DNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2.

More than 1600 human transcription factors orchestrate the transcriptional machinery to control gene expression and cell fate. Their function is conveyed through intrinsically disordered regions (IDRs) containing activation or repression domains but lacking quantitative structural ensemble models prevents their mechanistic decoding. Here we integrate single-molecule FRET and NMR spectroscopy with molecular simulations showing that DNA binding can lead to complex changes in the IDR ensemble and accessibility. The C-terminal IDR of pioneer factor Sox2 is highly disordered but its conformational dynamics are guided by weak and dynamic charge interactions with the folded DNA binding domain. Both DNA and nucleosome binding induce major rearrangements in the IDR ensemble without affecting DNA binding affinity. Remarkably, interdomain interactions are redistributed in complex with DNA leading to variable exposure of two activation domains critical for transcription. Charged intramolecular interactions allowing for dynamic redistributions may be common in transcription factors and necessary for sensitive tuning of structural ensembles.

Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs.

DNA regulation, replication and repair are processes fundamental to all known organisms and the sliding clamp proliferating cell nuclear antigen (PCNA) is central to all these processes. S-phase delaying protein 1 (Spd1) from S. pombe, an intrinsically disordered protein that causes checkpoint activation by inhibiting the enzyme ribonucleotide reductase, has one of the most divergent PCNA binding motifs known. Using NMR spectroscopy, in vivo assays, X-ray crystallography, calorimetry, and Monte Carlo simulations, an additional PCNA binding motif in Spd1, a PIP-box, is revealed. The two tandemly positioned, low affinity sites exchange rapidly on PCNA exploiting the same binding sites. Increasing or decreasing the binding affinity between Spd1 and PCNA through mutations of either motif compromised the ability of Spd1 to cause checkpoint activation in yeast. These results pinpoint a role for PCNA in Spd1-mediated checkpoint activation and suggest that its tandemly positioned short linear motifs create a neatly balanced competition-based system, involving PCNA, Spd1 and the small ribonucleotide reductase subunit, Suc22R2. Similar mechanisms may be relevant in other PCNA binding ligands where divergent binding motifs so far have gone under the PIP-box radar.

Molecular switching in transcription through splicing and proline-isomerization regulates stress responses in plants.

The Arabidopsis thaliana DREB2A transcription factor interacts with the negative regulator RCD1 and the ACID domain of subunit 25 of the transcriptional co-regulator mediator (Med25) to integrate stress signals for gene expression, with elusive molecular interplay. Using biophysical and structural analyses together with high-throughput screening, we reveal a bivalent binding switch in DREB2A containing an ACID-binding motif (ABS) and the known RCD1-binding motif (RIM). The RIM is lacking in a stress-induced DREB2A splice variant with retained transcriptional activity. ABS and RIM bind to separate sites on Med25-ACID, and NMR analyses show a structurally heterogeneous complex deriving from a DREB2A-ABS proline residue populating cis- and trans-isomers with remote impact on the RIM. The cis-isomer stabilizes an α-helix, while the trans-isomer may introduce energetic frustration facilitating rapid exchange between activators and repressors. Thus, DREB2A uses a post-transcriptionally and post-translationally modulated switch for transcriptional regulation.

Structural characterization of human tryptophan hydroxylase 2 reveals that L-Phe is superior to L-Trp as the regulatory domain ligand.

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the brain. Consequently, regulation of TPH2 is relevant for serotonin-related diseases, yet the regulatory mechanism of TPH2 is poorly understood and structural and dynamical insights are missing. We use NMR spectroscopy to determine the structure of a 47 N-terminally truncated variant of the regulatory domain (RD) dimer of human TPH2 in complex with L-Phe, and show that L-Phe is the superior RD ligand compared with the natural substrate, L-Trp. Using cryo-EM, we obtain a low-resolution structure of a similarly truncated variant of the complete tetrameric enzyme with dimerized RDs. The cryo-EM two-dimensional (2D) class averages additionally indicate that the RDs are dynamic in the tetramer and likely exist in a monomer-dimer equilibrium. Our results provide structural information on the RD as an isolated domain and in the TPH2 tetramer, which will facilitate future elucidation of TPH2's regulatory mechanism.

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2.

Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nanosecond and faster dynamics. Here we asked what effects the L49I and I57V substitutions, either individually or together, have on the slow conformational dynamics of CI2. We used 15 N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics, and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1°C. As the temperature is increased the population of the excited state decreases. Structural changes in the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The substitutions in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the substituted residues and the well-ordered water molecules links subtle structural changes around the substituted residues to the region in the protein that experience slow conformational changes.

Molecular insights into alginate ß-lactoglobulin A multivalencies-The foundation for their amorphous aggregates and coacervation.

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of ß-lactoglobulin A (ß-LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. ß-LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on ß-LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to ß-LgA, as concluded from saturation transfer difference and 1 H-ligand-targeted NMR analyses. Molecular docking using Glide within the Schrödinger suite 2016-1 revealed the orientation of AOSs to only vary slightly at the preferred ß-LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the ß-LgA complexes may help explain the first steps toward disordered ß-LgA alginate coacervate structures.

Deciphering the Alphabet of Disorder-Glu and Asp Act Differently on Local but Not Global Properties.

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences between glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing database of experimentally verified IDPs (DisProt) and show that glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while the Glu/Asp variants support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity, where glutamates can support increased helicity, important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in, e.g., transactivation domains and ion-binding.

A context-dependent and disordered ubiquitin-binding motif.

Ubiquitin is a small, globular protein that is conjugated to other proteins as a posttranslational event. A palette of small, folded domains recognizes and binds ubiquitin to translate and effectuate this posttranslational signal. Recent computational studies have suggested that protein regions can recognize ubiquitin via a process of folding upon binding. Using peptide binding arrays, bioinformatics, and NMR spectroscopy, we have uncovered a disordered ubiquitin-binding motif that likely remains disordered when bound and thus expands the palette of ubiquitin-binding proteins. We term this motif Disordered Ubiquitin-Binding Motif (DisUBM) and find it to be present in many proteins with known or predicted functions in degradation and transcription. We decompose the determinants of the motif showing it to rely on features of aromatic and negatively charged residues, and less so on distinct sequence positions in line with its disordered nature. We show that the affinity of the motif is low and moldable by the surrounding disordered chain, allowing for an enhanced interaction surface with ubiquitin, whereby the affinity increases ~ tenfold. Further affinity optimization using peptide arrays pushed the affinity into the low micromolar range, but compromised context dependence. Finally, we find that DisUBMs can emerge from unbiased screening of randomized peptide libraries, featuring in de novo cyclic peptides selected to bind ubiquitin chains. We suggest that naturally occurring DisUBMs can recognize ubiquitin as a posttranslational signal to act as affinity enhancers in IDPs that bind to folded and ubiquitylated binding partners.

Double Mutant of Chymotrypsin Inhibitor 2 Stabilized through Increased Conformational Entropy.

The conformational heterogeneity of a folded protein can affect not only its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here, we have examined whether changed native-state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics of amide groups in the backbone and the methyl groups in the side chains with molecular dynamics simulations to quantify the native-state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: a reduction in conformational dynamics (and entropy estimated from this) of the native state of the L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native-state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

Quantification of Conformational Entropy Unravels Effect of Disordered Flanking Region in Coupled Folding and Binding.

Intrinsic disorder (ID) constitutes a new dimension to the protein structure-function relationship. The ability to undergo conformational changes upon binding is a key property of intrinsically disordered proteins and remains challenging to study using conventional methods. A 1994 paper by R. S. Spolar and M. T. Record presented a thermodynamic approach for estimating changes in conformational entropy based on heat capacity changes, allowing quantification of residues folding upon binding. Here, we adapt the method for studies of intrinsically disordered proteins. We integrate additional data to provide a broader experimental foundation for the underlying relations and, based on >500 protein-protein complexes involving disordered proteins, reassess a key relation between polar and nonpolar surface area changes, previously determined using globular protein folding. We demonstrate the improved suitability of the adapted method to studies of the folded αα-hub domain RST from radical-induced cell death 1, whose interactome is characterized by ID. From extensive thermodynamic data, quantifying the conformational entropy changes upon binding, and comparison to the NMR structure, the adapted method improves accuracy for ID-based studies. Furthermore, we apply the method, in conjunction with NMR, to reveal hitherto undetected effects of interaction-motif context. Thus, inclusion of the disordered context of the DREB2A RST-binding motif induces structuring of the binding motif, resulting in major enthalpy-entropy compensation in the interaction interface. This study, also evaluating additional interactions, demonstrates the strength of the ID-adapted Spolar-Record thermodynamic approach for dissection of structural features of ID-based interactions, easily overlooked in traditional studies, and for translation of these into mechanistic knowledge.

