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Publications (10 of 17) Show all publications
Born, A., Nichols, P. J., Henen, M. A., Chi, C. N., Strotz, D., Bayer, P., . . . Vögeli, B. (2019). Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery. Biomolecular NMR Assignments, 13(1), 85-89
Open this publication in new window or tab >>Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery
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2019 (English)In: Biomolecular NMR Assignments, ISSN 1874-2718, E-ISSN 1874-270X, Vol. 13, no 1, p. 85-89Article in journal (Refereed) Published
Abstract [en]

Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain H-1, C-13 and N-15 NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.

Place, publisher, year, edition, pages
SPRINGER, 2019
Keywords
Pin1, Prolyl isomerase, NMR, Chemical shift assignments, Allostery
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-382507 (URN)10.1007/s12104-018-9857-9 (DOI)000463649500018 ()30353504 (PubMedID)
Available from: 2019-04-30 Created: 2019-04-30 Last updated: 2019-04-30Bibliographically approved
Nichols, P. J., Born, A., Henen, M. A., Strotz, D., Chi, C. N., Güntert, P. & Vögeli, B. (2018). Extending the Applicability of Exact Nuclear Overhauser Enhancements to Large Proteins and RNA. ChemBioChem (Print), 19(16), 1695-1701
Open this publication in new window or tab >>Extending the Applicability of Exact Nuclear Overhauser Enhancements to Large Proteins and RNA
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2018 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 16, p. 1695-1701Article, review/survey (Refereed) Published
Abstract [en]

Distance-dependent nuclear Overhauser enhancements (NOEs) are one of the most popular and important experimental restraints for calculating NMR structures. Despite this, they are mostly employed as semiquantitative upper distance bounds, and this discards the wealth of information that is encoded in the cross-relaxation rate constant. Information that is lost includes exact distances between protons and dynamics that occur on the sub-millisecond timescale. Our recently introduced exact measurement of the NOE (eNOE) requires little additional experimental effort relative to other NMR observables. So far, we have used eNOEs to calculate multistate ensembles of proteins up to approximately 150 residues. Here, we briefly revisit eNOE methodology and present two new directions for the use of eNOEs: applications to large proteins and RNA.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2018
Keywords
dynamics, NMR spectroscopy, nuclear Overhauser effect, proteasomes, RNA
National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-365167 (URN)10.1002/cbic.201800237 (DOI)000441744200002 ()29883016 (PubMedID)
Available from: 2018-11-09 Created: 2018-11-09 Last updated: 2018-11-09Bibliographically approved
Chi, C. N., Strotz, D., Riek, R. & Vögeli, B. R. (2018). NOE-derived methyl distances from a 360 kDa proteasome complex. Chemistry - A European Journal, 24(9), 2270-2276
Open this publication in new window or tab >>NOE-derived methyl distances from a 360 kDa proteasome complex
2018 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 24, no 9, p. 2270-2276Article in journal (Refereed) Published
Abstract [en]

Nuclear magnetic resonance spectroscopy is the prime tool to probe structure and dynamics of biomolecules at atomic resolution. A serious challenge for that method is the size limit imposed on molecules to be studied. Standard studies are typically restricted to ca. 30-40 kDa. More recent developments lead to spin relaxation measurements in methyl groups in single proteins or protein complexes as large as a mega-Dalton, which directly allow the extraction of angular information or experiments with paramagnetic samples. However, these probes are all of indirect nature in contrast to the most intuitive and easy-to-interpret structural/dynamics restraint, the internuclear distance, which can be measured by nuclear Overhauser enhancement (NOE). Here, we demonstrate time-averaged distance measurements on the 360 kDa half proteasome from Thermoplasma acidophilium. The approach is based on exact quantification of the NOE (eNOE). Our findings open up an avenue for such measurements on very large molecules. These restraints will help in a detailed determination of conformational changes upon perturbation such as ligand binding.

Keywords
eNOE, exact NOE, large molecule NMR, nuclear Overhauser effect, proteasome
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-337644 (URN)10.1002/chem.201705551 (DOI)000424792600032 ()29265588 (PubMedID)
Funder
Wenner-Gren Foundations
Available from: 2018-01-03 Created: 2018-01-03 Last updated: 2018-04-11Bibliographically 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
Jemth, P., Karlsson, E., Vogeli, B., Guzovsky, B., Andersson, E., Hultqvist, G., . . . Chi, C. N. (2018). Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins. Science Advances, 4(10), Article ID eaau4130.
Open this publication in new window or tab >>Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins
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2018 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 4, no 10, article id eaau4130Article in journal (Refereed) Published
Abstract [en]

In every established species, protein-protein interactions have evolved such that they are fit for purpose. However, the molecular details of the evolution of new protein-protein interactions are poorly understood. We have used nuclear magnetic resonance spectroscopy to investigate the changes in structure and dynamics during the evolution of a protein-protein interaction involving the intrinsically disordered CREBBP (CREB-binding protein) interaction domain (CID) and nuclear coactivator binding domain (NCBD) from the transcriptional coregulators NCOA (nuclear receptor coactivator) and CREBBP/p300, respectively. The most ancient low-affinity "Cambrian-like" [540 to 600 million years (Ma) ago] CID/NCBD complex contained less secondary structure and was more dynamic than the complexes from an evolutionarily younger "Ordovician-Silurian" fish ancestor (ca. 440 Ma ago) and extant human. The most ancient Cambrian-like CID/NCBD complex lacked one helix and several interdomain interactions, resulting in a larger solvent-accessible surface area. Furthermore, the most ancient complex had a high degree of millisecond-to-microsecond dynamics distributed along the entire sequences of both CID and NCBD. These motions were reduced in the Ordovician-Silurian CID/NCBD complex and further redistributed in the extant human CID/NCBD complex. Isothermal calorimetry experiments show that complex formation is enthalpically favorable and that affinity is modulated by a largely unfavorable entropic contribution to binding. Our data demonstrate how changes in structure and motion conspire to shape affinity during the evolution of a protein-protein complex and provide direct evidence for the role of structural, dynamic, and frustrational plasticity in the evolution of interactions between intrinsically disordered proteins.

