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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., Competing Interests: Conflict of interest: The authors declare that they have no conflict of interest with the contents of this article., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Intrinsically disordered protein regions may fold upon binding to an interaction partner. It is often argued that such coupled binding and folding enables the combination of high specificity with low affinity. The basic tenet is that an unfavorable folding equilibrium will make the overall binding weaker while maintaining the interaction interface. While theoretically solid, we argue that this concept may be misleading for intrinsically disordered proteins. In fact, experimental evidence suggests that interactions of disordered regions usually involve extended conformations. In such cases, the disordered region is exceptionally unlikely to fold into a bound conformation in the absence of its binding partner. Instead, these disordered regions can bind to their partners in multiple different conformations and then fold into the native bound complex, thus, if anything, increasing the affinity through folding. We concede that (de)stabilization of native structural elements such as helices will modulate affinity, but this could work both ways, decreasing or increasing the stability of the complex. Moreover, experimental data show that intrinsically disordered binding regions display a range of affinities and specificities dictated by the particular side chains and length of the disordered region and not necessarily by the fact that they are disordered. We find it more likely that intrinsically disordered regions are common in protein-protein interactions because they increase the repertoire of binding partners, providing an accessible route to evolve interactions rather than providing a stability-affinity trade-off., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Ever since Ranganathan and coworkers subjected the covariation of amino acid residues in the postsynaptic density-95/Discs large/Zonula occludens 1 (PDZ) domain family to a statistical correlation analysis, PDZ domains have represented a paradigmatic family to explore single domain protein allostery. Nevertheless, several theoretical and experimental studies in the past two decades have contributed contradicting results with regard to structural localization of the allosteric networks, or even questioned their actual existence in PDZ domains. In this review, we first describe theoretical and experimental approaches that were used to probe the energetic network(s) in PDZ domains. We then compare the proposed networks for two well-studied PDZ domains namely the third PDZ domain from PSD-95 and the second PDZ domain from PTP-BL. Our analysis highlights the contradiction between the different methods and calls for additional work to better understand these allosteric phenomena., (© The Author(s) 2019. Published by Oxford University Press.)

The PDZ domain is one of the most common protein-protein interaction domains in mammalian species. While several studies have demonstrated the importance of phosphorylation in interactions involving PDZ domains, there is a paucity of detailed mechanistic data addressing how the PDZ interaction is affected by phosphorylation. Here, we address this question by equilibrium and kinetic binding experiments using PDZ2 from protein tyrosine phosphatase L1 and its interaction with a peptide from the natural ligand RIL. The results show that phosphorylation of a serine residue in the RIL peptide has dual and opposing effects: it increases both the association and dissociation rate constants, which leads to an overall weakening of binding. Furthermore, we performed binding experiments with a RIL peptide in which the serine was replaced by a glutamate, a commonly used method to mimic phosphorylation in proteins. Strikingly, both the affinity and the ionic strength dependence of the affinity differed markedly for the phosphoserine and glutamate peptides. These results show that, in this particular case, glutamate is a poor mimic of serine phosphorylation., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The role of intermediates in the folding reaction of single-domain proteins is a controversial issue. It was previously shown by different methods that an on-pathway intermediate is populated in the presence of sodium sulphate during the folding of the FF domain from HYPA/FBP11. Here we demonstrate using analysis of the amplitudes of kinetic traces that this burst-phase folding intermediate is present at different salt concentration and at various pH, and is also found in roughly 30 site-directed mutants. The intermediate appears robust to changing conditions and thus fulfils an important criterion for a productive molecular species on the folding reaction pathway.