scholarly journals The dynamic properties of a nuclear coactivator binding domain are evolutionarily conserved

2021 ◽  
Author(s):  
Elin Karlsson ◽  
Frieda A Sorgenfrei ◽  
Eva Andersson ◽  
Jakob Dogan ◽  
Per Jemth ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Dynamic Properties ◽  
Binding Domain ◽  
Disordered Proteins ◽  
Structural Constraints ◽  
Hydrophobic Core ◽  
Creb Binding Protein ◽  
Biophysical Properties ◽  
Interaction Domain ◽  
Intrinsically Disordered

Evolution of proteins is constrained by their structure and function. While there is a consensus that the plasticity of intrinsically disordered proteins relaxes the structural constraints on evolution there is a paucity of data on the molecular details of these processes. The Nuclear co-activator binding domain (NCBD) from CREB-binding protein is a protein-protein interaction domain, which contains a hydrophobic core but is verging on being intrinsically disordered. These highly dynamic 'borderline' properties of NCBD makes it an interesting model system for evolutionary structure-function investigation. We have here compared the structure and biophysical properties of an ancient version of NCBD present in a bilaterian animal ancestor living around 600 million years ago with extant human NCBD. Using a combination of NMR spectroscopy, circular dichroism and kinetic methods we show that NCBD has retained its structure and dynamic biophysical properties in the ligand-free state in the evolutionary lineage leading from the bilaterian ancestor to humans. Our findings suggest that the dynamic properties of NCBD are subject to positive selection and thus important for its function, which includes mediating several distinct protein-protein interactions.

scholarly journals Co-Evolution of Intrinsically Disordered Proteins with Folded Partners Witnessed by Evolutionary Couplings

2018 ◽  
Vol 19 (11) ◽  
pp. 3315 ◽  
Author(s):  
Rita Pancsa ◽  
Fruzsina Zsolyomi ◽  
Peter Tompa
Keyword(s):  
Large Scale ◽  
Intrinsically Disordered Proteins ◽  
Protein Structures ◽  
Disordered Proteins ◽  
Cellular Interaction ◽  
Structural Constraints ◽  
Protein Residues ◽  
Intrinsically Disordered ◽  
Evolutionary Changes ◽  
Folded Proteins

Although improved strategies for the detection and analysis of evolutionary couplings (ECs) between protein residues already enable the prediction of protein structures and interactions, they are mostly restricted to conserved and well-folded proteins. Whereas intrinsically disordered proteins (IDPs) are central to cellular interaction networks, due to the lack of strict structural constraints, they undergo faster evolutionary changes than folded domains. This makes the reliable identification and alignment of IDP homologs difficult, which led to IDPs being omitted in most large-scale residue co-variation analyses. By preforming a dedicated analysis of phylogenetically widespread bacterial IDP–partner interactions, here we demonstrate that partner binding imposes constraints on IDP sequences that manifest in detectable interprotein ECs. These ECs were not detected for interactions mediated by short motifs, rather for those with larger IDP–partner interfaces. Most identified coupled residue pairs reside close (<10 Å) to each other on the interface, with a third of them forming multiple direct atomic contacts. EC-carrying interfaces of IDPs are enriched in negatively charged residues, and the EC residues of both IDPs and partners preferentially reside in helices. Our analysis brings hope that IDP–partner interactions difficult to study could soon be successfully dissected through residue co-variation analysis.

scholarly journals Enhancing Binding Affinity of an Intrinsically Disordered Protein by α-Methylation of Key Amino Acid Residues

2019 ◽  
Author(s):  
Valentin Bauer ◽  
Boris Schmidtgall ◽  
Gergő Gógl ◽  
Jozica Dolenc ◽  
Judit Osz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Intrinsically Disordered Protein ◽  
Selective Inhibition ◽  
Disordered Proteins ◽  
Creb Binding Protein ◽  
Alpha Helices ◽  
Intrinsically Disordered ◽  
Order Of Magnitude

Intrinsically disordered proteins (IDPs), which undergo folding upon binding to their targets, are critical players in protein interaction networks. Here we demonstrate that incorporation of non-canonical alpha-methylated amino acids into the unstructured activation domain of the transcriptional coactivator ACTR can stabilize helical conformations and strengthen binding interactions with the nuclear coactivator binding domain (NCBD) of CREB-binding protein (CBP). A combinatorial alpha-methylation scan of the ACTR sequence converged on two substitutions at positions 1055 and 1076 that increase affinity for both NCBD and the full length 270 kDa CBP by one order of magnitude. The first X-ray structure of the modified ACTR domain bound to NCBD revealed that the key alpha-methylated amino acids were localized within alpha-helices. Biophysical studies showed that the observed changes in binding energy are the result of long-range interactions and redistribution of enthalpy and entropy. This proof-of-concept study establishes a potential strategy for selective inhibition of protein-protein interactions involving IDPs in cells.<br>

scholarly journals Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and Its Domains and How This Is Modulated upon Interaction with Cdk2/cyclin A

