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Ivarsson, Ylva
Publications (10 of 48) Show all publications
Ali, M. & Ivarsson, Y. (2018). High-throughput discovery of functional disordered regions. Molecular Systems Biology, 14(5), Article ID e8377.
Open this publication in new window or tab >>High-throughput discovery of functional disordered regions
2018 (English)In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 14, no 5, article id e8377Article in journal, Editorial material (Other academic) Published
Abstract [en]

Partially or fully intrinsically disordered proteins are widespread in eukaryotic proteomes and play important biological functions. With the recognition that well defined protein structure is not a fundamental requirement for function come novel challenges, such as assigning function to disordered regions. In their recent work, Babu and colleagues (Ravarani etal,) took on this challenge by developing IDR-Screen, a robust high-throughput approach for identifying functions of disordered regions.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-361435 (URN)10.15252/msb.20188377 (DOI)000433500800005 ()29789308 (PubMedID)
Available from: 2018-12-10 Created: 2018-12-10 Last updated: 2018-12-10Bibliographically approved
Sundell, G., Arnold, R., Ali, M., Naksukpaiboon, P., Orts, J., Güntert, P., . . . Ivarsson, Y. (2018). Proteome‐wide analysis of phospho‐regulated PDZ domain interactions. Molecular Systems Biology, 14(8), Article ID e8129.
Open this publication in new window or tab >>Proteome‐wide analysis of phospho‐regulated PDZ domain interactions
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2018 (English)In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 14, no 8, article id e8129Article in journal (Refereed) Published
Abstract [en]

A key function of reversible protein phosphorylation is to regulate protein–protein interactions, many of which involve short linear motifs (3–12 amino acids). Motif‐based interactions are difficult to capture because of their often low‐to‐moderate affinities. Here, we describe phosphomimetic proteomic peptide‐phage display, a powerful method for simultaneously finding motif‐based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C‐terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD‐95/Dlg/ZO‐1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site‐specific phospho‐regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho‐regulation of motif‐based interactions on a large scale.

Keywords
PDZ domainphage displayphosphorylationprotein–protein interactionScribble
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-358712 (URN)10.15252/msb.20178129 (DOI)000444544200003 ()30126976 (PubMedID)
Funder
Swedish Research Council, 2012-05092Knut and Alice Wallenberg FoundationSwedish Research Council, 2016-04965Åke Wiberg FoundationCarl Tryggers foundation Wenner-Gren Foundations
Available from: 2018-08-30 Created: 2018-08-30 Last updated: 2018-11-28Bibliographically approved
Sundell, G., Vögeli, B., Ivarsson, Y. & Chi, C. N. (2018). The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1. Biochemistry, 57(1), 66-71
Open this publication in new window or tab >>The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1
2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 1, p. 66-71Article in journal (Refereed) Published
Abstract [en]

The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.

National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-337645 (URN)10.1021/acs.biochem.7b00965 (DOI)000419999100011 ()29144123 (PubMedID)
Funder
Wenner-Gren Foundations, WG-17Swedish Research Council, 2012-05092
Available from: 2018-01-03 Created: 2018-01-03 Last updated: 2018-04-04Bibliographically approved
Sundell, G., Vögeli, B., Ivarsson, Y. & Chi, C. N. (2018). The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity:: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1. Biochemistry, 57(1), 66-71
Open this publication in new window or tab >>The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity:: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1
2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 1, p. 66-71Article in journal (Refereed) Published
Abstract [en]

The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand–protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand–protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ–ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cistrans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.

