scholarly journals Partial structure, dampened mobility, and modest impact of a His tag in the SARS-CoV-2 Nsp2 C-terminal region

2021 ◽  
Author(s):  
Miguel Mompeán ◽  
Miguel Á. Treviño ◽  
Douglas V. Laurents
Keyword(s):  
Drug Targets ◽  
Intrinsically Disordered Proteins ◽  
Terminal Region ◽  
Disordered Proteins ◽  
X Ray Crystallography ◽  
Intrinsically Disordered ◽  
His Tag ◽  
Level Information ◽  
Human Proteins ◽  
A Minor

AbstractIntrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many viruses use their own IDPs to “hack” these processes to deactivate host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational preferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2, whose C-terminal region (CtR) is predicted to be disordered, interacts with human proteins that regulate translation initiation and endosome vesicle sorting. Molecules that block these interactions could be valuable leads for drug development. The 13Cβ and backbone 13CO, 1HN, 13Cα, and 15N nuclei of Nsp2’s 45-residue CtR were assigned and used to characterize its structure and dynamics in three contexts; namely: (1) retaining an N-terminal His tag, (2) without the His tag and with an adventitious internal cleavage, and (3) lacking both the His tag and the internal cleavage. Two five-residue segments adopting a minor extended population were identified. Overall, the dynamic behavior is midway between a completely rigid and a fully flexible chain. Whereas the presence of an N-terminal His tag and internal cleavage stiffen and loosen, respectively, neighboring residues, they do not affect the tendency of two regions to populate extended conformations.

scholarly journals Towards Targeting the Disordered SARS-CoV-2 Nsp2 C-terminal Region: Partial Structure and Dampened Mobility Revealed by NMR Spectroscopy

2020 ◽  
Author(s):  
Miguel Mompeán ◽  
Miguel Á. Treviño ◽  
Douglas V. Laurents
Keyword(s):  
Nmr Spectroscopy ◽  
Drug Targets ◽  
Intrinsically Disordered Proteins ◽  
Small Population ◽  
Terminal Region ◽  
Disordered Proteins ◽  
X Ray Crystallography ◽  
Intrinsically Disordered ◽  
Level Information ◽  
Human Proteins

AbstractIntrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many virus use their own IDPs to “hack” these processes to disactive host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational perferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2 interacts with human proteins that regulate translation initiation and endosome vesicle sorting, and the C-terminal region of this protein is predicted to be disordered. Molecules that block these interactions could be valuable leads for drug development. To enable inhibitor screening and to uncover conformational preferences and dynamics, we have expressed and purified the 13C,15N-labeled C-terminal region of Nsp2. The 13Cβ and backbone 13CO, 1HN, 13Cα and 15N nuclei were assigned by analysis of a series of 2D 1H-15N HSQC and 13C-15N CON as well as 3D HNCO, HNCA, CBCAcoNH and HncocaNH spectra. Overall, the chemical shift data confirm that this region is chiefly disordered, but contains two five-residue segments that adopt a small population of β-strand structure. Whereas the region is flexible on ms/ms timescales as gauged by T1ρ measurements, the {1H}-15N NOEs reveal a flexibility on ns/ps timescales that is midway between a fully flexible and a completely rigid chain.

scholarly journals Intrinsically Disordered Proteins as Regulators of Transient Biological Processes and as Untapped Drug Targets

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2118
Author(s):  
Yusuke Hosoya ◽  
Junko Ohkanda
Keyword(s):  
Drug Targets ◽  
Intrinsically Disordered Proteins ◽  
Dynamic Control ◽  
Disordered Proteins ◽  
Biological Processes ◽  
Phase Separations ◽  
Cellular Processes ◽  
Intrinsically Disordered ◽  
New Drug ◽  
Liquid Phase Separations

Intrinsically disordered proteins (IDPs) are critical players in the dynamic control of diverse cellular processes, and provide potential new drug targets because their dysregulation is closely related to many diseases. This review focuses on several medicinal studies that have identified low-molecular-weight inhibitors of IDPs. In addition, clinically relevant liquid–liquid phase separations—which critically involve both intermolecular interactions between IDPs and their posttranslational modification—are analyzed to understand the potential of IDPs as new drug targets.

scholarly journals Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding

2015 ◽  
Vol 112 (31) ◽  
pp. 9614-9619 ◽  
Author(s):  
Munehito Arai ◽  
Kenji Sugase ◽  
H. Jane Dyson ◽  
Peter E. Wright
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Terminal Region ◽  
Relaxation Dispersion ◽  
Disordered Proteins ◽  
Conformational Ensemble ◽  
Induced Fit ◽  
Conformational Selection ◽  
Intrinsically Disordered ◽  
Factor C ◽  
Binding Mechanisms

