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15 pags., 8 figs., 3 tabs., The phosphotransferase system (PTS), a metabolic pathway formed by five proteins, modulates the use of sugars in bacteria. The second protein in the chain is the histidine phosphocarrier, HPr, with the binding site at His15. The HPr kinase/phosphorylase (HPrK/P), involved in the bacterial use of carbon sources, phosphorylates HPr at Ser46, and it binds at its binding site. The regulator of sigma D protein (Rsd) also binds to HPr at His15. We have designed fragments of HPr, growing from its N-terminus and containing the His15. In this work, we obtained three fragments, HPr38, HPr58 and HPr70, comprising the first thirty-eight, fifty-eight and seventy residues of HPr, respectively. All fragments were mainly disordered, with evidence of a weak native-like, helical population around the binding site, as shown by fluorescence, far-ultraviolet circular dichroism, size exclusion chromatography and nuclear magnetic resonance. Although HPr38, HPr58 and HPr70 were disordered, they could bind to: (i) the N-terminal domain of first protein of the PTS, EIN; (ii) Rsd; and, (iii) HPrK/P, as shown by fluorescence and biolayer interferometry (BLI). The association constants for each protein to any of the fragments were in the low micromolar range, within the same range than those measured in the binding of HPr to each protein. Then, although acquisition of stable, native-like secondary and tertiary structures occurred at the last residues of the polypeptide, the ability to bind protein partners happened much earlier in the growing chain. Binding was related to the presence of the native-like structure around His15., This research has been funded by la Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana, Valencian Regional Government) [CIAICO 2021/0135 to JLN]. Some of the NMR equipment used in this work has been funded by Generalitat Valenciana (Spain) and co-financed with ERDF funds (OP ERDF of Generalitat Valenciana (Spain) 2014–2020). The funders did not have any role during acquisition and analyses of experiments or in the publication of this manuscript.

The oncoprotein Myc is a transcription factor regulating global gene expression and modulating cell proliferation, apoptosis, and metabolism. Myc has a nuclear localization sequence (NLS) comprising residues Pro320 to Asp328, to allow for nuclear translocation. We designed a peptide comprising such region and the flanking residues (Ala310-Asn339), NLS-Myc, to study, in vitro and in silico, the ability to bind importin alpha 3 (Imp alpha 3) and its truncated species (Delta Imp alpha 3) depleted of the importin binding domain (IBB), by using fluorescence, circular dichroism (CD), biolayer interferometry (BLI), nuclear magnetic resonance (NMR), and molecular simulations. NLS-Myc interacted with both importin species, with affinity constants of similar to 0.5 mu M (for Imp alpha 3) and similar to 60 nM (for Delta Imp alpha 3), as measured by BLI. The molecular simulations predicted that the anchoring of NLS-Myc took place in the major binding site of Imp alpha 3 for the NLS of cargo proteins. Besides clarifying the conformational behavior of the isolated NLS of Myc in solution, our results identified some unique properties in the binding of this localization sequence to the nuclear carrier Imp alpha 3, such as a difference in the kinetics of its release mechanism depending on the presence or absence of the IBB domain.

Plakophilin 1 (PKP1), a member of the armadillo repeat family of proteins, is a key structural component of cell-cell adhesion scaffolds, although it can also be found in other cell locations, including the cytoplasm and the nucleus. PADI4 (peptidyl-arginine deiminase 4) is one of the human isoforms of a family of enzymes engaged in the conversion of arginine to citrulline, and is present in monocytes, macrophages, granulocytes, and in several types of cancer cells. It is the only family member observed both within the nucleus and the cytoplasm under ordinary conditions. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with PADI4, by using several biophysical methods, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), isothermal titration calorimetry (ITC), and molecular simulations; furthermore, binding was also tested by Western-blot (WB) analyses. Our results show that there was binding between the two proteins, with a dissociation constant in the low micromolar range (∼ 1 μM). Molecular modelling provided additional information on the possible structure of the binding complex, and especially on the binding hot-spot predicted for PADI4. This is the first time that the interaction between these two proteins has been described and studied. Our findings could be of importance to understand the development of tumors, where PKP1 and PADI4 are involved. Moreover, our findings pave the way to describe the formation of neutrophil extracellular traps (NETs), whose construction is modulated by PADI4, and which mediate the proteolysis of cell-cell junctions where PKP1 intervenes.

Plakophilin 1 (PKP1), a member of the armadillo repeat family of proteins, is a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. However, PKP1 can be also found in the nucleus of several cells. NUPR1 is an intrinsically disordered protein (IDP) that localizes throughout the whole cell, and intervenes in the development and progression of several cancers. In this work, we studied the binding between PKP1 and NUPR1 by using several in vitro biophysical techniques and in cellulo approaches. The interaction occurred with an affinity in the low micromolar range (~10 μM), and involved the participation of at least one of the tryptophan residues of PKP1 (as shown by fluorescence and molecular docking). The binding region of NUPR1, mapped by NMR and molecular modelling, was a polypeptide patch at the 30s region of its sequence. The association between PKP1 and NUPR1 also occurred in cellulo and was localized in the nucleus, as tested by protein ligation assays (PLAs). We hypothesize that NUPR1 plays an active role in carcinogenesis modulating the function of PKP1.