A detailed understanding of intermolecular interactions requires highly localized probes. Monitoring changes in nuclear magnetic resonance (NMR) chemical shifts of proteins upon ligand titration is a common and convenient tool for obtaining residue- or even atom-specific information. It is well-known that the chemical shift is extremely sensitive to the local electronic environment, and its changes in magnitude as well as directionality can be used to infer structural and dynamic rearrangement.(1, 2) For example, if two ligands repopulate two states of a free protein in opposite ways, the chemical shift changes will also be of opposite directions, because the observed chemical shift is the population-weighted average of the values for the two individual states. This can be seen with two ligands of Pin1, fragments from Cdc25 and FFpSPR, that have opposite effects on the affinity in the catalytic site and thus exhibit shifts in opposite directions.(3, 4) Surprisingly, the standard procedure of chemical shift monitoring involves plotting a combined function of the absolute value change of backbone 1H and 15N shifts:(1)(1)where X is a scaling factor that regulates the weights of H and N shifts and is most commonly set to 1, 0.1, or 0.2. This approach dismisses the sign of both shifts, which carry valuable information. The full set of information about a combined chemical shift change may be given in a vector in the two-dimensional 1H/15N space, which would retain the magnitude and sign of both shifts. Here, we propose to use this approach for chemical shift mapping, as no additional experimental effort is required as compared to the standard procedure. We present the utility of the approach with the example of the first PDZ(PSD-95/Dlg/ZO-1) domain of Scribble, where relevant differences in binding modes are hidden when using eq 1 but are restored when following our proposed approach. In addition, we applied the approach to Cyclophilin a, a cistrans isomerase, in an attempt to capture fine differences between the interaction of the enzyme with a peptide in a cis or trans conformation and the wild type.PDZ domains make up a major class of protein–protein interacting domains present in the postsynaptic density of excitatory neurons.(5, 6) They are small, globular protein domains consisting of 90–100 amino acids and often occur as tandem repeats. PDZ domains function mainly by recognizing short C-terminal peptide motifs, although some also recognize internal peptide motifs.(7-9) The interaction with short C-terminal peptides, also known as canonical PDZ binding mode, has been well characterized; the last four amino acids of the peptide have been shown to be essential for recognition, and the upstream residues contribute to affinity and specificity.(10-12) For class I PDZ–peptide interaction, peptide positions 0 and −2 (numbering from the carboxylate C-terminus) have been deemed to be important determinants for affinity, while other peptide positions play additional roles in selectivity and enhancement of the binding affinity. There are hundreds of known or putative C-terminal peptide ligands present in the cell,(13) which are typically shared ligands between several PDZ domains. The mechanism through which PDZ domains distinguish between respective partners is not entirely clear. It is anticipated that phosphorylation of a particular ligand can trigger alternative signaling pathways quite different from those of the unphosphorylated ligands through disruption or promotion of specific interactions. For example, the Tiam1 PDZ domain binds both phosphorylated and unphosphorylated syndecan 1 as well as phosphorylated syndecan 3 ligands but not other isoforms of syndecans.(14) Phosphorylation of syndecan 1 promotes cell adhesion and ectodomain cleavage by disrupting the interaction between synthenin PDZ1 and phosphorylated syndecan 1.(15, 16) In summary, phosphorylation of PDZ ligands has been proposed as a regulatory mechanism.(14, 17, 18, 20) A priority in PDZ biology is to understand how a single PDZ domain can distinguish between all potential ligands. We recently found that the Scribble PDZ1 domain can interact with unphosphorylated and phosphorylated ligands representing the C-termini of different biological proteins. Depending on the phosphorylation site, phosphorylation may have a switching function, increasing the affinity for Scribble PDZ1 and decreasing the affinity for other PDZ domains.(19) Although we probed the molecular mechanism of interaction through NMR structure determination, with hindsight we realized that sign shift analysis provides an alternative and feasible approach for gaining a deeper understanding of the molecular details of the interactions. Here, we analyze the origin of the binding specificities by probing the directionality of chemical shift perturbation with heteronuclear single-quantum coherence spectra as well as slow millisecond dynamics.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-359433 (URN)10.1021/acs.biochem.7b00965 (DOI)
Available from: 2018-09-02 Created: 2018-09-02 Last updated: 2018-12-06Bibliographically approved
Wigington, C. P., Roy, J., Damle, N. P., Ulengin-Talkish, I., Cho, S. E., Davey, N. E., . . . Cyert, M. S. (2017). A novel role for the Calcineurin phosphatase at the nuclear pore. Paper presented at ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA. Molecular Biology of the Cell, 28(26), 3727-3727
Open this publication in new window or tab >>A novel role for the Calcineurin phosphatase at the nuclear pore
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2017 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Bethesda: American Society for Cell Biology (ASCB), 2017
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-352943 (URN)10.1091/mbc.E17-10-0618 (DOI)000426664301597 ()29237772 (PubMedID)
Conference
ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA
Note

See: Supplementary Material, 2017 ASCB-EMBO Meeting-Poster Abstracts, p. Monday-234-Monday-235, Meeting Abstract: P2238.

Available from: 2018-06-12 Created: 2018-06-12 Last updated: 2018-06-12Bibliographically approved
Davey, N. E., Seo, M.-H., Yadav, V. K., Jeon, J., Nim, S., Krystkowiak, I., . . . Ivarsson, Y. (2017). Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome. The FEBS Journal, 284(3), 485-498
Open this publication in new window or tab >>Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome
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2017 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 3, p. 485-498Article in journal (Refereed) Published
Abstract [en]

The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 mu M through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.

