Mechanism‐Dependent Modulation of Ultrafast Interfacial Water Dynamics in Intrinsically Disordered Protein Complexes

The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time‐resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinβ) with drastically different binding mechanisms, coupled folding–binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG‐Importinβ fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB‐Importinβ complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport.     

Intrinsically Disordered Tardigrade Proteins Self-Assemble into Fibrous Gels in Response to Environmental Stress

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade-unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS-8 from Hypsibius exemplaris. NMR spectroscopy reveals that the protein comprises an extended central helical domain flanked by disordered termini. Upon concentration, the protein is shown to successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature-dependent manner. The helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel. Soluble proteins can be encapsulated within cavities in the gel, maintaining their functional form. The ability to reversibly form fibrous gels may be associated with the enhanced protective properties of these proteins.     

Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water

Hyperpolarized water can selectively enhance NMR signals of rapidly exchanging protons in osteopontin (OPN), a metastasis‐associated intrinsically disordered protein (IDP), at near‐physiological pH and temperature. The transfer of magnetization from hyperpolarized water is limited to solvent‐exposed residues and therefore selectively enhances signals in ¹H‐¹⁵N correlation spectra. Binding to the polysaccharide heparin was found to induce the unfolding of preformed structural elements in OPN.     

Helical Propensity in an Intrinsically Disordered Protein Accelerates Ligand Binding

Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well‐ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence‐monitored stopped‐flow kinetic measurements we show that the secondary structure content in helix 1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.     

An Approach to NMR Assignment of Intrinsically Disordered Proteins

An efficient approach to NMR assignments in intrinsically disordered proteins is presented, making use of the good dispersion of cross peaks observed in [¹⁵N,¹³C′]‐ and [¹³C′,¹H^N]‐correlation spectra. The method involves the simultaneous collection of {3D (H)NCO(CAN)H and 3D (HACA)CON(CA)HA} spectra for backbone assignments via sequential H^N and H^α correlations and {3D (H)NCO(CACS)HS and 3D (HS)CS(CA)CO(N)H} spectra for side‐chain ¹H and ¹³C assignments, employing sequential ¹H data acquisitions with direct detection of both the amide and aliphatic protons. The efficacy of the approach for obtaining resonance assignments with complete backbone and side‐chain chemical shifts is demonstrated experimentally for the 61‐residue [¹³C,¹⁵N]‐labelled peptide of a voltage‐gated potassium channel protein of the Kv1.4 channel subunit. The general applicability of the approach for the characterisation of moderately sized globular proteins is also demonstrated.     

无序蛋白质在生物分子凝聚相形成与调控中的作用

生物分子凝聚形成生物体内的多种无膜细胞器,其独特的物理化学性质使其具有多样的生物学功能,包括感知外界环境的变化、调节蛋白在细胞内的浓度、调控信号转导途径以及选择性富集特定蛋白质和RNA等。同时,生物分子凝聚相的错误形成与调控会导致多种人类疾病,如神经退行性疾病、癌症和病毒性疾病等。无序蛋白质在生物分子凝聚相的形成和调控中发挥了重要作用。本文通过总结分析无序蛋白在生物分子凝聚相形成中的作用以及化学小分子对生物分子凝聚相的调控,探讨了通过靶向无序蛋白进行配体设计来获得调控生物分子凝聚相化学探针及药物的可能性,并展望了揭示无序蛋白及化学分子调控生物凝聚相机制应重点关注的问题。     

Mapping Multivalency and Differential Affinities within Large Intrinsically Disordered Protein Complexes with Segmental Motion Analysis

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time‐resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region‐specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes—a result with implications for the diversity of transport routes in nucleocytoplasmic transport.     