2018 ◽  
Author(s):  
Rebecca Beveridge ◽  
Lukasz Migas ◽  
Richard Kriwacki ◽  
Perdita E. Barran
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Intrinsically Disordered Proteins ◽  
Dynamic Properties ◽  
Cyclin A ◽  
Structural Heterogeneity ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Trimeric Complex ◽  
Conformational Landscape

Intrinsically disordered proteins have been reported to undergo ‘disorder to order’ transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states.

scholarly journals Multivalency enables unidirectional switch-like competition between intrinsically disordered proteins

2022 ◽  
Vol 119 (3) ◽  
pp. e2117338119
Author(s):  
Rebecca B. Berlow ◽  
H. Jane Dyson ◽  
Peter E. Wright
Keyword(s):  
Transcriptional Control ◽  
Intrinsically Disordered Proteins ◽  
Binding Protein ◽  
Critical Role ◽  
Molecular Switch ◽  
Negative Regulator ◽  
Disordered Proteins ◽  
Creb Binding Protein ◽  
Binding Motifs ◽  
Intrinsically Disordered

Intrinsically disordered proteins must compete for binding to common regulatory targets to carry out their biological functions. Previously, we showed that the activation domains of two disordered proteins, the transcription factor HIF-1α and its negative regulator CITED2, function as a unidirectional, allosteric molecular switch to control transcription of critical adaptive genes under conditions of oxygen deprivation. These proteins achieve transcriptional control by competing for binding to the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300 (CREB: cyclic-AMP response element binding protein). To characterize the mechanistic details behind this molecular switch, we used solution NMR spectroscopy and complementary biophysical methods to determine the contributions of individual binding motifs in CITED2 to the overall competition process. An N-terminal region of the CITED2 activation domain, which forms a helix when bound to TAZ1, plays a critical role in initiating competition with HIF-1α by enabling formation of a ternary complex in a process that is highly dependent on the dynamics and disorder of the competing partners. Two other conserved binding motifs in CITED2, the LPEL motif and an aromatic/hydrophobic motif that we term ϕC, function synergistically to enhance binding of CITED2 and inhibit rebinding of HIF-1α. The apparent unidirectionality of competition between HIF-1α and CITED2 is lost when one or more of these binding regions is altered by truncation or mutation of the CITED2 peptide. Our findings illustrate the complexity of molecular interactions involving disordered proteins containing multivalent interaction motifs and provide insight into the unique mechanisms by which disordered proteins compete for occupancy of common molecular targets within the cell.

scholarly journals Ion Mobility Mass Spectrometry Measures the Conformational Landscape of P27 and Its Domains and How This Is Modulated upon Interaction with Cdk2/cyclin A

2018 ◽  
Author(s):  
Rebecca Beveridge ◽  
Lukasz Migas ◽  
Richard Kriwacki ◽  
Perdita E. Barran
Keyword(s):  
Mass Spectrometry ◽  
Ion Mobility ◽  
Intrinsically Disordered Proteins ◽  
Dynamic Properties ◽  
Cyclin A ◽  
Structural Heterogeneity ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Trimeric Complex ◽  
Conformational Landscape

Intrinsically disordered proteins have been reported to undergo ‘disorder to order’ transitions upon binding to their partners in the cell. The extent of the ordering on binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behaviour for function, particularly in the C-terminal half of the protein, positioning it as an exemplary system to probe conformational diversity. Here we employ native ion mobility with mass spectrometry (IM-MS) to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. This stepwise approach reveals the conformational distributions of the constituent proteins and how they are restructured on complex formation; the trimeric Cdk2/cyclin A/p27-KID complex possesses significant structural heterogeneity cf. Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N and C termini of p27 interact with Cdk2/cyclin A in multiple closely associated states.

scholarly journals Evolutionary forces on different flavors of intrinsic disorder in the human proteome

2019 ◽  
Author(s):  
Sergio Forcelloni ◽  
Andrea Giansanti
Keyword(s):  
Natural Selection ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Structural Disorder ◽  
Disordered Proteins ◽  
Mutational Bias ◽  
Structural Constraints ◽  
Intrinsically Disordered ◽  
Evolutionary Forces ◽  
Functional Variants

ABSTRACTIn this study, we perform a systematic analysis of evolutionary forces (i.e., mutational bias and natural selection) that shape the codon usage bias of human genes encoding for different structural and functional variants of proteins. Well-structured proteins are expected to be more under control by natural selection than intrinsically disordered proteins because one or few mutations (even synonymous) in the genes can result in a protein that no longer folds correctly. On the contrary, intrinsically disordered proteins are generally thought to evolve more rapidly than well-folded proteins, primarily attributed to relaxed purifying natural selection due to the lack of structural constraints. Using different genetic tools, we find compelling evidence that intrinsically disordered proteins are the variant of human proteins on which both mutational bias and natural selection act more effectively, corroborating their essential role for evolutionary adaptability and protein evolvability. We speculate that intrinsically disordered proteins have a high tolerance to mutations (both neutral and adaptive) but also a selective propensity to preserve their structural disorder, i.e., flexibility and conformational dynamics under physiological conditions. Additionally, we confirm not only that intrinsically disordered proteins are preferentially encoded by GC-rich genes, but also that they are characterized by the highest fraction of CpG-sites in the sequences, implying a higher susceptibility to methylation resulting in C-T transition mutations. Our results provide new insight about protein evolution and human genetic diseases identifying intrinsically disordered proteins as reservoirs for evolutionary innovations.