Dynamic Na+/H+ exchanger 1 (NHE1) - calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation.

Calmodulin (CaM) engages in Ca2+-dependent interactions with numerous proteins, including a still incompletely understood physical and functional interaction with the human Na+/H+-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts stably expressing wildtype and mutant NHE1, we discovered multiple accessible states of this functionally important complex existing in different NHE1:CaM stoichiometries and structures. We determined the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities could be tuned by variations in NHE1:CaM ratio and calcium ([Ca2+]) and by phosphorylation of S648 in the first CaM-binding α-helix. In cells, Ca2+-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation and by mutation of the first CaM-binding α-helix, whereas it was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results demonstrate a diversity of NHE1:CaM interaction modes and suggest that CaM may contribute to NHE1 dimerization and thereby augment NHE1 regulation. We propose that a similar structural diversity is of relevance to many other CaM complexes.

Software for reconstruction of nonuniformly sampled NMR data.

Nonuniform sampling (NUS) of multidimensional NMR experiments is a powerful tool to obtain high-resolution spectra with less instrument time. With NUS, only a subset of the points needed for conventional Fourier transformation is recorded, and sophisticated algorithms are needed to reconstruct the missing data points. During the last decade, several software packages implementing the reconstruction algorithms have emerged and been refined and now result in spectra of almost similar quality as spectra from conventionally recorded and processed data. However, from the number of literature references to the reconstruction methods, many more multidimensional NMR spectra could presumably be recorded with NUS. To help researchers considering to start using NUS for their NMR experiments, we here review 13 different reconstruction methods found in five software packages (CambridgeCS, hmsIST, MddNMR, NESTA-NMR, and SMILE). We have compared how the methods run with the provided example scripts for reconstructing a nonuniform sampled three-dimensional 15 N-NOESY-HSQC at sampling densities from 5% to 50%. Overall, the spectra are all of similar quality above 20% sampling density. Thus, without any particular knowledge about the details of the reconstruction algorithms, significant reduction in the experiment time can be achieved. Below 20% sampling density, the intensities of particular weak peaks start being affected. MddNMR's IST with virtual echo and the SMILE algorithms still reproduced the spectra with the highest accuracy of peak intensities.

The intracellular lipid-binding domain of human Na+/H+ exchanger 1 forms a lipid-protein co-structure essential for activity.

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.

The PCNA interaction motifs revisited: thinking outside the PIP-box.

Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity. Combining NMR spectroscopy, affinity measurements and computational analyses, we corroborate that most PCNA-binding motifs reside in intrinsically disordered regions, that structure preformation is unrelated to affinity, and that the sequence-patterns that encode binding affinity extend substantially beyond the boundaries of the PIP-box. Our systematic multidisciplinary approach expands current views on PCNA interactions and reveals that the PIP-box affinity can be modulated over four orders of magnitude by positive charges in the flanking regions. Including the flanking regions as part of the motif is expected to have broad implications, particularly for interpretation of disease-causing mutations and drug-design, targeting DNA-replication and -repair.

The transmembrane autophagy cargo receptors ATI1 and ATI2 interact with ATG8 through intrinsically disordered regions with distinct biophysical properties.

Selective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8-interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8-interacting protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs). The N-terminal IDRs contain the functional AIMs, and we use nuclear magnetic resonance spectroscopy to directly observe the disorder-order transition of the AIM upon ATG8 binding. Our analyses also show that the IDRs of ATI1 and ATI2 are not equivalent, because ATI2 has properties of a fully disordered polypeptide, while ATI1 has properties more consistent with a collapsed pre-molten globule-like conformation, possibly as a consequence of a higher content of π-orbital-containing amino acid residues. Finally, we show that a sizable fraction of ATI2, but not ATI1, is phosphorylated in planta.

Characterization of Dynamic IDP Complexes by NMR Spectroscopy.