PADI4 is a peptidyl-arginine deiminase (PADI) involved in the conversion of arginine to citrulline. PADI4 is present in macrophages, monocytes, granulocytes, and several cancer cells. It is the only PADI family member observed within both the nucleus and the cytoplasm. PADI4 has a predicted nuclear localization sequence (NLS) comprising residues Pro56 to Ser83, to allow for nuclear translocation. Recent predictors also suggest that the region Arg495 to Ile526 is a possible NLS. To understand how PADI4 is involved in cancer, we studied the ability of intact PADI4 to bind importin α3 (Impα3), a nuclear transport factor that plays tumor-promoting roles in several cancers, and its truncated species (ΔImpα3) without the importin-binding domain (IBB), by using fluorescence, circular dichroism (CD), and isothermal titration calorimetry (ITC). Furthermore, the binding of two peptides, encompassing the first and the second NLS regions, was also studied using the same methods and molecular docking simulations. PADI4 interacted with both importin species, with affinity constants of ~1–5 µM. The isolated peptides also interacted with both importins. The molecular simulations predict that the anchoring of both peptides takes place in the major binding site of Impα3 for the NLS of cargo proteins. These findings suggest that both NLS regions were essentially responsible for the binding of PADI4 to the two importin species. Our data are discussed within the framework of a cell mechanism of nuclear transport that is crucial in cancer.

PADI4 (protein-arginine deiminase, also known as protein l-arginine iminohydrolase) is one of the human isoforms of a family of Ca

International audience; Chondroitin sulfate (CS) glycosaminoglycans are biologically active sulfated polysaccharides that pose an analytical challenge for their structural analysis and functional evaluation. In this study, we developed a hydrophilic interaction liquid chromatography separation method and its on-line coupling to mass spectrometry (MS) allowing efficient differentiation and sensitive detection of mono-, di-, and trisulfated CS disaccharides and their positional isomers, without requiring prior derivatization. The composition of the mobile phase in terms of pH and concentration showed great influence on the chromatographic separation and was varied to allow the distinction of each CS without signal overlap for a total analysis time of 25 min. This methodology was applied to determine the disaccharide composition of biological reaction media resulting from various enzymatic transformations of CS, such as enzymatic desulfation of CS disaccharides by a CS 4-O-endosulfatase, and depolymerization of the CS endocan by chondroitinase lyase ABC.

15 pags, 5 figs, 2 pags, Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because they have dynamical structure features and conventional drug design is not applicable. NUPR1 is an IDP playing an important role in pancreatic cancer. We previously reported that Trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumors growth. Unfortunately, TFP showed strong central nervous system side-effects. In this work, we undertook a multidisciplinary approach to optimize TFP, based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical and biological evaluations. A family of TFP-derived compounds was produced and the most active one, named ZZW-115, showed a dose-dependent tumor regression with no neurological effects and induced cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets., This work was supported by La Ligue Contre le Cancer, INCa, Canceropole PACA and INSERM (to JI); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to OA); Fondo de Investigaciones Sanitarias (PI15/00663 and PI18/00343 to OA); Spanish Ministry of Economy and Competitiveness (BFU201678232-P to AVC, CTQ2015-64445-R to JLN); Diputacion General de Aragon (Protein Targets Group B89 to AVC, and Digestive Pathology Group B01 to OA); Generalitat Valenciana (Prometeo 018/2013 to JLN); Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd); Fundacion Alfonso Martin-Escudero and Fondation de France (to PSC); China Scholarship Council (to WL and CH); Programme XU GUANGQI (to YX and JI); National Natural Science Foundation of China (81502920) and the Fundamental Research Funds for the Central Universities (106112017CDJQJ468823) (both to YX). BR acknowledges hospitality from the European Magnetic Resonance Center (CERM), Sesto Fiorentino, Florence, Italy.

Over a quarter million of protein phosphorylation sites have been identified so far, although the effects of site-specific phosphorylation on protein function and stability, as well as their possible impact in the phenotypic manifestation in genetic diseases are vastly unknown. We investigated here the effects of phosphorylating S82 in human NADP(H):quinone oxidoreductase 1, a representative example of disease-associated flavoprotein in which protein stability is coupled to the intracellular flavin levels. Additionally, the cancer-associated P187S polymorphism causes inactivation and destabilization of the enzyme. By using extensive in vitro and in silico characterization of phosphomimetic S82D mutations, we showed that S82D locally affected the flavin binding site of the wild-type (WT) and P187S proteins thus altering flavin binding affinity, conformational stability and aggregation propensity. Consequently, the phosphomimetic S82D may destabilize the WT protein intracellularly by promoting the formation of the degradation-prone apo-protein. Noteworthy, WT and P187S proteins respond differently to the phosphomimetic mutation in terms of intracellular stability, further supporting differences in molecular recognition of these two variants by the proteasomal degradation pathway. We propose that phosphorylation could have critical consequences on stability and function of human flavoproteins, important for our understanding of genotype-phenotype relationships in their related genetic diseases.

14 pags., 5 figs., 1 tab., 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., This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/ FEDER, EU) (RTI 2018-097991-B-I00 to JN and PGC 2018- 094548-B-I00 to AA and PM), and the Basque Government (IT1175-19 to AA and PM).

