Long-range modulation of chain motions within the intrinsically disordered transactivation domain of tumor suppressor p53

The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.     

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.     

Variation of free‐energy landscape of the p53 C‐terminal domain induced by acetylation: Enhanced conformational sampling

The C‐terminal domain (CTD) of tumor suppressor protein p53 is an intrinsically disordered region that binds to various partner proteins, where lysine of CTD is acetylated/nonacetylated and histidine neutralized/non‐neutralized. Because of the flexibility of the unbound CTD, a free‐energy landscape (FEL) is a useful quantity for determining its statistical properties. We conducted enhanced conformational sampling of CTD in the unbound state via virtual system coupled multicanonical molecular dynamics, in which the lysine was acetylated or nonacetylated and histidine was charged or neutralized. The fragments were expressed by an all‐atom model and were immersed in an explicit solvent. The acetylation and charge‐neutralization varied FEL greatly, which might be convenient to exert a hub property. The acetylation slightly enhanced alpha‐helix structures that are more compact than sheet/loop conformations. The charge‐neutralization produced hairpins. Additionally, circular dichroism experiments confirmed the computational results. We propose possible binding mechanisms of CTD to partners by investigating FEL. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.     

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.     

Folding of the Tau Protein on Microtubules

Microtubules are regulated by microtubule‐associated proteins. However, little is known about the structure of microtubule‐associated proteins in complex with microtubules. Herein we show that the microtubule‐associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β‐sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau.     

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY

Integral membrane proteins in bacteria are co‐translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N‐terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C‐terminal membrane targeting sequence. The central A domain binds to the translocon non‐specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome‐bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.     

The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins

Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well‐characterized folding upon binding to dynamic fuzzy complexes. To date, most studies concern the binding of an IDP to a structured protein, while the interaction between two IDPs is poorly understood. In this study, NMR, smFRET, and molecular dynamics (MD) simulation are combined to characterize the interaction between two IDPs, the C‐terminal domain (CTD) of protein 4.1G and the nuclear mitotic apparatus (NuMA) protein. It is revealed that CTD and NuMA form a fuzzy complex with remaining structural disorder. Multiple binding sites on both proteins were identified by molecular dynamics and mutagenesis studies. This study provides an atomic scenario in which two IDPs bearing multiple binding sites interact with each other in dynamic equilibrium. The combined approach employed here could be widely applicable for investigating IDPs and their dynamic interactions.     

Chemical synthesis of transactivation domain (TAD) of tumor suppressor protein p53 by native chemical ligation of three peptide segments

Chemical composition of tumor suppressor protein p53 is altered via multiple post-translational modifications which modulate its cellular lifetime and interactions with other biomolecules. Here we report total chemical synthesis of a 61-residue form of transactivation domain (TAD) of p53 based on native chemical ligation of three peptide segments. The experiments to characterize its binding to nuclear co-activator binding domain (NCBD) of CREB-binding protein confirmed native-like induced folding upon binding to NCBD. Thus, the synthetic approach described herein can be useful for the preparation of various post-translationally modified analogues of TAD-p53 for further functional biochemical and biophysical studies.     

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.     

Disorder-to-order transition of an intrinsically disordered region of sortase revealed by multiscale enhanced sampling

Molecular functions of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs), such as molecular recognition and cellular signaling, are ascribed to dynamic changes in the conformational space in response to binding of target molecules. Sortase, a transpeptitase in Gram-positive bacteria, has an IDR in a loop which undergoes a disordered-to-ordered transition (called "disordered loop"), accompanying a tilt of another loop ("dynamic loop"), upon binding of a signal peptide and a calcium ion. In this study, all-atom conformational ensembles of sortase were calculated for the four different binding states (with/without the peptide and with/without a calcium ion) by the multiscale enhanced sampling (MSES) simulation to examine how the binding of the peptide and/or calcium influences the conformational ensemble. The MSES is a multiscale and multicopy simulation method that allows an enhanced sampling of the all-atom model of large proteins including explicit solvent. A 100 ns MSES simulation of the ligand-free sortase using 20 replicas (in total 2 μs) demonstrated large flexibility in both the disordered and dynamic loops; however, their distributions were not random but had a clear preference which populates the N-terminal part of the disordered loop near the bound form. The MSES simulations of the three binding states clarified the allosteric mechanism of sortase: the N- and C-terminal parts of the disordered loop undergo a disorder-to-order transition independently of each other upon binding of the peptide and a calcium ion, respectively; however, upon binding of both ligands, the two parts work cooperatively to stabilize the bound peptide.     

Do Intrinsically Disordered Proteins Possess High Specificity in Protein–Protein Interactions?

