Conservation of Affinity Rather Than Sequence Underlies a Dynamic Evolution of the Motif-Mediated p53/MDM2 Interaction in Ray-Finned Fishes.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 µM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of "functional affinity" in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Intrinsically Disordered Flanking Regions Increase the Affinity of a Transcriptional Coactivator Interaction across Vertebrates.

Interactions between two proteins are often mediated by a disordered region in one protein binding to a groove in a folded interaction domain in the other one. While the main determinants of a certain interaction are typically found within a well-defined binding interface involving the groove, recent studies show that nonspecific contacts by flanking regions may increase the affinity. One example is the coupled binding and folding underlying the interaction between the two transcriptional coactivators NCOA3 (ACTR) and CBP, where the flanking regions of an intrinsically disordered region in human NCOA3 increases the affinity for CBP. However, it is not clear whether this flanking region-mediated effect is a peculiarity of this single protein interaction or if it is of functional relevance in a broader context. To further assess the role of flanking regions in the interaction between NCOA3 and CBP, we analyzed the interaction across orthologs and paralogs (NCOA1, 2, and 3) in human, zebra fish, and ghost shark. We found that flanking regions increased the affinity 2- to 9-fold in the six interactions tested. Conservation of the amino acid sequence is a strong indicator of function. Analogously, the observed conservation of increased affinity provided by flanking regions, accompanied by moderate sequence conservation, suggests that flanking regions may be under selection to promote the affinity between NCOA transcriptional coregulators and CBP.

Proteome-scale mapping of binding sites in the unstructured regions of the human proteome.

Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type-specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide-phage display library that tiles all disordered regions of the human proteome and allows the screening of ~ 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)-binding domains and confirmed the quality of the produced data by complementary biophysical or cell-based assays. Finally, we show how the amino acid resolution-binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteome-wide discovery of SLiM-based interactions.

Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions.

Low affinity and transient protein-protein interactions, such as short linear motif (SLiM)-based interactions, require dedicated experimental tools for discovery and validation. Here, we evaluated and compared biotinylated peptide pulldown and protein interaction screen on peptide matrix (PRISMA) coupled to mass-spectrometry (MS) using a set of peptides containing interaction motifs. Eight different peptide sequences that engage in interactions with three distinct protein domains (KEAP1 Kelch, MDM2 SWIB, and TSG101 UEV) with a wide range of affinities were tested. We found that peptide pulldown can be an effective approach for SLiM validation, however, parameters such as protein abundance and competitive interactions can prevent the capture of known interactors. The use of tandem peptide repeats improved the capture and preservation of some interactions. When testing PRISMA, it failed to provide comparable results for model peptides that successfully pulled down known interactors using biotinylated peptide pulldown. Overall, in our hands, we find that albeit more laborious, biotin-peptide pulldown was more successful in terms of validation of known interactions. Our results highlight that the tested affinity-capture MS-based methods for validation of SLiM-based interactions from cell lysates are suboptimal, and we identified parameters for consideration for method development.

Supertertiary protein structure affects an allosteric network.

The notion that protein function is allosterically regulated by structural or dynamic changes in proteins has been extensively investigated in several protein domains in isolation. In particular, PDZ domains have represented a paradigm for these studies, despite providing conflicting results. Furthermore, it is still unknown how the association between protein domains in supramodules, consitituting so-called supertertiary structures, affects allosteric networks. Here, we experimentally mapped the allosteric network in a PDZ:ligand complex, both in isolation and in the context of a supramodular structure, and show that allosteric networks in a PDZ domain are highly dependent on the supertertiary structure in which they are present. This striking sensitivity of allosteric networks to the presence of adjacent protein domains is likely a common property of supertertiary structures in proteins. Our findings have general implications for prediction of allosteric networks from primary and tertiary structures and for quantitative descriptions of allostery.

Divergent Evolution of a Protein-Protein Interaction Revealed through Ancestral Sequence Reconstruction and Resurrection.