NMR spectroscopy has proven to be a key method for studying intrinsically disordered proteins (IDPs). Nonetheless, traditional NMR methods developed for solving structures of ordered protein complexes are insufficient for the full characterization of dynamic IDP complexes, where the energy landscape is broader and more rugged. Furthermore, due to their high sensitivity to environmental changes, NMR studies of IDP complexes must be conducted with extra care and the observed NMR parameters thoroughly evaluated to enable disentanglement of binding events from ensemble distribution changes. In this chapter, written for the non-NMR expert, we start out by outlining sample preparation for IDP complexes, guide through the recording and evaluation of diagnostic 1H,15N-HSQC spectra, and delineate more sophisticated NMR strategies to follow for the particular type of complex. The most relevant experiments are then described in terms of aims, needs, pitfalls, analysis, and expected outcomes, with references to recent examples.

Cold Denaturation of the HIV-1 Protease Monomer.

The human immunodeficiency virus-1 (HIV-1) protease is a complex protein that in its active form adopts a homodimer dominated by ß-sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1 protease that is populated above 0 °C and therefore directly accessible to various spectroscopic approaches. Using nuclear magnetic resonance secondary chemical shifts, temperature coefficients, and protein dynamics, we suggest that the cold-denatured state populates a compact wet globule containing transient non-native-like α-helical elements. From the linearity of the temperature coefficients and the hydrodynamic radii, we propose that the overall architecture of the cold-denatured state is maintained over the temperature range studied.

Spatio-temporal control of cellular uptake achieved by photoswitchable cell-penetrating peptides.

The selective uptake of compounds into specific cells of interest is a major objective in cell biology and drug delivery. By incorporation of a novel, thermostable azobenzene moiety we generated peptides that can be switched optically between an inactive state and an active, cell-penetrating state with excellent spatio-temporal control.

Identification of bypass reactions leading to the formation of one central steroid degradation intermediate in metabolism of different bile salts in Pseudomonas sp. strain Chol1.

The bile salts cholate, deoxycholate, chenodeoxycholate and lithocholate are released from vertebrates into soil and water where environmental bacteria degrade these widespread steroid compounds. It was investigated whether different enzymes are required for the degradation of these tri-, di- and monohydroxylated bile salts in the model organism Pseudomonas sp. strain Chol1. Experiments with available and novel mutants showed that the degradation of the C5 -carboxylic side chain attached to the steroid skeleton is catalysed by the same set of enzymes. A difference was found for the degradation of partially degraded bile salts consisting of H-methylhexahydroindanone-propanoates (HIPs). With deoxycholate and lithocholate, which lack a hydroxy group at C7 of the steroid skeleton, an additional acyl-coenzyme A (CoA) dehydrogenase was required for ß-oxidation of the C3 -carboxylic side chain attached to the methylhexahydroindanone moiety. The ß-oxidation of this side chain could be measured in vitro. With cholate and deoxycholate, a reductive dehydroxylation of the C12-hydroxy group of HIP was required. Deletion of candidate genes for this reaction step revealed that a so-far unknown steroid dehydratase and a steroid oxidoreductase were responsible for this CoA-dependent reaction. These results showed that all bile salts are channelled into a common pathway via bypass reactions with 3'-hydroxy-HIP-CoA as central intermediate.

Mad2 Inhibitor-1 (M2I-1): A Small Molecule Protein-Protein Interaction Inhibitor Targeting the Mitotic Spindle Assembly Checkpoint.

The genetic integrity of each organism depends on the faithful segregation of its genome during mitosis. To meet this challenge, a cellular surveillance mechanism, termed the spindle assembly checkpoint (SAC), evolved that monitors the correct attachment of chromosomes and blocks progression through mitosis if corrections are needed. While the central role of the SAC for genome integrity is well established, its functional dissection has been hampered by the limited availability of appropriate small molecule inhibitors. Using a fluorescence polarization-based screen, we identify Mad2 inhibitor-1 (M2I-1), the first small molecule inhibitor targeting the binding of Mad2 to Cdc20, an essential protein-protein interaction (PPI) within the SAC. Based on computational and biochemical analyses, we propose that M2I-1 disturbs conformational dynamics of Mad2 critical for complex formation with Cdc20. Cellular studies revealed that M2I-1 weakens the SAC response, indicating that the compound might be active in cells. Thus, our study identifies the SAC specific complex formation between Mad2 and Cdc20 as a protein-protein interaction that can be targeted by small molecules.