Place, publisher, year, edition, pages
AMER ASSOC ADVANCEMENT SCIENCE, 2018
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-369756 (URN)10.1126/sciadv.aau4130 (DOI)000449221200069 ()30397651 (PubMedID)
Funder
Swedish Research Council
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2018-12-17Bibliographically approved
Strotz, D., Orts, J., Chi, C. N., Riek, R. & Vögeli, B. (2017). eNORA2 Exact NOE Analysis Program. Journal of Chemical Theory and Computation, 13(9), 4336-4346
Open this publication in new window or tab >>eNORA2 Exact NOE Analysis Program
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2017 (English)In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 13, no 9, p. 4336-4346Article in journal (Refereed) Published
Abstract [en]

We have recently developed an NMR protocol to extract exact distances between nuclei in proteins from an exact interpretation of NOESY buildup intensities (eNOEs). This enabled us to calculate multistate structural ensembles that exhibit realistic spatial sampling and long-range correlations. Our initial studies were laborious and required a deep understanding of the underlying spin dynamics. Here, we present a MatLab package that integrates all data processing steps required to convert intensities of assigned peaks in NOESY series into upper and lower distance limits for structure calculation. Those steps include organization of the data in object format, extraction of autorelaxation and cross-relaxation rate constants by fitting of diagonal peak decays and cross peak buildups, validation of the data, correction for spin diffusion, graphical display of the results, and generation of distance limits in CYANA compatible format. The analysis may be carried out using a full relaxation matrix or a simplified "divide and conquer" approach that allows for partial deuteration of protons. As the program does not require expertise beyond that of standard resonance assignment/structure calculation, it is suitable for experts and nonexperts alike.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-331631 (URN)10.1021/acs.jctc.7b00436 (DOI)000410867500034 ()28727914 (PubMedID)
Available from: 2017-10-16 Created: 2017-10-16 Last updated: 2018-01-25Bibliographically approved
Nichols, P. J., Born, A., Henen, M. A., Strotz, D., Orts, J., Olsson, S., . . . Vögeli, B. (2017). The Exact Nuclear Overhauser Enhancement: Recent Advances. Molecules, 22(7), Article ID 1176.
Open this publication in new window or tab >>The Exact Nuclear Overhauser Enhancement: Recent Advances
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2017 (English)In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 22, no 7, article id 1176Article, review/survey (Refereed) Published
Abstract [en]

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.

Keywords
NMR, allostery, biological macromolecules, conformational space, correlated dynamics, dynamics, exact NOE, proteins, structure calculation, structure ensemble
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-331630 (URN)10.3390/molecules22071176 (DOI)000406621300150 ()28708092 (PubMedID)
Funder
Wenner-Gren Foundations, WG-17
Available from: 2017-10-16 Created: 2017-10-16 Last updated: 2019-02-22Bibliographically approved
Toto, A., Pedersen, S. W., Karlsson, O. A., Moran, G. E., Andersson, E., Chi, C. N., . . . Jemth, P. (2016). Ligand binding to the PDZ domains of postsynaptic density protein 95. Protein Engineering Design & Selection, 29(5), 169-175
Open this publication in new window or tab >>Ligand binding to the PDZ domains of postsynaptic density protein 95
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2016 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 29, no 5, p. 169-175Article in journal (Refereed) Published
Abstract [en]

Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i) N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain.

Keywords
CRIPT, GluN2B, Kinetics, PDZ domain, PSD-95
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-298245 (URN)10.1093/protein/gzw004 (DOI)000376351600002 ()26941280 (PubMedID)
Funder
Swedish Research Council, 2012-5096
Available from: 2016-07-01 Created: 2016-07-01 Last updated: 2017-11-28Bibliographically approved
Karlsson, O. A., Ramirez, J., Öberg, D., Malmqvist, T., Engström, Å., Friberg, M., . . . Jemth, P. (2015). Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus. Scientific Reports, 5, Article ID 9382.
Open this publication in new window or tab >>Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 9382Article in journal (Refereed) Published
Abstract [en]

Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (K-d = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-258849 (URN)10.1038/srep09382 (DOI)000351373500005 ()25797137 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2015-07-23 Created: 2015-07-20 Last updated: 2017-12-04Bibliographically approved
Bach, A., Clausen, B. H., Moller, M., Vestergaard, B., Chi, C. N., Round, A., . . . Stromgaard, K. (2012). A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage. Proceedings of the National Academy of Sciences of the United States of America, 109(9), 3317-3322
Open this publication in new window or tab >>A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage
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2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 9, p. 3317-3322Article in journal (Refereed) Published
Abstract [en]

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

Keywords
drug discovery, ischemic stroke, protein-protein interactions
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-171422 (URN)10.1073/pnas.1113761109 (DOI)000300828200031 ()
Available from: 2012-03-19 Created: 2012-03-19 Last updated: 2017-12-07Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-4154-2378

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