Specific protein–protein interactions are critical to cellular function. Structural flexibility and disorder‐to‐order transitions upon binding enable intrinsically disordered proteins (IDPs) to overcome steric restrictions and form complementary binding interfaces, and thus, IDPs are widely considered to have high specificity and low affinity for molecular recognition. However, flexibility may also enable IDPs to form complementary binding interfaces with misbinding partners, resulting in a great number of nonspecific interactions. Consequently, it is questionable whether IDPs really possess high specificity. In this work, we investigated this question from a thermodynamic viewpoint. We collected mutant thermodynamic data for 35 ordered protein complexes and 43 disordered protein complexes. We found that the enthalpy–entropy compensation for disordered protein complexes was more complete than that for ordered protein complexes. We further simulated the binding processes of ordered and disordered protein complexes under mutations. Simulation data confirmed the observation of experimental data analyses and further revealed that disordered protein complexes possessed smaller changes in binding free energy than ordered protein complexes under the same mutation perturbations. Therefore, interactions of IDPs are more malleable than those of ordered proteins due to their structural flexibility in the complex. Our results provide new clues for exploring the relationship between protein flexibility, adaptability, and specificity.     

Multitude of binding modes attainable by intrinsically disordered proteins: a portrait gallery of disorder-based complexes

Proteins are constantly involved in the multitude of various interactions creating sophisticated networks which define and control all (or almost all) the biological processes taking place in any living organism. Intrinsically disordered proteins or regions play a number of crucial roles in mediating protein interactions. The lack of fixed structure protruding to the high level of intrinsic dynamics and almost unrestricted flexibility at various structure levels, being the major characteristics of intrinsically disordered proteins, provides them with unprecedented advantages over the ordered proteins. The binding modes attainable by disordered proteins are highly diverse, creating a multitude of unusual complexes. Although the majority of studied to date intrinsic disorder-based complexes are ordered or static entities originating due to the global or local disorder-to-order transitions, a new development is the discovery of dynamic complexes in which intrinsically disordered proteins continue to sample an ensemble of rapidly interconverting conformations mostly devoid of structure even in their bound state. The goal of this critical review is to illustrate binding plasticity of intrinsically disordered proteins by representing a portrait gallery of the disorder-based complexes (119 references).     

Replica exchange with guided annealing for accelerated sampling of disordered protein conformations

We critically examine a recently proposed convective replica exchange (cRE) method for enhanced sampling of protein conformation based on theoretical and numerical analysis. The results demonstrate that cRE and related replica exchange with guided annealing (RE‐GA) schemes lead to unbalanced exchange attempt probabilities and break detailed balance whenever the system undergoes slow conformational transitions (relative to the temperature diffusion timescale). Nonetheless, numerical simulations suggest that approximate canonical ensembles can be generated for systems with small conformational transition barriers. This suggests that RE‐GA maybe suitable for simulating intrinsically disordered proteins, an important class of newly recognized functional proteins. The efficacy of RE‐GA is demonstrated by calculating the conformational ensembles of intrinsically disordered kinase inducible domain protein. The results show that RE‐GA helps the protein to escape nonspecific compact states more efficiently and provides several fold speedups in generating converged and largely correct ensembles compared to the standard temperature RE. © 2014 Wiley Periodicals, Inc.     

Asymmetric Modulation of Protein Order–Disorder Transitions by Phosphorylation and Partner Binding

As for many intrinsically disordered proteins, order–disorder transitions in the N‐terminal oligomerization domain of the multifunctional nucleolar protein nucleophosmin (Npm‐N) are central to its function, with phosphorylation and partner binding acting as regulatory switches. However, the mechanism of this transition and its regulation remain poorly understood. In this study, single‐molecule and ensemble experiments revealed pathways with alternative sequences of folding and assembly steps for Npm‐N. Pathways could be switched by altering the ionic strength. Phosphorylation resulted in pathway‐specific effects, and decoupled folding and assembly steps to facilitate disorder. Conversely, binding to a physiological partner locked Npm‐N in ordered pentamers and counteracted the effects of phosphorylation. The mechanistic plasticity found in the Npm‐N order–disorder transition enabled a complex interplay of phosphorylation and partner‐binding steps to modulate its folding landscape.     

The native ensemble and folding of a protein molten-globule: functional consequence of downhill folding

The continually emerging functional significance of intrinsic disorder and conformational flexibility in proteins has challenged the long-standing dogma of a well-defined structure contributing to a specific function. Molten-globular states, a class of proteins with significant secondary-structure but a fluid hydrophobic core, is one such example. They have however been difficult to characterize due to the complexity of experimental data and lack of computational avenues. Here, we dissect the folding mechanism of the α-helical molten-globular protein NCBD from three fundamentally different approaches: statistical-mechanical variable barrier model, C(α)-based Gō-model and explicit water all-atom molecular dynamics (MD) simulations. We find that NCBD displays the characteristics of a one-state globally downhill folder but is significantly destabilized. Using simulation techniques, we generate a highly constrained but a heterogeneous native ensemble of the molten-globule for the first time that is consistent with experimental data including small angle X-ray scattering (SAXS), circular dichroism (CD), and nuclear magnetic resonance (NMR). The resulting native ensemble populates conformations reported in other bound-forms providing direct evidence to the mechanism of conformational selection for binding multiple partners in this domain. Importantly, our simulations reveal a connection between downhill folding and large conformational flexibility in this domain that has been evolutionarily selected and functionally exploited resulting in large binding promiscuity. Finally, the multimodel approach we employ here serves as a powerful methodology to study mechanisms and suggests that the thermodynamic features of molten-globules fall within the array of folding mechanisms available to small single-domain proteins.     

Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains

Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K(d) correlate with changes in k(off).