Valéry Ozenne

NMR characterization of long-range order in intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are predicted to represent a significant fraction of the human genome, and the development of meaningful molecular descriptions of these proteins remains a key challenge for contemporary structural biology. In order to describe the conformational behavior of IDPs, a molecular representation of the disordered state based on diverse sources of structural data that often exhibit complex and very different averaging behavior is required. In this study, we propose a combination of paramagnetic relaxation enhancements (PREs) and residual dipolar couplings (RDCs) to define both long-range and local structural features of IDPs in solution. We demonstrate that ASTEROIDS, an ensemble selection algorithm, faithfully reproduces intramolecular contacts, even in the presence of highly diffuse, ill-defined target interactions. We also show that explicit modeling of spin-label mobility significantly improves the reproduction of experimental PRE data, even in the case of highly disordered proteins. Prediction of the effects of transient long-range contacts on RDC profiles reveals that weak intramolecular interactions can induce a severe distortion of the profiles that compromises the description of local conformational sampling if it is not correctly taken into account. We have developed a solution to this problem that involves efficiently combining RDC and PRE data to simultaneously determine long-range and local structure in highly flexible proteins. This combined analysis is shown to be essential for the accurate interpretation of experimental data from alpha-synuclein, an important IDP involved in human neurodegenerative disease, confirming the presence of long-range order between distant regions in the protein.

Quantitative Description of Backbone Conformational Sampling of Unfolded Proteins at Amino Acid Resolution from NMR Residual Dipolar Couplings

An atomic resolution characterization of the structural properties of unfolded proteins that explicitly invokes the highly dynamic nature of the unfolded state will be extremely important for the development of a quantitative understanding of the thermodynamic basis of protein folding and stability. Here we develop a novel approach using residual dipolar couplings (RDCs) from unfolded proteins to determine conformational behavior on an amino acid specific basis. Conformational sampling is described in terms of ensembles of structures selected from a large pool of conformers. We test this approach, using extensive simulation, to determine how well the fitting of RDCs to reduced conformational ensembles containing few copies of the molecule can correctly reproduce the backbone conformational behavior of the protein. Having established approaches that allow accurate mapping of backbone dihedral angle conformational space from RDCs, we apply these methods to obtain an amino acid specific description of ubiquitin denatured in 8 M urea at pH 2.5. Cross-validation of data not employed in the fit verifies that an ensemble size of 200 structures is appropriate to characterize the highly fluctuating backbone. This approach allows us to identify local conformational sampling properties of urea-unfolded ubiquitin, which shows that the backbone sampling of certain types of charged or polar amino acids, in particular threonine, glutamic acid, and arginine, is affected more strongly by urea binding than amino acids with hydrophobic side chains. In general, the approach presented here establishes robust procedures for the study of all denatured and intrinsically disordered states.

Predictive Atomic Resolution Descriptions of Intrinsically Disordered hTau40 and α-Synuclein in Solution from NMR and Small Angle Scattering

The development of molecular descriptions of intrinsically disordered proteins (IDPs) is essential for elucidating conformational transitions that characterize common neurodegenerative disorders. We use nuclear magnetic resonance, small angle scattering, and molecular ensemble approaches to characterize the IDPs Tau and α-synuclein. Ensemble descriptions of IDPs are highly underdetermined due to the inherently large number of degrees of conformational freedom compared with available experimental measurements. Using extensive cross-validation we show that five different types of independent experimental parameters are predicted more accurately by selected ensembles than by statistical coil descriptions. The improvement increases in regions whose local sampling deviates from statistical coil, validating the derived conformational description. Using these approaches we identify enhanced polyproline II sampling in aggregation-nucleation sites, supporting suggestions that this region of conformational space is important for aggregation.

Mapping the Potential Energy Landscape of Intrinsically Disordered Proteins at Amino Acid Resolution

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.

Conformational Propensities of Intrinsically Disordered Proteins from NMR Chemical Shifts

The realization that a protein can be fully functional even in the absence of a stable three-dimensional structure has motivated a large number of studies describing the conformational behaviour of these proteins at atomic resolution. Here, we review recent advances in the determination of local structural propensities of intrinsically disordered proteins (IDPs) from experimental NMR chemical shifts. A mapping of the local structure in IDPs is of paramount importance in order to understand the molecular details of complex formation, in particular, for IDPs that fold upon binding or undergo structural transitions to pathological forms of the same protein. We discuss experimental strategies for the spectral assignment of IDPs, chemical shift prediction algorithms and the generation of representative structural ensembles of IDPs on the basis of chemical shifts. Additionally, we highlight the inherent degeneracies associated with the determination of IDP sub-state populations from NMR chemical shifts alone.