11 pags, 6 figs, 3 tabs., Background: The phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. It is formed by a protein cascade in which the first two proteins are general (namely enzyme I, EI, and the histidine phosphocarrier protein, HPr) and the others are sugar-specific permeases; the active site of HPr is His15. The HPr kinase/phosphorylase (HPrK/P), involved in the use of carbon sources in Gram-positive, phopshorylates HPr at a serine. The regulator of sigma D protein (Rsd) also binds to HPr. We are designing specific fragments of HPr, which can be used to interfere with those protein-protein interactions (PPIs), where the intact HPr intervenes. Methods: We obtained a fragment (HPr48) comprising the first forty-eight residues of HPr. HPr48 was disordered as shown by fluorescence, far-ultraviolet (UV) circular dichroism (CD), small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR)., Results: Secondary structure propensities, from the assigned backbone nuclei, further support the unfolded nature of the fragment. However, HPr48 was capable of binding to: (i) the N-terminal region of EI, EIN; (ii) the intact Rsd; and, (iii) HPrK/P, as shown by fluorescence, far-UV CD, NMR and biolayer interferometry (BLI). The association constants for each protein, as measured by fluorescence and BLI, were in the order of the low micromolar range, similar to those measured between the intact HPr and each of the other macromolecules., Conclusions: Although HPr48 is forty-eight-residue long, it assisted antibiotics to exert antimicrobial activity., General significance: HPr48 could be used as a lead compound in the development of new antibiotics, or, alternatively, to improve the efficiency of existing ones, This research was funded by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN, RTI2018-101969-J-I00 to AF]. Some of the NMR equipment used in this work have been funded by Generalitat Valenciana (Spain) and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana (Spain) 2014-2020).

9 pags, 3 figs, Cancer cells activate stress-response mechanisms to adapt themselves to a variety of stressful conditions. Among these protective mechanisms, those controlled by the stress-induced nuclear protein 1 (NUPR1 ) belong to the most conserved ones. NUPR1 is an 82-residue-long, monomeric, basic and intrinsically disordered protein (IDP), which was found to be invariably overexpressed in some, if not all, cancer tissues. Remarkably, we and others have previously showed that genetic inactivation of the Nupr1 gene antagonizes the growth of pancreatic cancer as well as several other tumors. With the use of a multidisciplinary strategy by combining biophysical, biochemical, bioinformatic, and biological approaches, a trifluoperazine-derived compound, named ZZW-115, has been identified as an inhibitor of the NUPR1 functions. The anticancer activity of the ZZW-115 was first validated on a large panel of cancer cells. Furthermore, ZZW-115 produced a dose-dependent tumor regression of the tumor size in xenografted mice. Mechanistically, we have demonstrated that NUPR1 binds to several importins. Because ZZW-115 binds NUPR1 through the region around the amino acid Thr68, which is located into the nuclear location signal (NLS) region of the protein, we demonstrated that treatment with ZZW-115 inhibits completely the translocation of NUPR1 from the cytoplasm to the nucleus by competing with importins., This work in the authors' laboratories was supported by La Ligue Contre le Cancer, INCa, Fondation de France, and INSERM (to JI); Fondation ARC (to PS); the Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to OA); the Fondo de Investigaciones Sanitarias (PI15/00663 and PI18/00343 to OA); the Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to AVC, RTI2018 097991-BIO to JLN); the Diputacion General de Aragon (Protein Targets Group B89 to AVC, and Digestive Pathology Group B01 to OA); the Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd); the Fundacion Alfonso Martin-Escudero and Fondation de France (to PSC); the China Scholarship Council (to WL and CH); Programme XU GUANGQI and CAI YUANPEI (to YX and JI); and the National Natural Science Foundation of China (81502920 to YX).

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 Ca2+-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 (alpha Tm), with a detectably higher affinity (similar to 10 mu M) of HcTnI-C27 than that of HcTnI-C27-H (similar to 15 mu M), consistent with an impaired Ca2+-desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to aTm, 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 Ca2+-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.

23 pags., 5 figs., 3 tabs. -- This article belongs to the Special Issue Structural, Functional and Folding Strategies of Oligomeric Proteins, The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence., This research was funded by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN, BIO2016-78020-R to ACA and CTQ2017-85425-P to JGHC]; JGHC thanks Fundación Séneca, Región de Murcia for funding (20933/PI/18).

11 pags., 6 figs., 2 tabs., Neurofibromin-1 (NF1) is a large, multidomain tumour suppressor encoded by the NF1 gene. The gene is mutated in neurofibromatosis type I, a disease characterized by malignant tumours of the nervous system and benign neurofibromas. The best-known activity of NF1 is the down-regulation of the mitogen-activated protein kinase pathway via its three-hundred-residue-long GTPase-activating protein (GAP) domain (the so-called GAP-related domain (NF1-GRD)). The NF1-GRD stimulates Ras GTPase activity in turning off signalling. Despite this activity, NF1-GRD has been demonstrated to bind to other different proteins, such as SPRED1 or MC1R. We have embarked on the biophysical and conformational characterization of NF1-GRD in solution by using several spectroscopic (namely fluorescence and circular dichroism (CD)) and biophysical techniques (namely size exclusion chromatography (SEC) and differential scanning calorimetry (DSC)). This biophysical characterization is crucial in deciphering NF1-GRD interactome and in finding biochemical features, modulating possible protein interactions. The native-like structure of NF1-GRD (as monitored by intrinsic fluorescence and far-UV CD) was strongly pH-dependent showing a pH-titration causing a substantial increase in its helicity. NF1-GRD had a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by intrinsic and ANS fluorescence, and CD. Chemical denaturations showed that NF1-GRD unfolded through an intermediate which has a substantial amount of solvent-exposed hydrophobic patches., This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN, BFU2016-78232-P to AVC]; Andalusian Regional Government [“Endocrinology and Metabolism Group” CTS- 202 to SMR]; Diputacion General de Aragon [“Protein targets and Bioactive Compounds group” E45-20R to AVC].