The postsynaptic density extends across the postsynaptic dendritic spine with discs large (DLG) as the most abundant scaffolding protein. DLG dynamically alters the structure of the postsynaptic density, thus controlling the function and distribution of specific receptors at the synapse. DLG contains three PDZ domains and one important interaction governing postsynaptic architecture is that between the PDZ3 domain from DLG and a protein called cysteine-rich interactor of PDZ3 (CRIPT). However, little is known regarding functional evolution of the PDZ3:CRIPT interaction. Here, we subjected PDZ3 and CRIPT to ancestral sequence reconstruction, resurrection, and biophysical experiments. We show that the PDZ3:CRIPT interaction is an ancient interaction, which was likely present in the last common ancestor of Eukaryotes, and that high affinity is maintained in most extant animal phyla. However, affinity is low in nematodes and insects, raising questions about the physiological function of the interaction in species from these animal groups. Our findings demonstrate how an apparently established protein-protein interaction involved in cellular scaffolding in bilaterians can suddenly be subject to dynamic evolution including possible loss of function.

Fuzziness and Frustration in the Energy Landscape of Protein Folding, Function, and Assembly.

Are all protein interactions fully optimized? Do suboptimal interactions compromise specificity? What is the functional impact of frustration? Why does evolution not optimize some contacts? Proteins and their complexes are best described as ensembles of states populating an energy landscape. These ensembles vary in breadth from narrow ensembles clustered around a single average X-ray structure to broader ensembles encompassing a few different functional "taxonomic" states on to near continua of rapidly interconverting conformations, which are called "fuzzy" or even "intrinsically disordered". Here we aim to provide a comprehensive framework for confronting the structural and dynamical continuum of protein assemblies by combining the concepts of energetic frustration and interaction fuzziness. The diversity of the protein structural ensemble arises from the frustrated conflicts between the interactions that create the energy landscape. When frustration is minimal after folding, it results in a narrow ensemble, but residual frustrated interactions result in fuzzy ensembles, and this fuzziness allows a versatile repertoire of biological interactions. Here we discuss how fuzziness and frustration play off each other as proteins fold and assemble, viewing their significance from energetic, functional, and evolutionary perspectives.We demonstrate, in particular, that the common physical origin of both concepts is related to the ruggedness of the energy landscapes, intramolecular in the case of frustration and intermolecular in the case of fuzziness. Within this framework, we show that alternative sets of suboptimal contacts may encode specificity without achieving a single structural optimum. Thus, we demonstrate that structured complexes may not be optimized, and energetic frustration is realized via different sets of contacts leading to multiplicity of specific complexes. Furthermore, we propose that these suboptimal, frustrated, or fuzzy interactions are under evolutionary selection and expand the biological repertoire by providing a multiplicity of biological activities. In accord, we show that non-native interactions in folding or interaction landscapes can cooperate to generate diverse functional states, which are essential to facilitate adaptation to different cellular conditions. Thus, we propose that not fully optimized structures may actually be beneficial for biological activities of proteins via an alternative set of suboptimal interactions. The importance of such variability has not been recognized across different areas of biology.This account provides a modern view on folding, function, and assembly across the protein universe. The physical framework presented here is applicable to the structure and dynamics continuum of proteins and opens up new perspectives for drug design involving not fully structured, highly dynamic protein assemblies.

How Fast Is Protein-Ligand Association?

There is increasing interest in studying protein interactions and their role in cell biology using kinetics. However, there is confusion about the proper terminology in terms of the distinction between rates and rate constants. We recommend a more stringent use of the words speed, fast, slow, rate, and rate constant.

Functional interplay between protein domains in a supramodular structure involving the postsynaptic density protein PSD-95.