Phosphorylation of human Tau protein by microtubule affinity-regulating kinase 2.

Tau protein plays an important role in neuronal physiology and Alzheimers neurodegeneration. Its abilities to aggregate abnormally, to bind to microtubules (MTs), and to promote MT assembly are all influenced by phosphorylation. Phosphorylation of serine residues in the KXGS motifs of Taus repeat domain, crucial for MT interactions and aggregation, is facilitated most efficiently by microtubule-associated protein/microtubule affinity-regulating kinases (MARKs). Here we applied high-resolution nuclear magnetic resonance analysis to study the kinetics of phosphorylation of Tau by MARK2 and its impact on the structure and microtubule binding of Tau. We demonstrate that MARK2 binds to the N-terminal tail of Tau and selectively phosphorylates three major and five minor serine residues in the repeat domain and C-terminal tail. Structural changes induced by phosphorylation of Tau by MARK2 are highly localized in the proximity of the phosphorylation site and do not affect the global conformation, in contrast to phosphorylation in the proline-rich region. Furthermore, single-residue analysis of binding of Tau to MTs provides support for a model in which Taus hot spots of MT interaction bind independently of each other and are differentially affected by phosphorylation.

Structural Impact of Tau Phosphorylation at Threonine 231

Phosphorylation of the microtubule-associated protein Tau influences the assembly and stabilization of microtubules and is deregulated in several neurodegenerative diseases. The high flexibility of Tau, however, has prevented an atomic-level description of its phosphorylation-induced structural changes. Employing an extensive set of distance and orientational restraints together with a novel ensemble calculation approach, we determined conformational ensembles of Tau fragments in the non-phosphorylated state and, when phosphorylated at T231/S235 or T231/S235/S237/S238, four important sites of phosphorylation in Alzheimer disease. Comparison of the molecular ensembles showed that phosphorylation of the regulatory T231 does not perturb the backbone conformation of the proximal microtubule-binding (225)KVAVVR(230) motif. Instead, phosphorylated T231 selectively engages in a salt bridge with R230 that can compete with the formation of intermolecular salt bridges to tubulin. Our study provides an ensemble description which will be useful for the analysis of conformational transitions in Tau and other intrinsically disordered proteins.

Modulation of the Intrinsic Helix Propensity of an Intrinsically Disordered Protein Reveals Long-Range Helix–Helix Interactions

Intrinsically disordered proteins (IDPs) are widespread and important in biology but defy the classical protein structure-function paradigm by being functional in the absence of a stable, folded conformation. Here we investigate the coupling between transient secondary and tertiary structure in the protein activator for thyroid hormone and retinoid receptors (ACTR) by rationally modulating the helical propensity of a partially formed α-helix via mutations. Eight mutations predicted to affect the population of a transient helix were produced and investigated by NMR spectroscopy. Chemical shift changes distant to the mutation site are observed in regions containing other transient helices indicating that distant helices are stabilized through long-range hydrophobic helix-helix interactions and demonstrating the coupling of transient secondary and tertiary structure. The long-range structure of ACTR is also probed using paramagnetic relaxation enhancements (PRE) and residual dipolar couplings, which reveal an additional long-range contact between the N- and C-terminal segments. Compared to residual dipolar couplings and PRE, modulation of the helical propensity by mutagenesis thus reveals a different set of long-range interactions that may be obscured by stronger interactions that dominate other NMR measurements. This approach thus offers a complementary and generally applicable strategy for probing long-range structure in disordered proteins.

Exploring the minimally frustrated energy landscape of unfolded ACBP.

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.

Direct Prediction of NMR Residual Dipolar Couplings from the Primary Sequence of Unfolded Proteins

Conformational analysis: an approach to the prediction of RDCs from disordered protein chains, integrating the effect of nearest neighbors and the alignment characteristics of the statistical coil, is reported. NMR residual dipolar couplings (RDC) are sensitive probes of conformational sampling in unfolded proteins.