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Publications (10 of 88) Show all publications
Karlsson, E., Andersson, E., Dogan, J., Gianni, S., Jemth, P. & Camilloni, C. (2019). A structurally heterogeneous transition state underlies coupled binding and folding of disordered proteins. Journal of Biological Chemistry, 294(4), 1230-1239
Open this publication in new window or tab >>A structurally heterogeneous transition state underlies coupled binding and folding of disordered proteins
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2019 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, no 4, p. 1230-1239Article in journal (Refereed) Published
Abstract [en]

Many intrinsically disordered proteins (IDPs) attain a well-defined structure in a coupled folding and binding reaction with another protein. Such reactions may involve early to late formation of different native structural regions along the reaction pathway. To obtain insights into the transition state for a coupled binding and folding reaction, we performed restrained molecular dynamics simulations using previously determined experimental binding phi(b) values of the interaction between two IDP domains: the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors (ACTR) and the nuclear co-activator binding domain (NCBD) of CREB-binding protein, each forming three well-defined alpha-helices upon binding. These simulations revealed that both proteins are largely disordered in the transition state for complex formation, except for two helices, one from each domain, that display a native-like structure. The overall transition state structure was extended and largely dynamic with many weakly populated contacts. To test the transition state model, we combined site-directed mutagenesis with kinetic experiments, yielding results consistent with overall diffuse interactions and formation of native intramolecular interactions in the third NCBD helix during the binding reaction. Our findings support the view that the transition state and, by inference, any encounter complex in coupled binding and folding reactions are structurally heterogeneous and largely independent of specific interactions. Furthermore, experimental phi(b) values and Bronsted plots suggested that the transition state is globally robust with respect to most mutations but can display more native-like features for some highly destabilizing mutations, possibly because of Hammond behavior or ground-state effects.

Keywords
intrinsically disordered protein, pre-steady-state kinetics, protein folding, protein-protein interaction, molecular dynamics
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-377701 (URN)10.1074/jbc.RA118.005854 (DOI)000457879500014 ()30514761 (PubMedID)
Funder
Swedish Research Council, 2016-04965
Available from: 2019-02-25 Created: 2019-02-25 Last updated: 2019-02-25Bibliographically approved
Ivarsson, Y. & Jemth, P. (2019). Affinity and specificity of motif-based protein-protein interactions. Current opinion in structural biology, 54, 26-33
Open this publication in new window or tab >>Affinity and specificity of motif-based protein-protein interactions
2019 (English)In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 54, p. 26-33Article, review/survey (Refereed) Published
Abstract [en]

It is becoming increasingly clear that eukaryotic cell physiology is largely controlled by protein protein interactions involving disordered protein regions, which usually interact with globular domains in a coupled binding and folding reaction. Several protein recognition domains are part of large families where members can interact with similar peptide ligands. Because of this, much research has been devoted to understanding how specificity can be achieved. A combination of interface complementarity, interactions outside of the core binding site, avidity from multidomain architecture and spatial and temporal regulation of expression resolves the conundrum. Here, we review recent advances in molecular aspects of affinity and specificity in such protein-protein interactions.

Place, publisher, year, edition, pages
Current Biology, 2019
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-362357 (URN)10.1016/j.sbi.2018.09.009 (DOI)000473554100005 ()30368054 (PubMedID)
Funder
Swedish Foundation for Strategic Research , SB16-0039Swedish Research Council, 2016-04965Swedish Research Council, 2016-04134
Available from: 2018-10-03 Created: 2018-10-03 Last updated: 2019-08-08Bibliographically approved
Karlsson, E., Andersson, E., Jones, N. C., Hoffmann, S. V., Jemth, P. & Kjaergaard, M. (2019). Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism. Biophysical Journal, 117(4), 729-742
Open this publication in new window or tab >>Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism
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2019 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 117, no 4, p. 729-742Article in journal (Refereed) Published
Abstract [en]

Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.