18 pags., 4 figs., 5 tabs. -- This article belongs to the Special Issue Folding and Design of α-Helical Proteins and Peptides: Theory Meets Nanomaterials, Biotechnology and Health, The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr is mainly disordered. We attempted to improve the affinity of HPr to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. The refore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function., This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI2018-097991-B-I00 to J.L.N., BFU2016-78232-P to A.V.-C., BES-2017-080739 to D.O.-A., and RTI2018-101969-J-I00 to A.F.). The NMR equipment used in this work was funded by Generalitat Valenciana (Spain) and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana (Spain) 2014–2020).

ALP thanks Professors Jose Manuel Sanchez-Ruiz and Beatriz Ibarra-Molero (both from the University of Granada) for providing access and advice on their home-built software for electrostatic calculations. BR acknowledges kind hospitality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy. This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-BI00 to JLN and RTI2018-096246-B-I00 to ALP]; FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades [Grant P18-RT-2413 to ALP]. Financial support from EU Horizon 2020 project EU FT-ICR MS (731077) as well as institutional (CZ.1.05/1.1.00/02.0109) and MS facility support (LM2018127 CIISB) are gratefully acknowledged. Funding for open charge: Universidad de Granada/CBUA., The phosphomimetic mutation S82D in the cancer-associated, FADdependent human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a decrease in flavin-adenine dinucleotide-binding affinity and intracellular stability. We test in this work whether the evolutionarily recent neutral mutation R80H in the vicinity of S82 may alter the strong functional effects of S82 phosphorylation through electrostatic interactions. We show using biophysical and bioinformatic analyses that the reverse mutation H80R prevents the effects of S82D phosphorylation on hNQO1 by modulating the local stability. Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation were milder, resembling the behaviour found in hNQO1 when this residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently neutral and evolutionarily divergent mutations may determine the functional response of mammalian orthologues towards phosphorylation., Spanish Government, European ERDF Funds (MCIU/AEI/FEDER, EU) RTI2018-097991-BI00 RTI2018-096246-B-I00, FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades P18-RT-2413, EU Horizon 2020 project EU FT-ICR MS 731077, Universidad de Granada/CBUA CZ.1.05/1.1.00/02.0109 LM2018127 CIISB

13 pags, 9 figs. -- Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbapap.2021.140670., RYBP (Ring1 and YY1 binding protein, UniProt ID: Q8N488) is an epigenetic factor with a key role during embryonic development; it does also show an apoptotic function and an ubiquitin binding activity. RYBP is an intrinsically disordered protein (IDP), with a Zn-finger domain at its N-terminal region, which folds upon binding to DNA. It is predicted that RYBP has a nuclear localization sequence (NLS), comprising residues Asn58 to Lys83, to allow for nuclear translocation. We studied in this work the ability of intact RYBP to bind Impα3 and its truncated species, ΔImpα3, without the importin binding domain (IBB), by using fluorescence and circular dichroism (CD). Furthermore, the binding of the peptide matching the isolated NLS region of RYBP (NLS-RYBP) was also studied using the same methods and isothermal titration calorimetry (ITC), and in silico molecular docking. Moreover, we carried out experiments with NLS-RYBP in the absence or in the presence of NaCl (140 mM). Our results show that RYBP interacted with Impα3 and ΔImpα3, causing protein precipitation. The NLS-RYBP also interacted with both importin species (dissociation constant in the low micromolar range), at low or high ionic strength, as shown by intrinsic fluorescence and ITC. These findings indicate that the NLS region, which was mainly unfolded in isolation in solution, was essentially responsible for the binding of RYBP to each of the importin species. Furthermore, the molecular simulations predict that the anchoring of NLS-RYBP takes place in the major binding site for the NLS of cargo proteins bound to Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in RYBP and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation., The authors thank two anonymous reviewers for helpful suggestions and discussions. BR acknowledges the kind use of computational resources of the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy. This research was funded by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN and BFU2016-78232-P to AVC]; Diputacion General de Aragon [Protein Targets and Bioactive Compounds Group E45_17R to AVC and Pre-doctoral Research Contract 2019 to AJA]; and Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd). JLN thanks Miguel Vidal (CIB, CSIC, Madrid) for the kindly gift of the RYBP vector.

12 pags, 7 figs. -- Supplementary material includes five figures containing: (i) the fluorescence spectrum of the mixture containing prothymosin α and ARM-PKP1 (Fig. S1); (ii) the fluorescence titration curve of SAMp73 and ARM-PKP1 in the presence of 100 mM NaCl (Fig. S2); (iii) the 2D 15N, 1H- HSQC-NMR spectra of isolated SAMp73 and in the presence of ARM-PKP1 in 100 mM NaCl; (iv) binding of ARM-PKP1 and SAMp73 as monitored by ITC in 100 mM of NaCl (Fig. S4); and (v) the fluorescence titrations of the fragments α1α2 and α4α5 with ARM-PKP1 (Fig. S5). Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbagen.2021.129914, Plakophilin 1 (PKP1) is a component of desmosomes, which are key structural components for cell-cell adhesion, and can also be found in other cell locations. The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing crucial roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); such domain, SAMp73, is involved in the interaction with other macromolecules., This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN; BFU2016-78232-P to AVC; BES-2017-080739 to DOA]; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, Investing in your future') [PI15/00663 and PI18/00349 to OA]; PAIDI program, Group BIO309 (Junta de Andalucia) to MEF-V; Diputacion General de Aragon [Protein Targets and Bioactive Compounds Group E45_20R to AVC, and Digestive Pathology Group B25_20R to OA]; and Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd).