Keywords
EVH1 domain, PDZ domain, Protein-protein interactions, short linear motifs, VHS domain
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-317956 (URN)10.1111/febs.13995 (DOI)000393601800011 ()28002650 (PubMedID)
Available from: 2017-03-31 Created: 2017-03-31 Last updated: 2017-11-29Bibliographically approved
Cho, S. E., Roy, J., Ivarsson, Y. & Cyert, M. S. (2017). Investigating the phospho-regulation of ER shaping protein RTN1A (Reticulon-1A) by the Calcineurin phosphatase.. Paper presented at ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA. Molecular Biology of the Cell, 28(26), 3727-3727
Open this publication in new window or tab >>Investigating the phospho-regulation of ER shaping protein RTN1A (Reticulon-1A) by the Calcineurin phosphatase.
2017 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Bethesda: American Society for Cell Biology (ASCB), 2017
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-352939 (URN)10.1091/mbc.e17-10-0618 (DOI)000426664300526 ()29237772 (PubMedID)
Conference
ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA
Note

See: Supplementary Material, 2017 ASCB-EMBO Meeting-Poster Abstracts, p. Sunday-336, Meeting Abstract: P1528.

Available from: 2018-06-12 Created: 2018-06-12 Last updated: 2018-06-12Bibliographically approved
Roy, J., Wigington, C. P., Damle, N. P., Ulengin-Talkish, I., Cho, S. E., Davey, N. E., . . . Cyert, M. S. (2017). Mapping the human calcineurin phosphatase signaling network through global identification of short linear motifs that mediate substrate recognition. Paper presented at ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA.. Molecular Biology of the Cell, 28(26), 3727-3727
Open this publication in new window or tab >>Mapping the human calcineurin phosphatase signaling network through global identification of short linear motifs that mediate substrate recognition
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2017 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Philadelphia: American Society for Cell Biology (ASCB), 2017
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-352941 (URN)10.1091/mbc.E17-10-0618 (DOI)000426664300551 ()29237772 (PubMedID)
Conference
ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA.
Note

See: Supplementary Material, 2017 ASCB-EMBO Meeting-Poster Abstracts, p. Sunday-351-Sunday-352, Meeting Abstract: P1553.

Available from: 2018-06-12 Created: 2018-06-12 Last updated: 2018-06-12Bibliographically approved
Wu, C.-G., Chen, H., Guo, F., Yadav, V. K., Mcilwain, S. J., Rowse, M., . . . Xing, Y. (2017). PP2A-B' holoenzyme substrate recognition, regulation and role in cytokinesis. Cell Discovery, 3, Article ID 17027.
Open this publication in new window or tab >>PP2A-B' holoenzyme substrate recognition, regulation and role in cytokinesis
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2017 (English)In: Cell Discovery, ISSN 2056-5968, Vol. 3, article id 17027Article in journal (Refereed) Published
Abstract [en]

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B'-family PP2A regulatory subunits and holoenzymes. The B'-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B'α-binding motifs serve as common binding sites for B' subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B'-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B' holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B' that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B' holoenzymes in various cellular processes.

Keywords
CIP2A, PP2A-B′ holoenzyme, SLiMs, centrosome, cytokinesis, midbody
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:uu:diva-337721 (URN)10.1038/celldisc.2017.27 (DOI)000414925200001 ()28884018 (PubMedID)
Funder
Swedish Research Council, 2012-05092 2016-04965Carl Tryggers foundation , CTS15:226
Available from: 2018-01-04 Created: 2018-01-04 Last updated: 2018-02-12Bibliographically approved
Wigington, C. P., Roy, J., Damle, N. P., El Cho, S., Davey, N., Ivarsson, Y., . . . Cyert, M. S. (2017). Uncovering Novel Substrates and Functions for the Calcineurin Phosphatase in Human Cells. Paper presented at Annual Meeting of the American-Society-for-Pharmacology-and-Experimental-Therapeutics (ASPET) at Experimental Biology Meeting, APR 22-26, 2017, Chicago, IL. The FASEB Journal, 31(1), Article ID 771.5.
Open this publication in new window or tab >>Uncovering Novel Substrates and Functions for the Calcineurin Phosphatase in Human Cells
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2017 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 31, no 1, article id 771.5Article in journal, Meeting abstract (Other academic) Published
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-335024 (URN)000405461403004 ()
Conference
Annual Meeting of the American-Society-for-Pharmacology-and-Experimental-Therapeutics (ASPET) at Experimental Biology Meeting, APR 22-26, 2017, Chicago, IL
Available from: 2017-12-01 Created: 2017-12-01 Last updated: 2018-01-13Bibliographically approved
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