Place, publisher, year, edition, pages
CELL PRESS, 2019
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-393728 (URN)10.1016/j.bpj.2019.07.014 (DOI)000482097100011 ()31378314 (PubMedID)
Funder
Swedish Research Council, 2016-04965
Available from: 2019-09-27 Created: 2019-09-27 Last updated: 2019-09-27Bibliographically approved
Ivarsson, Y. & Jemth, P. (2019). Editorial overview: Folding and binding. Current opinion in structural biology, 54, 139-140
Open this publication in new window or tab >>Editorial overview: Folding and binding
2019 (English)In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 54, p. 139-140Article in journal, Editorial material (Other academic) Published
Place, publisher, year, edition, pages
CURRENT BIOLOGY LTD, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390346 (URN)10.1016/j.sbi.2019.02.006 (DOI)000473554100018 ()30901729 (PubMedID)
Available from: 2019-08-08 Created: 2019-08-08 Last updated: 2019-08-08Bibliographically approved
Åberg, E., Karlsson, O. A., Andersson, E. & Jemth, P. (2018). Binding Kinetics of the Intrinsically Disordered p53 Family Transactivation Domains and MDM2. Journal of Physical Chemistry B, 122(27), 6899-6905
Open this publication in new window or tab >>Binding Kinetics of the Intrinsically Disordered p53 Family Transactivation Domains and MDM2
2018 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 122, no 27, p. 6899-6905Article in journal (Refereed) Published
Abstract [en]

Because of their prominent roles in cell-cycle regulation and cancer, the interaction between MDM2 and the intrinsically disordered transactivation domain (TAD) of p53 is exceptionally well-studied. However, although there are numerous computational studies on the interaction mechanism, there is a paucity of experimental data regarding the kinetics and mechanism. We have used stopped flow fluorescence to investigate the binding reaction between MDM2 and TAD from p53 as well as from its paralogs p63 and p73, and in particular, focused on the salt dependence of the interaction. The observed kinetics are consistent with a two-state mechanism within the time frame of the stopped flow methodology; thus, any conformational changes including the previously identified MDM2 lid dynamics must occur on a time scale <5 ms at 10 °C. The association rate constants are similar for the three TADs, and differences in the dissociation rate constants determine the various affinities with MDM2. In contrast to previous studies, we found a relatively small ionic-strength dependence for all three interactions, highlighting the large variation in the role of electrostatics among binding reactions of intrinsically disordered proteins (IDPs). The basal association rate constants in the absence of electrostatic interactions were relatively high (≥2 × 106 M–1 s–1 at 10 °C), suggesting that a large number of initial contacts may lead to a productive complex. Our findings support an emerging picture of “conformational funneling” occurring in the initial stages of interactions involving IDPs and that these early binding events can rely on hydrophobic as well as charge–charge interactions.

National Category
Biochemistry and Molecular Biology Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-361528 (URN)10.1021/acs.jpcb.8b03876 (DOI)000439002900006 ()29878773 (PubMedID)
Funder
Swedish Research Council, 2016-04134
Available from: 2018-09-25 Created: 2018-09-25 Last updated: 2018-09-25Bibliographically approved
Sereikaite, V., Jensen, T. M. T., Bartling, C. R. O., Jemth, P., Pless, S. A. & Stromgaard, K. (2018). Probing Backbone Hydrogen Bonds in Proteins by Amide-to-Ester Mutations. ChemBioChem (Print), 19(20), 2136-2145
Open this publication in new window or tab >>Probing Backbone Hydrogen Bonds in Proteins by Amide-to-Ester Mutations
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2018 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 20, p. 2136-2145Article, review/survey (Refereed) Published
Abstract [en]

All proteins contain characteristic backbones formed of consecutive amide bonds, which can engage in hydrogen bonds. However, the importance of these is not easily addressed by conventional technologies that only allow for side-chain substitutions. By contrast, technologies such as nonsense suppression mutagenesis and protein ligation allow for manipulation of the protein backbone. In particular, replacing the backbone amide groups with ester groups, that is, amide-to-ester mutations, is a powerful tool to examine backbone-mediated hydrogen bonds. In this minireview, we showcase examples of how amide-to-ester mutations can be used to uncover pivotal roles of backbone-mediated hydrogen bonds in protein recognition, folding, function, and structure.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2018
Keywords
amide-to-ester mutations, hydrogen bonds, protein backbone, proteins, structure and function
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-369617 (URN)10.1002/cbic.201800350 (DOI)000447635300002 ()30073762 (PubMedID)
Funder
Swedish Research Council
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2018-12-17Bibliographically 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
Gautier, C., Visconti, L., Jemth, P. & Gianni, S. (2017). Addressing the role of the alpha-helical extension in the folding of the third PDZ domain from PSD-95. Scientific Reports, 7, Article ID 12593.
Open this publication in new window or tab >>Addressing the role of the alpha-helical extension in the folding of the third PDZ domain from PSD-95
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 12593Article in journal (Refereed) Published
Abstract [en]

