Project description:It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions that can both contribute to function. Although equally interesting, the disordered regions are difficult to study, because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. NMR spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, a task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe the structural properties of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. Ataxin-3 is a 42-kDa protein containing a globular N-terminal Josephin domain and a C-terminal tail that comprises 13 polyglutamine repeats within a low complexity region. We developed a strategy that allowed us to achieve 87% assignment of the NMR spectrum using a mixed protocol based on high-dimensionality, high-resolution experiments and different labeling schemes. Thanks to the almost complete spectral assignment, we proved that the C-terminal tail is flexible, with extended helical regions, and interacts only marginally with the rest of the protein. We could also, for the first time to our knowledge, observe the structural propensity of the polyglutamine repeats within the context of the full-length protein and show that its structure is stabilized by the preceding region.
Project description:Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabilize microtubules in neuronal axons at all stages of development. In Alzheimer's and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphorylation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed picture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to define specific conformational biases that can easily be rationalized as enhancing amyloidogenic propensity. Representative structures for the native and hyperphosphorylated tau ensembles were generated by refinement of a broad sample of conformations generated by low-computational complexity modeling, based on agreement with the TRESI-HDX profiles.
Project description:It is generally known that, unlike structured proteins, intrinsically disordered proteins, IDPs, exhibit various structures and conformers, the so-called conformational ensemble, CoE. This study aims to better understand the conformers that make up the IDP ensemble by decomposing the CoE into groups separated by their radius of gyration, Rg. A common approach to studying CoE for IDPs is to use low-resolution techniques, such as small-angle scattering, and combine those with computer simulations on different length scales. Herein, the well-studied antimicrobial saliva protein histatin 5 was utilized as a model peptide for an IDP; the average intensity curves were obtained from small-angle X-ray scattering; and compared with fully atomistic, explicit water, molecular dynamics simulations; then, the intensity curve was decomposed with respect to the different Rg values; and their secondary structure propensities were investigated. We foresee that this approach can provide important information on the CoE and the individual conformers within; in that case, it will serve as an additional tool for understanding the IDP structure-function relationship on a more detailed level.
Project description:Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2-5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin.
Project description:There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.
Project description:Cdt1 is a protein critical for DNA replication licensing and is well-established to be a binding partner of the minichromosome maintenance (MCM) complex. Cdt1 has also been demonstrated to have an emerging, "moonlighting" role at the kinetochore via direct binding to microtubules and to the Ndc80 complex. However, it is not known how the structure and conformations of Cdt1 could allow for these multiple, completely unique sets of protein complexes. And while there exist multiple robust methods to study entirely folded or entirely unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. It this work, we employ multiple orthogonal biophysical and computational techniques to provide a detailed structural characterization of human Cdt1 92-546. DSF and DSCD show both folded winged helix (WH) domains of Cdt1 are relatively unstable. CD and NMR show the N-terminal and the linker regions are intrinsically disordered. Using DLS and SEC-MALS, we show that Cdt1 is polydisperse, monomeric at high concentrations, and without any apparent inter-molecular self-association. SEC-SAXS of the monomer in solution enabled computational modeling of the protein in silico. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble. Using experimental SAXS data, we filtered for conformations which did and did not fit our data. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with our SAXS data. The best fit models have the N-terminal and linker regions extended into solution and the two folded domains close to each other in apparent "folded over" conformations. The best fit Cdt1 conformations are consistent with a function as a scaffold protein which may be sterically blocked without the presence of binding partners. Our studies also provide a template for combining experimental and computational biophysical techniques to study mixed-folded proteins.
Project description:Regions of intrinsic disorder play crucial roles in biological systems, yet they often elude characterization by conventional biophysical techniques. To capture conformational distributions across different timescales, we employed a freezing approach coupled with solid-state NMR analysis. Using segmentally isotopically labeled α-synuclein (α-syn), we investigated the conformational preferences of the six alanines, three glycines, and a single site (L8) in the disordered amino terminus under three distinct conditions: in 8 M urea, as a frozen monomer in buffer, and within the disordered regions flanking the amyloid core. The experimental spectra varied significantly among these conditions and deviated from those of a statistical coil. In 8 M urea, monomeric α-syn exhibited the most restricted conformational sampling, rarely accessing chemical shifts characteristic of α-helices or β-strands. In buffer, monomeric α-syn showed broader conformational sampling, favoring α-helical conformations and, to a lesser extent, random coil states. Notably, amino acids in the disordered regions flanking the amyloid core demonstrated the most extensive conformational sampling, with broad peaks encompassing the entire range of possible chemical shifts and a marked preference for highly extended β-strand conformations. Collectively, this work demonstrates that intrinsically disordered regions exhibit distinct conformational preferences, which are influenced not only by the chemical environment but also by the conformations of adjacent protein sequences. The differences in the conformational ensembles of the disordered amino terminus may explain why the monomer and the amyloid form of α-syn interact with different biomolecules inside cells.
Project description:We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.
Project description:Many intrinsically disordered proteins switch between unfolded and folded-like forms in the presence of their binding partner. The possibility of a pre-equilibrium between the two macrostates is challenging to discern given the complex conformational landscape. Here, we show that CytR, a disordered DNA-binding domain, samples a folded-like excited state in its native ensemble through equilibrium multi-probe spectroscopy, kinetics and an Ising-like statistical mechanical model. The population of the excited state increases upon stabilization of the native ensemble with an osmolyte, while decreasing with increasing temperatures. A conserved proline residue, the mutation of which weakens the binding affinity to the target promoter, is found to uniquely control the population of the minor excited state. Semi-quantitative statistical mechanical modeling reveals that the conformational diffusion coefficient of disordered CytR is an order of magnitude slower than the estimates from folded domains. The osmolyte and proline mutation smoothen and roughen up the landscape, respectively, apart from modulation of populations. Our work uncovers general strategies to probe for excited structured states in disordered ensembles, and to measure and modulate the roughness of the disordered landscapes, inter-conversion rates of species and their populations.
Project description:Intrinsically disordered proteins (IDPs) constitute a large portion of "Dark Proteome" - difficult to characterize or yet to be discovered protein structures. Here we used conformationally constrained α-methylated amino acids to bias the conformational ensemble in the free unstructured activation domain of transcriptional coactivator ACTR. Different sites and patterns of substitutions were enabled by chemical protein synthesis and led to distinct populations of α-helices. A specific substitution pattern resulted in a substantially higher binding affinity to nuclear coactivator binding domain (NCBD) of CREB-binding protein, a natural binding partner of ACTR. The first X-ray structure of the modified ACTR domain - NCBD complex visualized a unique conformation of ACTR and confirmed that the key α-methylated amino acids are localized within α-helices in the bound state. This study demonstrates a strategy for characterization of individual conformational states of IDPs.