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin &alpha, with several human isoforms, among them, importin &alpha, 3 (Imp&alpha, 3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Imp&alpha, 3 and its depleted species, ∆Imp&alpha, 3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp &alpha, 3 with a low micromolar affinity (~5 &mu, M). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ∆Imp&alpha, 3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Imp&alpha, 3, confirming that the IBB could act as an auto-inhibition region of Imp&alpha, 3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin &alpha, of which there are several human isoforms, among them, importin &alpha, 3 (Imp&alpha, 3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54&ndash, 74) of NUPR1 towards Imp&alpha, 3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Imp&alpha, 3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Imp&alpha, 3 with low micromolar affinity (1.7&ndash, 27 &mu, M). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important, furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional&ndash, proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Imp&alpha, 3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.

11 Pags. Issue editorial. The definitive version is available at: https://www.sciencedirect.com/science/journal/00039861, Ductile and flexible proteins and regions, known as intrinsically disordered proteins (IDPs) and regions (IDRs), have key roles in all organisms. In the last two decades numerous studies have shown to be heavily involved in essential cellular processes and to be related with the complexity of organisms, being the number higher in eukaryote proteomes compared with prokaryotes. Also IDPs and IDRs are abundant in viruses. These proteins are crucial in development, adaptation and evolution of organisms and are involved in many diseases. Despite their elusive features and difficulties of experimental studies, the structural and functional data about IDPs and IDRs have increased in the last years, and computational and bioinformatics analysis have also contributed to that. Recent investigations have revealed novel aspects of IDPs and IDRs that contribute to progress in this field. In this special issue authors will contribute to give an overview about the current advances and challenges in this promising subject. The main topics deal with: (a) use of NMR, EPR, and other biophysical techniques in IDP structural characterization, (b) physical-basis of order-to order transition and allosteric mechanisms affecting IDPs, c) structural organization of IDPs in solution and membranes, (d) roles of IDPs in virus infection, algal metabolism, and plant development and stress tolerance.

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS type I is regulated by protein-protein interactions with a 65-residue-long protein (IF7). IF7 binds initially to GS through residues at its N terminus. In this work, we studied the conformational preferences of the N-terminal region of IF7 (IF7pep, residues Ala7-Ala29), its binding to GS and its functional properties. Isolated IF7pep populated a nascent helix in aqueous solution. IF7pep was bound to GS with an affinity constant of 0.4 μM, and a 1:1 stoichiometry. IF7pep did not inactivate GS, suggesting that there were other IF7 regions important to carry out the inactivating function. Binding of IF7pep to GS was electrostatically-driven and it did not follow a kinetic two-state model.

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65-residue-long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pKa values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C-terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pKa values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random-coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N-terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.

International audience; Recently, a novel CS/DS 4-O-endosulfatase was identified from a marine bacterium and its catalytic mechanism was investigated further (Wang, W., et. al (2015) J. Biol. Chem.290, 7823–7832; Wang, S., et. al (2019) Front. Microbiol.10, 1309). In the study herein, we provide new insight about the structural characteristics of the substrate which determine the activity of this enzyme. The substrate specificities of the 4-O-endosulfatase were probed by using libraries of structure-defined CS/DS oligosaccharides issued from synthetic and enzymatic sources. We found that this 4-O-endosulfatase effectively remove the 4-O-sulfate of disaccharide sequences GlcUAβ1-3GalNAc(4S) or GlcUAβ1-3GalNAc(4S,6S) in all tested hexasaccharides. The sulfated GalNac residue is resistant to the enzyme when adjacent uronic residues are sulfated as shown by the lack of enzymatic desulfation of GlcUAβ1-3GalNAc(4S) connected to a disaccharide GlcUA(2S)β1-3GalNAc(6S) in an octasaccharide. The 3-O-sulfation of GlcUA was also shown to hinder the action of this enzyme. The 4-O-endosulfatase exhibited an oriented action from the reducing to the non-reducing whatever the saturation or not of the non-reducing end. Finally, the activity of the 4-O-endosulfatase decreases with the increase in substrate size. With the deeper understanding of this novel 4-O-endosulfatase, such chondroitin sulfate (CS)/dermatan sulfate (DS) sulfatase is a useful tool for exploring the structure–function relationship of CS/DS.

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS is regulated, among other mechanisms, by protein-protein interactions with a 65-residue-long, intrinsically disordered protein (IDP), named IF7. IDPs explore diverse conformations in their free states and, in some cases, in their molecular complexes. We used both nuclear magnetic resonance (NMR) at 11.7 T and small angle X-ray scattering (SAXS) to study the size and the dynamics in the picoseconds-to-nanosecond (ps-ns) timescale of: (i) isolated IF7; and (ii) the IF7/GS complex. Our SAXS findings, together with MD results, show: (i) some of the possible IF7 structures in solution; and, (ii) that the presence of IF7 affected the structure of GS in solution. The joint use of SAXS and NMR shows that movements of each amino acid of IF7 were uncorrelated with those of its neighbors. Residues of IF7 with the largest values of the relaxation rates (R1, R2 and ηxy), in the free and bound species, were mainly clustered around: (i) the C terminus of the protein; and (ii) Ala30. These residues, together with Arg8 (which is a hot-spot residue in the interaction with GS), had a restricted mobility in the presence of GS. The C-terminal region, which appeared more compact in our MD simulations of isolated IF7, seemed to be involved in non-native contacts with GS that help in the binding between the two macromolecules. Ministerio de Economía y Competitividad RTI 2018-097991- BI00