PDZ domains are one of the most important protein-protein interaction domains in human. While presenting a conserved three dimensional structure, a substantial number of PDZ domains display structural extensions suggested to be involved in their folding and binding mechanisms. The C-terminal a-helix extension (alpha 3) of the third PDZ domain from PSD-95 (PDZ3) has been reported to have a role in function of the domain as well as in the stabilization of the native fold. Here we report an evaluation of the effect of the truncation of this additional helix on the folding and unfolding kinetics of PDZ3. Fluorescent variants of full length and truncated PDZ3 were produced and stopped-flow fluorescence measurements were made under different experimental conditions (pH, ionic strength and temperature) to investigate the folding kinetics of the respective variant. The results show that folding of PDZ3 is robust and that the mechanism is only marginally affected by the truncation, which contributes to a destabilization of the native state, but otherwise do not change the overall observed kinetics. Furthermore, the increase in the unfolding rate constants, but not the folding rate constant upon deletion of alpha 3 suggests that the a-helical extension is largely unstructured in the folding transition state.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-337114 (URN)10.1038/s41598-017-12827-0 (DOI)000412138800029 ()28974728 (PubMedID)
Funder
EU, Horizon 2020, 675341
Available from: 2017-12-21 Created: 2017-12-21 Last updated: 2017-12-21Bibliographically approved
Hultqvist, G., Åberg, E., Camilloni, C., Sundell, G., Andersson, E., Dogan, J., . . . Jemth, P. (2017). Emergence and evolution of an interaction between intrinsically disordered proteins. eLIFE, 6, Article ID e16059.
Open this publication in new window or tab >>Emergence and evolution of an interaction between intrinsically disordered proteins
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2017 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e16059Article in journal (Refereed) Published
Abstract [en]

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 (K(d similar to)5 mu M). At the time of the first vertebrate-specific whole genome duplication, the affinity had increased (K-d\similar to 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.

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-322818 (URN)10.7554/eLife.16059 (DOI)000400663100001 ()
Available from: 2017-09-12 Created: 2017-09-12 Last updated: 2018-01-28Bibliographically approved
Åberg, E., Saccoccia, F., Grabherr, M., Ore, W. Y., Jemth, P. & Hultqvist, G. (2017). Evolution of the p53-MDM2 pathway. BMC Evolutionary Biology, 17, Article ID 177.
Open this publication in new window or tab >>Evolution of the p53-MDM2 pathway
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2017 (English)In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 17, article id 177Article in journal (Refereed) Published
Abstract [en]

Background: The p53 signalling pathway, which controls cell fate, has been extensively studied due to its prominent role in tumor development. The pathway includes the tumor supressor protein p53, its vertebrate paralogs p63 and p73, and their negative regulators MDM2 and MDM4. The p53/p63/p73-MDM system is ancient and can be traced in all extant animal phyla. Despite this, correct phylogenetic trees including both vertebrate and invertebrate species of the p53/p63/p73 and MDM families have not been published. Results: Here, we have examined the evolution of the p53/p63/p73 protein family with particular focus on the p53/ p63/p73 transactivation domain (TAD) and its co-evolution with the p53/p63/p73- binding domain (p53/p63/p73BD) of MDM2. We found that the TAD and p53/p63/p73BD share a strong evolutionary connection. If one of the domains of the protein is lost in a phylum, then it seems very likely to be followed by loss of function by the other domain as well, and due to the loss of function it is likely to eventually disappear. By focusing our phylogenetic analysis to p53/p63/ p73 and MDM proteins from phyla that retain the interaction domains TAD and p53/p63/p73BD, we built phylogenetic trees of p53/p63/p73 and MDM based on both vertebrate and invertebrate species. The trees follow species evolution and contain a total number of 183 and 98 species for p53/p63/p73 and MDM, respectively. We also demonstrate that the p53/p63/p73 and MDM families result from whole genome duplications. Conclusions: The signaling pathway of the TAD and p53/p63/p73BD in p53/p63/p73 and MDM, respectively, dates back to early metazoan time and has since then tightly co-evolved, or disappeared in distinct lineages.

Place, publisher, year, edition, pages
BioMed Central, 2017
Keywords
p53, MDM, Co-evolution, Phylogeny
National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-334047 (URN)10.1186/s12862-017-1023-y (DOI)000407013500001 ()28774266 (PubMedID)
Available from: 2017-11-21 Created: 2017-11-21 Last updated: 2018-01-28Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-1516-7228

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