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Proteins interact with other proteins, with nucleic acids, lipids, carbohydrates and various small molecules in the living cell. These interactions have been quantified and structurally characterized in numerous stud-ies such that we today have a comprehensive picture of protein structure and function. However, proteins are dynamic and even folded proteins are likely more heterogeneous than they appear in most descrip-tions. One property of proteins that relies on dynamics and heterogeneity is allostery, the ability of a pro-tein to change structure and function upon ligand binding to an allosteric site. Over the last decades the concept of allostery was broadened to embrace all types of long-range interactions across a protein including purely entropic changes without a conformational change in single protein domains. But with this re-definition came a problem: How do we measure allostery? In this opinion, we discuss some caveats arising from the quantitative description of single-domain allostery from an experimental perspective and how the limitations cannot be separated from the definition of allostery per se. Furthermore, we attempt to tie together allostery with the concept of frustration in an effort to investigate the links between these two complex, and yet general, properties of proteins. We arrive at the conclusion that the sensitivity to perturbation of allosteric networks in single protein domains is too large for the networks to be of sig-nificant biological relevance.

Code used for generating Figure 2D,E,F and supp Figure 2,3,4

Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners., This work was supported by grants from the Knut and Alice Wallenberg foundation (evolution of new genes and proteins) to L.A., P.J. and M.S. and from the Swedish Research Council (grant 2017-03827) to M.S.

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.

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., Science Advances, 4 (10), ISSN:2375-2548

Identification list of all p53/p63/p73 sequences that are in the phylogenetic tree in Fig. 2a. The species included are itemized according to phyla and paralog where the Latin name, sequence ID and database is listed. (PDF 81Â kb)

Sequences Logos based on the multiple sequence alignment of p53/p63/p73 TAD. The color-coding is according to the eBioX alignment tool. (PDF 13Â kb)

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.

Sequence Logos based on the multiple sequence alignment of MDM p53/p63/p73BD. The color-coding is according to the eBioX alignment tool. (PDF 25Â kb)

Identification list of all MDM sequences that are in the phylogenetic tree in Fig. 2B. The species included are itemized according to phyla and paralog where the Latin name, sequence ID and database are listed. (PDF 74Â kb)

Phylogenetic tree with support values based on multiple sequence alignment of the MDM protein family only including species with the p53/p63/p73 BD. Support values are presented as numbers between 0 and 1 in a color gradient between red and blue. (PDF 21Â kb)

Alignment of the p53/p63/p73BD in the MDM protein family. This alignment together with the alignment of the rest of the protein (not shown) was used to generate the phylogenetic tree. The color-coding is according to the eBioX alignment tool. (PDF 3715Â kb)

Phylogenetic tree with support values based on multiple sequence alignment of the p53/p63/p73 protein family only including species with the TAD. Support values are presented as numbers between 0 and 1 in a color gradient between red and blue. (PDF 94Â kb)

Alignment of the TAD in the p53/p63/p73 protein family. This alignment together with the alignment of the rest of the protein (not shown) was used to generate the phylogenetic tree. The color-coding is according to the eBioX alignment tool. (PDF 106Â kb)

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., Author summary Barring patterns on individual feathers are widespread phenomena in a number of wild bird species. Still, the genetic background and molecular mechanisms that give rise to barring remains poorly understood. Sex-linked barring is a striping pattern present on individual feathers in domestic chickens, which can be utilized as a model species to gain an understanding of the underlying ‘mode of action’ of biological pattern formation. Our findings suggest that regulatory mutations in the tumor suppressor gene CDKN2A first resulted in a primitive barring pattern and that two missense mutations in the same gene occurred later and independently, causing the more distinct barring pattern of extant chicken breeds. A plausible mechanism is that the altered expression of CDKN2A causes melanocyte progenitor cells to prematurely stop dividing and instead differentiate into pigment-producing cells. The temporary lack of melanocytes expresses itself as a white bar until the progenitor cells are replenished and pigment is produced to form a pigmented bar. It is remarkable that a good proportion of the world-wide production of animal protein is based on chicken that are carrying a functionally important missense mutation in the CDKN2A tumor suppressor gene.

The barred beauty- how a tumour suppressor locus can delight your eyes. Avian Model Systems 9

8 p.-5 fig.-1 sch., Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group H-3-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries., This work was supported by European Commission Grants QLK-CT-1999.00536 (Program “Biologically Active Novel Glycosaminoglycans”) and BIO4-CT98-0538 (Program “Heparan Sulfate Sequencing Demonstration”), by Swedish Research Council Grant 13401, and by a grant from Polysackaridforskning AB.

The only experimental strategy to address the structure of folding transition states, theso-called Φ value analysis, relies on the synergy between site directed mutagenesisand the measurement of reaction kinetics. Despite its importance, the Φ value analysishas been often criticized and its power to pinpoint structural information has beenquestioned. In this Hypothesis we demonstrate that comparing the Φ values betweenproteins not only allows highlighting the robustness of folding pathways, but alsoprovides per se a strong validation of the method.

Glutathione peroxidase (GPX) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). GPX therefore has potential therapeutic value as an antioxidant, but its pharmacological development has been limited because GPX uses a selenocysteine as its catalytic group and it is difficult to generate selenium-containing proteins with traditional recombinant DNA technology. Here, we show that naturally occurring proteins can be modified to generate GPX activity. The rat theta-class glutathione transferase T2-2 (rGST T2-2) presents an ideal scaffold for the design of a novel GPX catalyst because it already binds GSH and contains a serine close to the substrate binding site, which can be chemically modified to bind selenium. The modified Se-rGST T2-2 efficiently catalyzes the reduction of hydrogen peroxide, and the GPX activity surpasses the activities of some natural GPXs.

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 (Kd = 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., Scientific Reports, 5, ISSN:2045-2322