19 pags., 6 figs., 4 tabs., Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR., This work was supported by La Ligue Contre le Cancer, INCa, Canceropole PACA and INSERM to JI; Fondation ARC to PS. La Ligue Contre le Cancer (Equipe Labellisée) to VG and JI; Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017) to OA; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, ‘Investing in your future’) (PI15/00663 and PI18/00343) to OA; Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to AVC, RTI2018-097991-BI00 to JLN); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to AVC, and Digestive Pathology Group B25_17R to OA); Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd); Fondation de France to PSC; China Scholarship Council to WL and CH; Programme XU GUANGQI to YX and JI; and National Natural Science Foundation of China (81502920), the Fundamental Research Funds for the Central Universities (106112017CDJQJ468823) to YX. Part of this work was performed using the France-BioImaging infrastructure supported by the Agence Nationale de la Recherche (ANR-10-INBS-04-01, call “Investissements d’Avenir”).

The discovery of new small-molecule chemical entities capable of modulating protein function usually begins with the high-throughput screening (HTS) of collections of compounds, or fragments of these, in order to select for molecules interacting with a target biomolecule (usually a protein). The process is the same whether the protein is well folded or is an intrinsically disordered one (IDP); however, subtle details may arise depending on the capability of the experimental technique to grasp particular structural and functional features of IDPs. For a full characterization of such interactions, the thermodynamic, stoichiometric, kinetic, and, quite often, structural details must be described. The set of biophysical methods now at hand constitutes a powerful toolbox for designing and implementing HTS assays and performing a full characterization of the interaction of hit and lead compounds selected by HTS. The aim of this chapter is to provide an overview of some high-throughput techniques used during the screening, identification, and validation of ligands. Fluorescence-based methods, which offer an attractive combination of high-throughput and small-cost but low-resolution structural aspects, and nuclear magnetic resonance (NMR)–based methods, which offer a combination of high-medium throughput and comprehensive structural insight, are discussed in detail, with recent examples applied to IDPs. We also describe briefly the use of surface plasmon resonance (SPR) and its complementarities with the techniques previously mentioned.

[Background]: Eukaryotic cells have a continuous transit of macromolecules between the cytoplasm and the nucleus. Several carrier proteins are involved in this transport. One of them is importin α, which must form a complex with importin β to accomplish its function, by domain-swapping its 60-residue-long N terminus. There are several human isoforms of importin α; among them, importin α3 has a particularly high flexibility., [Methods]: We studied the conformational stability of intact importin α3 (Impα3) and its truncated form, where the 64-residue-long, N-terminal importin-β-binding domain (IBB) has been removed (ΔImpα3), in a wide pH range, with several spectroscopic, biophysical, biochemical methods and with molecular dynamics (MD)., [Results]: Both species acquired native-like structure between pH 7 and 10.0, where Impα3 was a dimer (with an apparent self-association constant of ~10 μM) and ΔImpα3 had a higher tendency to self-associate than the intact species. The acquisition of secondary, tertiary and quaternary structure, and the burial of hydrophobic patches, occurred concomitantly. Both proteins unfolded irreversibly at physiological pH, by using either temperature or chemical denaturants, through several partially folded intermediates. The MD simulations support the presence of these intermediates., [Conclusions]: The thermal stability of Impα3 at physiological pH was very low, but was higher than that of ΔImpα3. Both proteins were stable in a narrow pH range, and they unfolded at physiological pH populating several intermediate species., [General significance]: The low conformational stability explains the flexibility of Impα3, which is needed to carry out its recognition of complex cargo sequences., This work was supported by Spanish Ministry of Economy and Competitiveness and European FEDER Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN and JG, BFU2016-75471-C2-1-P to CA, PGC2018-094548-B-I00 to AA and BIO2016-78020-R to ACA], Basque Country [IT-1175-19 to AA], GVA and EU-FEDER funds “Una forma de hacer Europa” [GVA-IDIFEDER 2018/020] and Portuguese FCT [UIDB/04565/2020 and SAICTPAC/0019/2015 to AC and MP]. AUC SV assays were performed at the Molecular Interactions Facility at the CIB Margarita Salas. CD-G acknowledges Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD) FCT reference: SFRH/PD/BD/135154/2017. BR acknowledges the kind hospitality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy.

Background: NUPR1 is a multifunctional intrinsically disordered protein (IDP) involved, among other functions, in chromatin remodelling, and development of pancreatic ductal adenocarcinoma (PDAC). It interacts with several biomolecules through hydrophobic patches around residues Ala33 and Thr68. The drug trifluoperazine (TFP), which hampers PDAC development in xenografted mice, also binds to those regions. Because of the large size of the hot-spot interface of NUPR1, small molecules could not be adequate to modulate its functions. Methods: We explored how amphipathic helical-designed peptides were capable of interacting with wild-type NUPR1 and the Thr68Gln mutant, inhibiting the interaction with NUPR1 protein partners. We used in vitro biophysical techniques (fluorescence, circular dichroism (CD), nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC)), in silico studies (docking and molecular dynamics (MD)), and in cellulo protein ligation assays (PLAs) to study the interaction. Results: Peptide dissociation constants towards wild-type NUPR1 were ~ 3 μM, whereas no interaction was observed with the Thr68Gln mutant. Peptides interacted with wild-type NUPR1 residues around Ala33 and residues at the C terminus, as shown by NMR. The computational results clarified the main determinants of the interactions, providing a mechanism for the ligand-capture that explains why peptide binding was not observed for Thr68Gln mutant. Finally, the in cellulo assays indicated that two out of four peptides inhibited the interaction of NUPR1 with the C-terminal region of the Polycomb RING protein 1 (C-RING1B). Conclusions: Designed peptides can be used as lead compounds to inhibit NUPR1 interactions. General significance: Peptides may be exploited as drugs to target IDPs.

