Publications by authors named "Gustav N Sundell"

8 Publications

  • Page 1 of 1

A Consensus Binding Motif for the PP4 Protein Phosphatase.

Mol Cell 2019 12 1;76(6):953-964.e6. Epub 2019 Oct 1.

Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.
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http://dx.doi.org/10.1016/j.molcel.2019.08.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981294PMC
December 2019

Proteome-wide analysis of phospho-regulated PDZ domain interactions.

Mol Syst Biol 2018 08 20;14(8):e8129. Epub 2018 Aug 20.

Department of Chemistry - BMC, Uppsala University, Uppsala, Sweden

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100724PMC
http://dx.doi.org/10.15252/msb.20178129DOI Listing
August 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 2018 01 27;57(1):66-71. Epub 2017 Nov 27.

Department of Medical Biochemistry and Microbiology, Uppsala University , BMC Box 582, SE-75123 Uppsala, Sweden.

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 N/H 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.
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http://dx.doi.org/10.1021/acs.biochem.7b00965DOI Listing
January 2018

Emergence and evolution of an interaction between intrinsically disordered proteins.

Elife 2017 04 11;6. Epub 2017 Apr 11.

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

Protein-protein interactions involving intrinsically disordered proteins are important for cellular function and common in all organisms. However, it is not clear how such interactions emerge and evolve on a molecular level. We performed phylogenetic reconstruction, resurrection and biophysical characterization of two interacting disordered protein domains, CID and NCBD. CID appeared after the divergence of protostomes and deuterostomes 450-600 million years ago, while NCBD was present in the protostome/deuterostome ancestor. The most ancient CID/NCBD formed a relatively weak complex (∼5 µM). At the time of the first vertebrate-specific whole genome duplication, the affinity had increased (∼200 nM) and was maintained in further speciation. Experiments together with molecular modeling using NMR chemical shifts suggest that new interactions involving intrinsically disordered proteins may evolve via a low-affinity complex which is optimized by modulating direct interactions as well as dynamics, while tolerating several potentially disruptive mutations.
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http://dx.doi.org/10.7554/eLife.16059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5419745PMC
April 2017

Improved affinity at the cost of decreased specificity: a recurring theme in PDZ-peptide interactions.

Sci Rep 2016 10 3;6:34269. Epub 2016 Oct 3.

Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.

The E6 protein from human papillomavirus (HPV) plays an important role during productive infection and is a potential drug target. We have previously designed a high affinity bivalent protein binder for the E6 protein, a fusion between a helix from the E6 associated protein and PDZØ9, an engineered variant (L391F/K392M) of the second PDZ domain from synapse associated protein 97 (SAP97 PDZ2). How the substitutions improve the affinity of SAP97 PDZ2 for HPV E6 is not clear and it is not known to what extent they affect the specificity for cellular targets. Here, we explore the specificity of wild type SAP97 PDZ2 and PDZØ9 through proteomic peptide phage display. In addition, we employ a double mutant cycle of SAP97 PDZ2 in which the binding kinetics for nine identified potential cellular peptide ligands are measured and compared with those for the C-terminal E6 peptide. The results demonstrate that PDZØ9 has an increased affinity for all peptides, but at the cost of specificity. Furthermore, there is a peptide dependent coupling free energy between the side chains at positions 391 and 392. This corroborates our previous allosteric model for PDZ domains, involving sampling of intramolecular energetic pathways.
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http://dx.doi.org/10.1038/srep34269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5046105PMC
October 2016

Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin.

FEBS Lett 2016 Jan 8;590(1):3-12. Epub 2016 Jan 8.

Department of Chemistry - BMC, Uppsala University, Sweden.

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance.
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http://dx.doi.org/10.1002/1873-3468.12037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819696PMC
January 2016

Structural basis of Sorcin-mediated calcium-dependent signal transduction.

Sci Rep 2015 Nov 18;5:16828. Epub 2015 Nov 18.

Institute of Molecular Biology and Pathology CNR; Dept. Biochemical Sciences, Sapienza University, P.le A. Moro 5, 00185, Rome, Italy.

Sorcin is an essential penta-EF hand calcium binding protein, able to confer the multi-drug resistance phenotype to drug-sensitive cancer cells and to reduce Endoplasmic Reticulum stress and cell death. Sorcin silencing blocks cell cycle progression in mitosis and induces cell death by triggering apoptosis. Sorcin participates in the modulation of calcium homeostasis and in calcium-dependent cell signalling in normal and cancer cells. The molecular basis of Sorcin action is yet unknown. The X-ray structures of Sorcin in the apo (apoSor) and in calcium bound form (CaSor) reveal the structural basis of Sorcin action: calcium binding to the EF1-3 hands promotes a large conformational change, involving a movement of the long D-helix joining the EF1-EF2 sub-domain to EF3 and the opening of EF1. This movement promotes the exposure of a hydrophobic pocket, which can accommodate in CaSor the portion of its N-terminal domain displaying the consensus binding motif identified by phage display experiments. This domain inhibits the interaction of sorcin with PDCD6, a protein that carries the Sorcin consensus motif, co-localizes with Sorcin in the perinuclear region of the cell and in the midbody and is involved in the onset of apoptosis.
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http://dx.doi.org/10.1038/srep16828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649501PMC
November 2015

Interaction analysis through proteomic phage display.

Biomed Res Int 2014 11;2014:176172. Epub 2014 Sep 11.

Department of Chemistry-BMC, Uppsala University, P.O. Box 576, 751 23 Uppsala, Sweden.

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.
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http://dx.doi.org/10.1155/2014/176172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177731PMC
June 2015