Conformational Ensemble of Disordered Proteins Probed by Solvent Paramagnetic Relaxation Enhancement (sPRE)

Characterization of the conformational ensemble of disordered proteins is highly important for understanding protein folding and aggregation mechanisms, but remains a computational and experimental challenge owing to the dynamic nature of these proteins. New observables that can provide unique insights into transient residual structures in disordered proteins are needed. Here using denatured ubiquitin as a model system, NMR solvent paramagnetic relaxation enhancement (sPRE) measurements provide an accurate and highly sensitive probe for detecting low populations of residual structure in a disordered protein. Furthermore, a new ensemble calculation approach based on sPRE restraints in conjunction with residual dipolar couplings (RDCs) and small‐angle X‐ray scattering (SAXS) is used to define the conformational ensemble of disordered proteins at atomic resolution. The approach presented should be applicable to a wide range of dynamic macromolecules.     

Forced Folding of a Disordered Protein Accesses an Alternative Folding Landscape

Intrinsically disordered proteins (IDPs) are involved in diverse cellular functions. Many IDPs can interact with multiple binding partners, resulting in their folding into alternative ligand‐specific functional structures. For such multi‐structural IDPs, a key question is whether these multiple structures are fully encoded in the protein sequence, as is the case in many globular proteins. To answer this question, here we employed a combination of single‐molecule and ensemble techniques to compare ligand‐induced and osmolyte‐forced folding of α‐synuclein. Our results reveal context‐dependent modulation of the protein′s folding landscape, suggesting that the codes for the protein′s native folds are partially encoded in its primary sequence, and are completed only upon interaction with binding partners. Our findings suggest a critical role for cellular interactions in expanding the repertoire of folds and functions available to disordered proteins.     

Studying Intrinsically Disordered Proteins under True In Vivo Conditions by Combined Cross‐Polarization and Carbonyl‐Detection NMR Spectroscopy

Under physiological conditions, studies of intrinsically disordered proteins (IDPs) by conventional NMR methods based on proton detection are severely limited by fast amide‐proton exchange with water. ¹³C detection has been proposed as a solution to the exchange problem, but is hampered by low sensitivity. We propose a new pulse sequence combining proton–nitrogen cross‐polarization and carbonyl detection to record high‐resolution, high‐sensitivity NMR spectra of IDPs under physiological conditions. To demonstrate the efficacy of this approach, we recorded a high‐quality N–CO correlation spectrum of α‐synuclein in bacterial cells at 37 °C.     

Structural and Dynamic Characterization of the Molecular Hub Early Region 1A (E1A) from Human Adenovirus

The small‐DNA human adenovirus encodes one of the most versatile molecular hubs, the E1A protein. This protein is essential for productive viral infection in human cells and a vast amount of biologically relevant data are available on its interactions with host proteins. Up to now, however, no high‐resolution structural and dynamic information on E1A is available despite its important biological role. Among the different spliced variants of E1A, two are expressed at high level in the early stage of infection. These are 243 and 289 residues isoforms. Herein, we present their NMR characterization, showing that they are both highly disordered, but also demonstrate a certain heterogeneous behavior in terms of structural and dynamic properties. Furthermore, we present the characterization of the isolated domain of the longer variant, known as CR3. This study opens the way to understanding at the molecular level how E1A functions.     

Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

The kinase inhibitory domain of the cell cycle regulatory protein p27^Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of ¹³C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.     

Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C-Terminal Domain of Histone H1.0

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T¹¹⁸-H1.0 (T¹¹⁸PKK motif) and T¹⁴⁰-H1.0 (T¹⁴⁰PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T¹¹⁸-H1.0 and pT¹¹⁸-H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104–115 and a short α/3₁₀ helix (residues 119–123), that are almost perpendicular in T¹¹⁸-H1.0 but have a poorly defined orientation in pT¹¹⁸-H1.0. Peptides T¹⁴⁰-H1.0 and pT¹⁴⁰-H1.0 form very similar α helices between residues 141–147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT¹¹⁸-H1.0 and pT¹⁴⁰-H1.0 has pK_a values below the intrinsic values, which can be explained by non-specific charge–charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT¹⁴⁰ pK_a being closer to the intrinsic pK_a value than the pT¹¹⁸ pK_a. Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.