International audience; NUPR1 is a protumoral multifunctional intrinsically disordered protein, which is activated during the acute phases of pancreatitis, interacting with several biomolecules through residues around Ala33 and Thr68. Because of the large size of this hot-spot, designed small molecules could be insufficient to modulate all NUPR1 functions. In this work, we studied NUPR1 interactions with dendrimers by using biophysical techniques and in silico methods. Our results, obtained with different functionalized dendrimers (anionic, cationic and neutral) and several of their generations, indicate that NUPR1 was bound to the dendrimers. Functionalities at the dendrimer periphery modulated the affinity for NUPR1, and for any dendrimer, the affinity increased with generation. The affinities of most of the dendrimers were in the range 4-40 × 103 M-1, and those of the [Gn]-PhCO2Na dendrimers were similar to those of NUPR1 for its natural partners (0.1-1 × 106 M-1). In all dendrimers, the residues of NUPR1 first affected upon binding were located around Ala33, indicating that NUPR1 employs the same hot-spot to recognize any natural or synthetic molecule.

13 pags., 7 figs., Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 μM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R, R, R and η), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region., This work was supported bySpanish Ministry of Economy and Competitiveness [CTQ2015-64445-R (to JLN) ]with European ERDF funds;by the French La Ligue Contre le Cancer, INCa, Canceropole PACA, DGOS (la bellisation SIRIC) and INSERM (to JLI). The NMR equipment used in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OPERDF of Comunitat Valenciana 2014-2020). BR acknowledges kind hospitality in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy.

The assembly of the protein complex of cytochrome c oxidase (COX), which participates in the mitochondrial respiratory chain, requires a large number of accessory proteins (the so-called assembly factors). Human COX assembly factor 3 (hCOA3), also known as MITRAC12 or coiled-coil domain-containing protein 56 (CCDC56), interacts with the first subunit protein of COX to form its catalytic core and promotes its assemblage with the other units. Therefore, hCOA3 is involved in COX biogenesis in humans and can be exploited as a drug target in patients with mitochondrial dysfunctions. However, to be considered a molecular target, its structure and conformational stability must first be elucidated. We have embarked on the description of such features by using spectroscopic and hydrodynamic techniques, in aqueous solution and in the presence of detergents, together with computational methods. Our results show that hCOA3 is an oligomeric protein, forming aggregates of different molecular masses in aqueous solution. Moreover, on the basis of fluorescence and circular dichroism results, the protein has (i) its unique tryptophan partially shielded from solvent and (ii) a relatively high percentage of secondary structure. However, this structure is highly flexible and does not involve hydrogen bonding. Experiments in the presence of detergents suggest a slightly higher content of nonrigid helical structure. Theoretical results, based on studies of the primary structure of the protein, further support the idea that hCOA3 is a disordered protein. We suggest that the flexibility of hCOA3 is crucial for its interaction with other proteins to favor mitochondrial protein translocation and assembly of proteins involved in the respiratory chain.

10 pags., 6 figs., 1 tab., Plakophilin 1 (PKP1) is a member of the armadillo repeat family of proteins. It serves as a scaffold component of desmosomes, which are key structural components for cell–cell adhesion. We have embarked on the biophysical and conformational characterization of the ARM domain of PKP1 (ARM-PKP1) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC)). ARM-PKP1 was a monomer in solution at physiological pH, with a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by fluorescence and CD. The presence or absence of disulphide bridges did not affect its low stability. The protein unfolded through an intermediate which has lost native-like secondary structure. ARM-PKP1 acquired a native-like structure in a narrow pH range (between pH 6.0 and 8.0), indicating that its adherent properties might only work in a very narrow pH range., This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/ERDF, EU) [RTI2018-097991-B-I00 to JLN, BIO2016-78020-R to ACA, CTQ2017-85425-P to JGHC] and by the PAIDI program, Group BIO309 (Junta de Andalucía) to MEFV. JGHC thanks Fundación Séneca, Región de Murcia for funding (20933/PI/18).

7 pags., 4 figs., BRMS1 is a 246-residue-long protein belonging to the family of metastasis suppressors. It is a predominantly nuclear protein, although it can also function in the cytoplasm. At its C terminus, it has a region that is predicted to be a nuclear localization sequence (NLS); this region, NLS2, is necessary for metastasis suppression. We have studied in vitro and in silico the conformational preferences in aqueous solution of a peptide (NLS2-pep) that comprises the NLS2 of BRMS1, to test whether it has a preferred conformation that could be responsible for its function. Our spectroscopic (far-UV circular dichroism, DOSY-NMR and 2D-NMR) and computational (all-atom molecular dynamics) results indicate that NLS2-pep was disordered in aqueous solution. Furthermore, it did not acquire a structure even when experiments were performed in a more hydrophobic environment, such as the one provided by 2,2,2-trifluoroethanol (TFE). The hydrodynamic radius of the peptide in water was identical to that of a random-coil sequence, in agreement with both our molecular simulations and other theoretical predictions. Thus, we suggest that NLS2 is a disordered region, with non pre-formed structure, that participates in metastasis suppression., This work was supported by Spanish Ministry of Economy and Competitiveness and European Funds [CTQ2015-64445-R (to JLN)].Partial support was received from Susan G. Komen for the Cure [SAC11037], the National Foundation for Cancer Research and theAmerican Cancer Society [PF-16-227-O1-CSM]. DP-U acknowledges theuse of the“Manuel Rico”NMR laboratory, LMR (CSIC), a Spanish large-scale national NMR facility ICTS R-LRB. BR acknowledges kind hospi-tality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy

