Structure, dynamics, and stability of the globular domain of human linker histone H1.0 and the role of positive charges.

Linker histone H1 (H1) is an abundant chromatin-binding protein that acts as an epigenetic regulator binding to nucleosomes and altering chromatin structures and dynamics. Nonetheless, the mechanistic details of its function remain poorly understood. Recent work suggest that the number and position of charged side chains on the globular domain (GD) of H1 influence chromatin structure and hence gene repression. Here, we solved the solution structure of the unbound GD of human H1.0, revealing that the structure is almost completely unperturbed by complex formation, except for a loop connecting two antiparallel ß-strands. We further quantified the role of the many positive charges of the GD for its structure and conformational stability through the analysis of 11 charge variants. We find that modulating the number of charges has little effect on the structure, but the stability is affected, resulting in a difference in melting temperature of 26 K between GD of net charge +5 versus +13. This result suggests that the large number of positive charges on H1-GDs have evolved for function rather than structure and high stability. The stabilization of the GD upon binding to DNA can thus be expected to have a pronounced electrostatic component, a contribution that is amenable to modulation by posttranslational modifications, especially acetylation and phosphorylation.

Insight into Calcium-Binding Motifs of Intrinsically Disordered Proteins.

Motifs within proteins help us categorize their functions. Intrinsically disordered proteins (IDPs) are rich in short linear motifs, conferring them many different roles. IDPs are also frequently highly charged and, therefore, likely to interact with ions. Canonical calcium-binding motifs, such as the EF-hand, often rely on the formation of stabilizing flanking helices, which are a key characteristic of folded proteins, but are absent in IDPs. In this study, we probe the existence of a calcium-binding motif relevant to IDPs. Upon screening several carefully selected IDPs using NMR spectroscopy supplemented with affinity quantification by colorimetric assays, we found calcium-binding motifs in IDPs which could be categorized into at least two groups-an Excalibur-like motif, sequentially similar to the EF-hand loop, and a condensed-charge motif carrying repetitive negative charges. The motifs show an affinity for calcium typically in the ~100 µM range relevant to regulatory functions and, while calcium binding to the condensed-charge motif had little effect on the overall compaction of the IDP chain, calcium binding to Excalibur-like motifs resulted in changes in compaction. Thus, calcium binding to IDPs may serve various structural and functional roles that have previously been underreported.

Interactions by Disorder - A Matter of Context.

Living organisms depend on timely and organized interactions between proteins linked in interactomes of high complexity. The recent increased precision by which protein interactions can be studied, and the enclosure of intrinsic structural disorder, suggest that it is time to zoom out and embrace protein interactions beyond the most central points of physical encounter. The present paper discusses protein-protein interactions in the view of structural disorder with an emphasis on flanking regions and contexts of disorder-based interactions. Context constitutes an overarching concept being of physicochemical, biomolecular, and physiological nature, but it also includes the immediate molecular context of the interaction. For intrinsically disordered proteins, which often function by exploiting short linear motifs, context contributes in highly regulatory and decisive manners and constitute a yet largely unrecognized source of interaction potential in a multitude of biological processes. Through selected examples, this review emphasizes how multivalency, charges and charge clusters, hydrophobic patches, dynamics, energetic frustration, and ensemble redistribution of flanking regions or disordered contexts are emerging as important contributors to allosteric regulation, positive and negative cooperativity, feedback regulation and negative selection in binding. The review emphasizes that understanding context, and in particular the role the molecular disordered context and flanking regions take on in protein interactions, constitute an untapped well of energetic modulation potential, also of relevance to drug discovery and development.

Random coil chemical shifts for serine, threonine and tyrosine phosphorylation over a broad pH range.

Phosphorylation is one of the main regulators of cellular signaling typically occurring in flexible parts of folded proteins and in intrinsically disordered regions. It can have distinct effects on the chemical environment as well as on the structural properties near the modification site. Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins (IDPs) and the reliability of the analysis depends on an appropriate choice of random coil model. Random coil chemical shifts and sequence correction factors were previously determined for an Ac-QQXQQ-NH2-peptide series with X being any of the 20 common amino acids. However, a matching dataset on the phosphorylated states has so far only been incompletely determined or determined only at a single pH value. Here we extend the database by the addition of the random coil chemical shifts of the phosphorylated states of serine, threonine and tyrosine measured over a range of pH values covering the pKas of the phosphates and at several temperatures (www.bio.ku.dk/sbinlab/randomcoil). The combined results allow for accurate random coil chemical shift determination of phosphorylated regions at any pH and temperature, minimizing systematic biases of the secondary chemical shifts. Comparison of chemical shifts using random coil sets with and without inclusion of the phosphoryl group, revealed under/over estimations of helicity of up to 33%. The expanded set of random coil values will improve the reliability in detection and quantification of transient secondary structure in phosphorylation-modified IDPs.

Extreme disorder in an ultrahigh-affinity protein complex.

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.