Structural analysis of flexible macromolecular systems such as intrinsically disordered or multidomain proteins with flexible linkers is a difficult task as high-resolution techniques are barely applicable. A new approach, ensemble optimization method (EOM), is proposed to quantitatively characterize flexible proteins in solution using small-angle X-ray scattering (SAXS). The flexibility is taken into account by allowing for the coexistence of different conformations of the protein contributing to the experimental scattering pattern. These conformers are selected using a genetic algorithm from a pool containing a large number of randomly generated models covering the protein configurational space. Quantitative criteria are developed to analyze the EOM selected models and to determine the optimum number of conformers in the ensemble. Simultaneous fitting of multiple scattering patterns from deletion mutants, if available, provides yet more detailed local information about the structure. The efficiency of EOM is demonstrated in model and practical examples on completely or partially unfolded proteins and on multidomain proteins interconnected by linkers. In the latter case, EOM is able to distinguish between rigid and flexible proteins and to directly assess the interdomain contacts.

Natively unfolded proteins play key roles in normal and pathological biochemical processes. Despite their importance for function, this category of proteins remains beyond the reach of classical structural biology because of their inherent conformational heterogeneity. We present a description of the intrinsic conformational sampling of unfolded proteins based on residue-specific ͞ propensities from loop regions of a folded protein database and simple volume exclusion. This approach is used to propose a structural model of the 57-aa, natively disordered region of the nucleocapsid-binding domain of Sendai virus phosphoprotein. Structural ensembles obeying these simple rules of conformational sampling are used to simulate averaged residual dipolar couplings (RDCs) and small-angle x-ray scattering data. This protein is particularly informative because RDC data from the equally sized folded and unfolded domains both report on the unstructured region, allowing a quantitative analysis of the degree of order present in this part of the protein. Close agreement between experimental and simulated RDC and small-angle x-ray scattering data validates this simple model of conformational sampling, providing a precise description of local structure and dynamics and average dimensions of the ensemble of sampled structures. RDC data from two urea-unfolded systems are also closely reproduced. The demonstration that conformational behavior of unfolded proteins can be accurately predicted from the primary sequence by using a simple set of rules has important consequences for our understanding of the structure and dynamics of the unstructured state.

Proteins with intrinsically disordered domains are implicated in a vast range of biological processes, especially in cell signaling and regulation. Having solved the quaternary structure of the folded domains in the tumor suppressor p53 by a multidisciplinary approach, we have now determined the average ensemble structure of the intrinsically disordered N-terminal transactivation domain (TAD) by using residual dipolar couplings (RDCs) from NMR spectroscopy and small-angle x-ray scattering (SAXS). Remarkably, not only were we able to measure RDCs of the isolated TAD, but we were also able to do so for the TAD in both the full-length tetrameric p53 protein and in its complex with a specific DNA response element. We determined the orientation of the TAD ensemble relative to the core domain, found that the TAD was stiffer in the proline-rich region (residues 64 -92), which has a tendency to adopt a polyproline II (PPII) structure, and projected the TAD away from the core. We located the TAD in SAXS experiments on a complex between tetrameric p53 and four Taz2 domains that bind tightly to the TAD (residues 1-57) and acted as ''reporters.'' The p53-Taz2 complex was an extended cross-shaped structure. The quality of the SAXS data enabled us to model the disordered termini and the folded domains in the complex with DNA. The core domains enveloped the response element in the center of the molecule, with the Taz2-bound TADs projecting outward from the core.hybrid methods ͉ natively unfolded ͉ protein ͉ residual dipolar coupling ͉ small-angle x-ray scattering T he tumor suppressor p53 is a multifunctional protein that plays vital roles in maintaining the integrity of the human genome, controlling apoptosis, cell-cycle arrest, and DNA repair (1). p53 is a homotetramer, with folded tetramerization and core domains that are linked together and flanked by intrinsically disordered (or natively unfolded) domains at the N and C termini (1, 2). As such, with 37% of its structure intrinsically disordered, p53 is typical of the structural content of the human proteome. More than 30% of eukaryotic genomes encode contiguous unfolded regions longer than 30 aa in length, and up to 80% in cancer-associated proteins (3). This new class of intrinsically disordered proteins (IDPs) is involved in a vast range of cellular processes, including molecular recognition, transcription and transposition, packaging, repair and replication, as well as signaling, cell cycle control, multiprotein complex assembly, and endocytosis. Many partly or fully disordered proteins undergo conformational transitions to folded forms only on interaction with a target ligand (4). An intrinsically disordered domain is possibly an essential structural feature that facilitates promiscuous binding to many partner proteins and is also readily accessible for posttranslational modification that modulates binding.Solving the structures of proteins with intrinsically disordered domains now represents a major stumbling block in relating structure and biological function. Class...