15 pags., 6 figs., 3 tabs. -- Open Access funded by Creative Commons Atribution Licence 4.0, p53 is frequently mutated in human cancers. Its levels are tightly regulated by the E3 ubiquitin ligase MDM2. The complex between MDM2 and p53 is largely formed by the interaction between the N-terminal domain of MDM2 and the N-terminal transactivation (TA) domain of p53 (residues 15–29). We investigated the kinetic and thermodynamic basis of the MDM2/p53 interaction by using wild-type and mutant variants of the TA domain. We focus on the effects of phosphorylation at positions Thr18 and Ser20 including their substitution with phosphomimetics. Conformational propensities of the isolated peptides were investigated using in silico methods and experimentally by circular dichroism and H-NMR in aqueous solution. Both experimental and computational analyses indicate that the p53 peptides are mainly disordered in aqueous solution, with evidence of nascent helix around the Ser20-Leu25 region. Both phosphorylation and the phosphomimetics at Thr18 result in a decrease in the binding affinity by ten- to twenty-fold when compared to the wild-type. Phosphorylation and phosphomimetics at Ser20 result in a smaller decrease in the affinity. Mutation of Lys24 and Leu25 also disrupts the interaction. Our results may be useful for further development of peptide-based drugs targeting the MDM2/p53 interaction., This work was supported by Spanish Ministry of Economy and Competitiveness [CTQ 2015–64445-R (to JLN)]. JLN acknowledges the regional government Generalitat Valenciana for a BEST short-stay fellowship in the Department of Pharmacology, Cambridge, UK. LSI acknowledges support of a Senior Fellowship from the Medical Research Foundation (UK).

Group II introns are self-splicing mobile genetic retroelements. The spliced intron RNA and the intron-encoded protein (IEP) form ribonucleoprotein particles (RNPs) that recognize and invade specific DNA target sites. The IEP is a reverse transcriptase/maturase that may bear a C-terminal endonuclease domain enabling the RNP to cleave the target DNA strand to prime reverse transcription. However, some mobile introns, such as RmInt1, lack the En domain but nevertheless retrohome efficiently to transient single-stranded DNA target sites at a DNA replication fork. Their mobility is associated with host DNA replication, and they use the nascent lagging strand as a primer for reverse transcription. We searched for proteins that interact with RmInt1 RNPs and direct these RNPs to the DNA replication fork. Co-immunoprecipitation assays suggested that DnaN (the β-sliding clamp), a component of DNA polymerase III, interacts with the protein component of the RmInt1 RNP. Pulldown assays, far-western blots and biolayer interferometry supported this interaction. Peptide binding assays also identified a putative DnaN-interacting motif in the RmInt1 IEP structurally conserved in group II intron IEPs. Our results suggest that intron RNP interacts with the β-sliding clamp of the DNA replication machinery, favouring reverse splicing into the transient ssDNA at DNA replication forks., Spanish Ministerio de Economía, Industria y Competitividad including ERDF (European Regional Development Funds), Plan Nacional de I+D+i Research Grants [BIO2014-51953-P, BIO2017-82244-P to N.T.; CTQ2015-64445-R to J.L.N.]; Generalitat Valenciana [Prometeo 018/2013 to J.L.N.]; Agence Nationale de la Recherche [ANR-15-CE11-0003-01 to MM]; Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS) [ECTZ18552 to MM]; ITMO Cancer [18CN047-00 to MM]; Grenoble Instruct Center [ISBG: UMS 3518 CNRS-CEA-UJF-EMBL]; FRISBI [ANR-10-INSB-05-02]; GRAL [ANR-10-LABX-49-01]; Grenoble Partnership for Structural Biology (PSB). Funding for open access charge: Plan Nacional de I+D+i Research Grant [BIO2017-82244-P].

3 pags, 1 fig, Intrinsically disordered proteins (IDPs) do not have a well-defined structure, but they have key biological tasks in cancer development. By using the disordered cancer-related protein NUPR1 as a proof-of-concept, we have developed a new multidisciplinary approach to tackle drug-design against IDPs, using it to repurpose drugs for treating pancreatic adenocarcinoma (PDAC)., This work in the authors' laboratories was supported by [La Ligue Contre le Cancer], INCa, [Canceropole PACA] and [INSERM] to JLI and LP; [Miguel Servet Program from Instituto de Salud Carlos III] under grant [CPII13/00017] to OA; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') under grants [PI15/00663 and PI18/00349] to OA; Ministerio de Economia, Industria y Competitividad, Gobierno de Espana under grants [BFU2016-78232-P to AVC, CTQ2015-64445-R to JLN]; [Diputacion General de Aragon] under grants [Protein Targets and Bioactive Compound Group to AVC, and Digestive Pathology Group to OA]; [Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas] (CIBERehd) toOAand AVC; [Programme XUGUANGQI] to YX and JLI; and [National Natural Science Foundation of China (81502920), the Fundamental Research Funds for the Central Universities] under grant [106112017CDJQJ468823] to YX. The Fundacion Alfonso Martin-Escudero and Fondation de France supported to PSC.