scholarly journals Solvent-dependent segmental dynamics in intrinsically disordered proteins

2019 ◽  
Vol 5 (6) ◽  
pp. eaax2348 ◽  
Author(s):  
Nicola Salvi ◽  
Anton Abyzov ◽  
Martin Blackledge
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Dynamic Properties ◽  
Nmr Relaxation ◽  
Bulk Water ◽  
Water Dynamics ◽  
Disordered Proteins ◽  
Segmental Dynamics ◽  
Intrinsically Disordered ◽  
Dynamics Simulations ◽  
And Function

Protein and water dynamics have a synergistic relationship, which is particularly important for intrinsically disordered proteins (IDPs), although the details of this coupling remain poorly understood. Here, we combine temperature-dependent molecular dynamics simulations using different water models with extensive nuclear magnetic resonance (NMR) relaxation to examine the importance of distinct modes of solvent and solute motion for the accurate reproduction of site-specific dynamics in IDPs. We find that water dynamics play a key role in motional processes internal to “segments” of IDPs, stretches of primary sequence that share dynamic properties and behave as discrete dynamic units. We identify a relationship between the time scales of intrasegment dynamics and the lifetime of hydrogen bonds in bulk water. Correct description of these motions is essential for accurate reproduction of protein relaxation. Our findings open important perspectives for understanding the role of hydration water on the behavior and function of IDPs in solution.

scholarly journals Adenoviral E1A Exploits Flexibility and Disorder to Target Cellular Proteins

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1541
Author(s):  
Maria Grazia Murrali ◽  
Isabella C. Felli ◽  
Roberta Pierattelli
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Direct Interaction ◽  
Disordered Proteins ◽  
Resonance Spectroscopy ◽  
Creb Binding Protein ◽  
Adenovirus E1a ◽  
Intrinsically Disordered ◽  
Functional Aspects ◽  
Conformational Freedom

Direct interaction between intrinsically disordered proteins (IDPs) is often difficult to characterize hampering the elucidation of their binding mechanism. Particularly challenging is the study of fuzzy complexes, in which the intrinsically disordered proteins or regions retain conformational freedom within the assembly. To date, nuclear magnetic resonance spectroscopy has proven to be one of the most powerful techniques to characterize at the atomic level intrinsically disordered proteins and their interactions, including those cases where the formed complexes are highly dynamic. Here, we present the characterization of the interaction between a viral protein, the Early region 1A protein from Adenovirus (E1A), and a disordered region of the human CREB-binding protein, namely the fourth intrinsically disordered linker CBP-ID4. E1A was widely studied as a prototypical viral oncogene. Its interaction with two folded domains of CBP was mapped, providing hints for understanding some functional aspects of the interaction with this transcriptional coactivator. However, the role of the flexible linker connecting these two globular domains of CBP in this interaction was never explored before.

scholarly journals Enhancing Binding Affinity of an Intrinsically Disordered Protein by α-Methylation of Key Amino Acid Residues

2019 ◽  
Author(s):  
Valentin Bauer ◽  
Boris Schmidtgall ◽  
Gergő Gógl ◽  
Jozica Dolenc ◽  
Judit Osz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Intrinsically Disordered Protein ◽  
Selective Inhibition ◽  
Disordered Proteins ◽  
Creb Binding Protein ◽  
Alpha Helices ◽  
Intrinsically Disordered ◽  
Order Of Magnitude

Intrinsically disordered proteins (IDPs), which undergo folding upon binding to their targets, are critical players in protein interaction networks. Here we demonstrate that incorporation of non-canonical alpha-methylated amino acids into the unstructured activation domain of the transcriptional coactivator ACTR can stabilize helical conformations and strengthen binding interactions with the nuclear coactivator binding domain (NCBD) of CREB-binding protein (CBP). A combinatorial alpha-methylation scan of the ACTR sequence converged on two substitutions at positions 1055 and 1076 that increase affinity for both NCBD and the full length 270 kDa CBP by one order of magnitude. The first X-ray structure of the modified ACTR domain bound to NCBD revealed that the key alpha-methylated amino acids were localized within alpha-helices. Biophysical studies showed that the observed changes in binding energy are the result of long-range interactions and redistribution of enthalpy and entropy. This proof-of-concept study establishes a potential strategy for selective inhibition of protein-protein interactions involving IDPs in cells.<br>

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