Cell scaffolding and signaling are governed by protein-protein interactions. Although a particular interaction is often defined by two specific domains binding to each other, this interaction often occurs in the context of other domains in multidomain proteins. How such adjacent domains form supertertiary structures and modulate protein-protein interactions has only recently been addressed and is incompletely understood. The postsynaptic density protein PSD-95 contains a three-domain supramodule, denoted PSG, which consists of PDZ, Src homology 3 (SH3), and guanylate kinase-like domains. The PDZ domain binds to the C terminus of its proposed natural ligand, CXXC repeat-containing interactor of PDZ3 domain (CRIPT), and results from previous experiments using only the isolated PDZ domain are consistent with the simplest scenario for a protein-protein interaction; namely, a two-state mechanism. Here we analyzed the binding kinetics of the PSG supramodule with CRIPT. We show that PSG binds CRIPT via a more complex mechanism involving two conformational states interconverting on the second timescale. Both conformational states bound a CRIPT peptide with similar affinities but with different rates, and the distribution of the two conformational states was slightly shifted upon CRIPT binding. Our results are consistent with recent structural findings of conformational changes in PSD-95 and demonstrate how conformational transitions in supertertiary structures can shape the ligand-binding energy landscape and modulate protein-protein interactions.

Templated folding of intrinsically disordered proteins.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed "templated folding," whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Mapping the transition state for a binding reaction between ancient intrinsically disordered proteins.

Intrinsically disordered protein domains often have multiple binding partners. It is plausible that the strength of pairing with specific partners evolves from an initial low affinity to a higher affinity. However, little is known about the molecular changes in the binding mechanism that would facilitate such a transition. We previously showed that the interaction between two intrinsically disordered domains, NCBD and CID, likely emerged in an ancestral deuterostome organism as a low-affinity interaction that subsequently evolved into a higher-affinity interaction before the radiation of modern vertebrate groups. Here we map native contacts in the transition states of the low-affinity ancestral and high-affinity human NCBD/CID interactions. We show that the coupled binding and folding mechanism is overall similar but with a higher degree of native hydrophobic contact formation in the transition state of the ancestral complex and more heterogeneous transient interactions, including electrostatic pairings, and an increased disorder for the human complex. Adaptation to new binding partners may be facilitated by this ability to exploit multiple alternative transient interactions while retaining the overall binding and folding pathway.

Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery.

Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted 'energetic coupling' describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein-ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.

An Early Association between the α-Helix of the TEAD Binding Domain of YAP and TEAD Drives the Formation of the YAP:TEAD Complex.

The Hippo pathway is an evolutionarily conserved signaling pathway that is involved in the control of organ size and development. The TEAD transcription factors are the most downstream elements of the Hippo pathway, and their transcriptional activity is regulated via the interaction with different co-regulators such as YAP. The structure of the YAP:TEAD complex shows that YAP binds to TEAD via two distinct secondary structure elements, an α-helix and an Ω-loop, and site-directed mutagenesis experiments revealed that the Ω-loop is the "hot spot" of this interaction. While much is known about how YAP and TEAD interact with each other, little is known about the mechanism leading to the formation of a complex between these two proteins. Here we combine site-directed mutagenesis with pre-steady-state kinetic measurements to show that the association between these proteins follows an apparent one-step binding mechanism. Furthermore, linear free energy relationships and a Φ analysis suggest that binding-induced folding of the YAP α-helix to TEAD occurs independently of and before formation of the Ω-loop interface. Thus, the binding-induced folding of YAP appears not to conform to the concomitant formation of tertiary structure (nucleation-condensation) usually observed for coupled binding and folding reactions. Our findings demonstrate how a mechanism reminiscent of the classical framework (diffusion-collision) mechanism of protein folding may operate in disorder-to-order transitions involving intrinsically disordered proteins.

A structurally heterogeneous transition state underlies coupled binding and folding of disordered proteins.

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 Φb 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 α-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 Φb values and Brønsted 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.

The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A.

Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the CDKN2A tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The B0 allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the B1 and B2 alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are cis-regulatory mutations causing a higher CDKN2A expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that CDKN2A is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner.

Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism.

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.

Probing Backbone Hydrogen Bonds in Proteins by Amide-to-Ester Mutations.

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.

Evolution of the p53-MDM2 pathway.

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.

Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain.

Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (∼10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured state. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein-protein interactions.

Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.