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 R-synuclein, an important IDP involved in human neurodegenerative disease, confirming the presence of long-range order between distant regions in the protein.

martin.blackledge@ibs.fr.

PTP1B, a validated therapeutic target for diabetes and obesity, plays a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We defined a novel mechanism of allosteric inhibition that targets the C-terminal, non-catalytic segment of PTP1B. We present the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small molecule inhibitor, MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.

Despite their importance for biological activity, slower molecular motions beyond the nanosecond range remain poorly understood. We have assembled an unprecedented set of experimental NMR data, comprising up to 27 residual dipolar couplings per amino acid, to define the nature and amplitude of backbone motion in protein G using the Gaussian axial fluctuation model in three dimensions. Slower motions occur in the loops, and in the ␤-sheet, and are absent in other regions of the molecule, including the ␣-helix. In the ␤-sheet an alternating pattern of dynamics along the peptide sequence is found to form a long-range network of slow motion in the form of a standing wave extending across the ␤-sheet, resulting in maximal conformational sampling at the interaction site. The alternating nodes along the sequence match the alternation of strongly hydrophobic side chains buried in the protein core. Confirmation of the motion is provided through extensive crossvalidation and by independent hydrogen-bond scalar coupling analysis that shows this motion to be correlated. These observations strongly suggest that dynamical information can be transmitted across hydrogen bonds and have important implications for understanding collective motions and long-range information transfer in proteins.protein dynamics ͉ slow motions ͉ correlated M olecular dynamics, manifest in backbone and side-chain mobilities, play a crucial role in protein stability and function (1-4). The accurate characterization and understanding of protein motions thus adds an additional dimension to the structural information derived from genomics projects (5, 6). Although local backbone fluctuations on the picosecond to nanosecond time scale have been the subject of detailed characterization using NMR (7, 8) and molecular dynamics simulations (2), slower motions, in the submicrosecond to second range, remain poorly understood. Relaxation dispersion has been used to successfully identify sites of conformational exchange between states experiencing different chemical shifts in peptides (9) and proteins (10), but specific geometric motional models are often difficult to extract from these data. Slow time scales are, however, of particular interest because functionally important biological processes, including enzyme catalysis (11), signal transduction (12), ligand binding, and allosteric regulation (13), as well as collective motions involving groups of atoms or whole amino acids (14), are expected to occur in this time range. Residual dipolar couplings (RDCs) report on averages over longer time scales (up to the millisecond range) and therefore encode key information for understanding slower protein motions over a very broad time scale (15,16). Recent studies have exploited the orientational averaging properties of RDCs to characterize the amplitude and direction of motions of NH vectors (17)(18)(19) or to study local variations in position and dynamics of the amide proton (20,21). Despite this activity, key questions remain concerning the nature and amplitude of ...

Intrinsically unstructured proteins play key biochemical roles in a vast range of normal and pathological processes. To study these systems, it is necessary to invoke an ensemble of rapidly interconverting conformations. Residual dipolar couplings (RDCs) are particularly powerful probes of the behavior of unfolded proteins, reporting on time and ensemble-averaged conformations up to and beyond the millisecond time scale. In this study, we present a novel interpretation of RDCs in unfolded systems that simultaneously defines long-range structural order and local conformational sampling. This approach is used to describe the structure and dynamics of alpha-Synuclein (alphaS), a protein that is strongly implicated in the development of Parkinson's disease (PD), allowing unambiguous detection of strongly populated conformers containing long-range contacts between the N- and C-terminal domains. The structural model combines two features required for the description of alphaS in solution: local conformational fluctuation based on random sampling of residue-specific phi/psi distributions, and long-range contacts induced by the presence of nonbonding interactions between domains that are distant in primary sequence. Both aspects are found to be necessary for the reproduction of the nonaveraged RDCs from alphaS. Although RDCs have previously been shown to report on local conformational preferences in unstructured proteins, this study shows the additional sensitivity of these measurements to the presence of long-range order in highly flexible systems.