Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α

Neira, José L.; Palomino‐Schätzlein, Martina; Ricci, Caterina; Ortore, María Grazia; Rizzuti, Bruno; Iovanna, Juan

doi:10.1016/j.bbapap.2019.07.005

Cited by 10 publications

(13 citation statements)

References 103 publications

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“…We acquired SAXS experiments to confirm that wild-type NUPR1 at these conditions (50 mM Tris buffer, pH 7.2) showed the same conformational features as those found previously under different conditions (50 mM acetic buffer, 50 mM, pH 4.5) (Neira et al, 2019). SAXS experiments were performed on a Rigaku 3-pinhole PSAXS-L equipment operating at 45 kV and 0.88 mA, and with samples at 25°C in Tris buffer (50 mM, pH 7.2) with a NUPR1 concentration of 4.3 mg/ml (∼460 μM).…”

Section: Small Angle X-ray Scattering (Saxs) and Dlssupporting

confidence: 70%

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

Bonucci

Palomino‐Schätzlein

Molina

et al. 2021

Front. Mol. Biosci.

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The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.

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Section: Small Angle X-ray Scattering (Saxs) and Dlssupporting

confidence: 70%

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

Bonucci

Palomino‐Schätzlein

Molina

et al. 2021

Front. Mol. Biosci.

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Section: Methodsmentioning

confidence: 99%

“…The HSQC spectra of double-labeled HcTnI-C27 were acquired as described ( 61 ) at 14.1 T with 40 μM free peptide or 20 μM of double-labeled peptide and 104 μM (protomer) of αTm in the binding buffer. Other experimental details are as described previously ( 61 ). The 1 H carrier was at the water frequency, which was removed by using the WATERGATE sequence ( 58 ); the spectral width in this dimension was 9 ppm.…”

Section: Methodsmentioning

confidence: 99%

The muscle-relaxing C-terminal peptide from troponin I populates a nascent helix, facilitating binding to tropomyosin with a potent therapeutic effect

Hornos

Feng

Rizzuti

et al. 2021

Journal of Biological Chemistry

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The conserved C-terminal end segment of troponin I (TnI) plays a critical role in regulating muscle relaxation. This function is retained in the isolated C-terminal 27 amino acid peptide (residues 184–210) of human cardiac TnI (HcTnI-C27): When added to skinned muscle fibers, HcTnI-C27 reduces the Ca 2+ -sensitivity of activated myofibrils and facilitates relaxation without decreasing the maximum force production. However, the underlying mechanism of HcTnI-C27 function is unknown. We studied the conformational preferences of HcTnI-C27 and a myopathic mutant, Arg192His, (HcTnI-C27-H). Both peptides were mainly disordered in aqueous solution with a nascent helix involving residues from Trp191 to Ile195, as shown by NMR analysis and molecular dynamics simulations. The population of nascent helix was smaller in HcTnI-C27-H than in HcTnI-C27, as shown by circular dichroism (CD) titrations. Fluorescence and isothermal titration calorimetry (ITC) showed that both peptides bound tropomyosin (αTm), with a detectably higher affinity (∼10 μM) of HcTnI-C27 than that of HcTnI-C27-H (∼15 μM), consistent with an impaired Ca 2+ -desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to αTm, HcTnI-C27 acquired a weakly stable helix-like conformation involving residues near Trp191, as shown by transferred nuclear Overhauser effect spectroscopy and hydrogen/deuterium exchange experiments. With the potent Ca 2+ -desensitization effect of HcTnI-C27 on skinned cardiac muscle from a mouse model of hypertrophic cardiomyopathy, the data support that the C-terminal end domain of TnI can function as an isolated peptide with the intrinsic capacity of binding tropomyosin, providing a promising therapeutic approach to selectively improve diastolic function of the heart.

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“…Upon binding to their partners, intrinsically disordered proteins span a continuum in the extent of structuredness, from fully folded to partially ordered to fully disordered. The "extreme" fuzzy complexes in the latter case have been characterized when the partners are another disordered protein or a nucleic acid [1][2][3][4][5] . A third class of partners for disordered proteins comprise membranes [6][7][8][9] .…”

Section: Introductionmentioning

confidence: 99%

“…Many disordered proteins are enriched in charged residues 11 , and interactions between opposite charged residues are crucial features of extreme fuzzy complexes between disordered proteins 1,2 . Likewise the interactions between basic residues of proteins and acidic phosphate groups of nucleic acids are crucial for their high-affinity, fuzzy association [3][4][5] . The inner leaflet of the plasma membrane is highly acidic, due to asymmetric distribution of charged lipids including phosphatidylserine, phosphatidylinositol, and the latter's phosphorylated variants 12 , and thus forms a target for polybasic proteins including signaling molecules 7 .…”

Section: Introductionmentioning

confidence: 99%

Extreme Fuzzy Association of an Intrinsically Disordered Protein with Acidic Membranes

Hicks

Escobar

Cross

et al. 2020

Preprint

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Many physiological and pathophysiological processes, including Mycobacterium tuberculosis (Mtb) cell division, may involve fuzzy membrane association by proteins via intrinsically disordered regions. The fuzziness is extreme when the conformation and pose of the bound protein and the composition of the proximal lipids are all highly dynamic. Here we tackled the challenge in characterizing the extreme fuzzy membrane association of the disordered, cytoplasmic N-terminal region (NT) of ChiZ, an Mtb divisome protein, by combining solution and solid-state NMR spectroscopy and molecular dynamics simulations. In a typical pose, NT is anchored to acidic membranes by Arg residues in the midsection. Competition for Arg interactions between lipids and acidic residues, all in the first half of NT, makes the second half more prominent in membrane association. This asymmetry is accentuated by membrane tethering of the downstream transmembrane helix. These insights into sequence-interaction relations may serve as a paradigm for understanding fuzzy membrane association.

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Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α

Cited by 10 publications

References 103 publications

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

The muscle-relaxing C-terminal peptide from troponin I populates a nascent helix, facilitating binding to tropomyosin with a potent therapeutic effect

Extreme Fuzzy Association of an Intrinsically Disordered Protein with Acidic Membranes

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