy assay. Each mutant was also tested in two biological assays to provide a correlation between fine specificity and function. Galectin-3 R186S, which has selectively lost affinity for LacNAc, a disaccharide moiety commonly found on glycoprotein glycans, has lost the ability to activate neutrophil leukocytes and intracellular targeting into vesicles. K176L has increased affinity for β-galactosides substituted with GlcNAcβ1–3, as found in poly-N-acetyllactosaminoglycans, and increased potency to activate neutrophil leukocytes even though it has lost other aspects of galectin-3 fine specificity. G182A has altered carbohydrate-binding fine specificity and altered intracellular targeting into vesicles, a possible link to the intracellular galectin-3-mediated anti-apoptotic effect known to be lost by this mutant. Finally, the mutants have helped to define the differences in fine specificity shown by Xenopus, mouse, and human galectin-3 and, as such, the evidence for adaptive change during evolution.

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SummaryDss1 (also known as Sem1) is a conserved, intrinsically disordered protein with a remarkably broad functional diversity. It is a proteasome subunit but also associates with the BRCA2, RPA, Csn12-Thp1, and TREX-2 complexes. Accordingly, Dss1 functions in protein degradation, DNA repair, transcription, and mRNA export. Here in Schizosaccharomyces pombe, we expand its interactome further to include eIF3, the COP9 signalosome, and the mitotic septins. Within its intrinsically disordered ensemble, Dss1 forms a transiently populated C-terminal helix that dynamically interacts with and shields a central binding region. The helix interfered with the interaction to ATP-citrate lyase but was required for septin binding, and in strains lacking Dss1, ATP-citrate lyase solubility was reduced and septin rings were more persistent. Thus, even weak, transient interactions within Dss1 may dynamically rewire its interactome.

Intrinsic disorder is important for protein regulation, yet its role in regulation of ion transport proteins is essentially uninvestigated. The ubiquitous plasma membrane carrier protein Na(+)/H(+) Exchanger isoform 1 (NHE1) plays pivotal roles in cellular pH and volume homeostasis, and its dysfunction is implicated in several clinically important diseases. This study shows, for the first time for any carrier protein, that the distal part of the C-terminal intracellular tail (the cdt, residues V686-Q815) from human (h) NHE1 is intrinsically disordered. Further, we experimentally demonstrated the presence of a similar region of intrinsic disorder (ID) in NHE1 from the teleost fish Pleuronectes americanus (paNHE1), and bioinformatic analysis suggested ID to be conserved in the NHE1 family. The sequential variation in structure propensity as determined by NMR, but not the amplitude, was largely conserved between the h- and paNHE1cdt. This suggests that both proteins contain molecular recognition features (MoRFs), i.e., local, transiently formed structures within an ID region. The functional relevance of the most conserved MoRF was investigated by introducing a point mutation that significantly disrupted the putative binding feature. When this mutant NHE1 was expressed in full length NHE1 in AP1 cells, it exhibited impaired trafficking to the plasma membrane. This study demonstrated that the distal regulatory domain of NHE1 is intrinsically disordered yet contains conserved regions of transient structure. We suggest that normal NHE1 function depends on a protein recognition element within the ID region that may be linked to NHE1 trafficking via an acidic ER export motif.

Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na + /H + -exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer p Thr over p Ser, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of p T779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.

BackgroundExtracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding.Methods and resultsHere, we identify the human Na+/H+ exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D-domains and by two non-canonical F-sites located in the disordered intracellular tail of hNHE1, mutation of which reduced cellular hNHE1-ERK1/2 co-localization, as well as reduced cellular ERK1/2 activation. Time-resolved NMR spectroscopy revealed that ERK2 phosphorylated the disordered tail of hNHE1 at six sites in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner.ConclusionsThis work characterizes a new type of scaffolding complex, which we term a “shuffle complex”, between the disordered hNHE1-tail and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0252-7) contains supplementary material, which is available to authorized users.

Intrinsically disordered proteins (IDPs) are involved in many pivotal cellular processes including phosphorylation and signalling. The structural and functional effects of phosphorylation of IDPs remain poorly understood and difficult to predict. Thus, a need exists to identify motifs that confer phosphorylation-dependent perturbation of the local preferences for forming e.g. helical structures as well as motifs that do not. The disordered distal tail of the Na/H exchanger 1 (NHE1) is six-times phosphorylated (S693, S723, S726, S771, T779, S785) by the mitogen activated protein kinase 2 (MAPK1, ERK2). Using NMR spectroscopy, we found that two out of those six phosphorylation sites had a stabilizing effect on transient helices. One of these was further investigated by circular dichroism and NMR spectroscopy as well as by molecular dynamic simulations, which confirmed the stabilizing effect and resulted in the identification of a short linear motif for helix stabilisation: [S/T]-P-{3}-[R/K] where [S/T] is the phosphorylation-site. By analysing IDP and phosphorylation site databases we found that the motif is significantly enriched around known phosphorylation sites, supporting a potential wider-spread role in phosphorylation-mediated regulation of intrinsically disordered proteins. The identification of such motifs is important for understanding the molecular mechanism of cellular signalling, and is crucial for the development of predictors for the structural effect of phosphorylation; a tool of relevance for understanding disease-promoting mutations that for example interfere with signalling for instance through constitutive active and often cancer-promoting signalling.