Intrinsically disordered proteins (IDPs) frequently function in protein interaction networks that regulate crucial cellular signaling pathways. Many IDPs undergo transitions from disordered conformational ensembles to folded structures upon binding to their cellular targets. Several possible binding mechanisms for coupled folding and binding have been identified: folding of the IDP after association with the target (“induced fit”), or binding of a prefolded state in the conformational ensemble of the IDP to the target protein (“conformational selection”), or some combination of these two extremes. The interaction of the intrinsically disordered phosphorylated kinase-inducible domain (pKID) of the cAMP-response element binding (CREB) protein with the KIX domain of a general transcriptional coactivator CREB-binding protein (CBP) provides an example of the induced-fit mechanism. Here we show by NMR relaxation dispersion experiments that a different intrinsically disordered ligand, the transactivation domain of the transcription factor c-Myb, interacts with KIX at the same site as pKID but via a different binding mechanism that involves elements of conformational selection and induced fit. In contrast to pKID, the c-Myb activation domain has a strong propensity for spontaneous helix formation in its N-terminal region, which binds to KIX in a predominantly folded conformation. The C-terminal region of c-Myb exhibits a much smaller helical propensity and likely folds via an induced-fit process after binding to KIX. We propose that the intrinsic secondary structure propensities of pKID and c-Myb determine their binding mechanisms, consistent with their functions as inducible and constitutive transcriptional activators.

scholarly journals Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

2022 ◽  
Author(s):  
Spencer Smyth ◽  
Zhenfu Zhang ◽  
Alaji Bah ◽  
Thomas Tsangaris ◽  
Jennifer Dawson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Intrinsically Disordered Proteins ◽  
Regulatory Protein ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Terminal Region ◽  
Disordered Proteins ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Intrinsically Disordered

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamics characterization of their ensembles remain challenging, both in isolation and they form dynamic fuzzy complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Forster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a nonuniform segmental flexibility around six different labelling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-microsecond timescales. Upon hyperphosphorylation, which induces folding of ~40 residues in 4E-BP2, the quenching rates decreased at labelling sites closest to the phosphorylation sites and within the folded domain, and increased at the other sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs were significantly reduced upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step towards a mechanistic understanding of this important IDP via integrative modelling.

scholarly journals Therapeutic Interventions of Cancers Using Intrinsically Disordered Proteins as Drug Targets: c-Myc as Model System

2017 ◽  
Vol 16 ◽  
pp. 117693511769940 ◽  
Author(s):  
Deepak Kumar ◽  
Nitin Sharma ◽  
Rajanish Giri
Keyword(s):  
Drug Targets ◽  
Intrinsically Disordered Proteins ◽  
Hot Spot ◽  
Intrinsic Disorder ◽  
Therapeutic Interventions ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
In Vivo Experiments

The concept of protein intrinsic disorder has taken the driving seat to understand regulatory proteins in general. Reports suggest that in mammals nearly 75% of signalling proteins contain long disordered regions with greater than 30 amino acid residues. Therefore, intrinsically disordered proteins (IDPs) have been implicated in several human diseases and should be considered as potential novel drug targets. Moreover, intrinsic disorder provides a huge multifunctional capability to hub proteins such as c-Myc and p53. c-Myc is the hot spot for understanding and developing therapeutics against cancers and cancer stem cells. Our past understanding is mainly based on in vitro and in vivo experiments conducted using c-Myc as whole protein. Using the reductionist approach, c-Myc oncoprotein has been divided into structured and disordered domains. A wealth of data is available dealing with the structured perspectives of c-Myc, but understanding c-Myc in terms of disordered domains has just begun. Disorderness provides enormous flexibility to proteins in general for binding to numerous partners. Here, we have reviewed the current progress on understanding c-Myc using the emerging concept of IDPs.

scholarly journals Prediction of MoRFs in Protein Sequences with MLPs Based on Sequence Properties and Evolution Information

Entropy ◽  
2019 ◽  
Vol 21 (7) ◽  
pp. 635 ◽  
Author(s):  
Hao He ◽  
Jiaxiang Zhao ◽  
Guiling Sun
Keyword(s):  
Neural Networks ◽  
Drug Targets ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Evolutionary Information ◽  
Sliding Windows ◽  
Intrinsically Disordered ◽  
Functional Regions ◽  
Interaction Partners ◽  
Mlp Neural Networks

Molecular recognition features (MoRFs) are one important type of intrinsically disordered proteins functional regions that can undergo a disorder-to-order transition through binding to their interaction partners. Prediction of MoRFs is crucial, as the functions of MoRFs are associated with many diseases and can therefore become the potential drug targets. In this paper, a method of predicting MoRFs is developed based on the sequence properties and evolutionary information. To this end, we design two distinct multi-layer perceptron (MLP) neural networks and present a procedure to train them. We develop a preprocessing process which exploits different sizes of sliding windows to capture various properties related to MoRFs. We then use the Bayes rule together with the outputs of two trained MLP neural networks to predict MoRFs. In comparison to several state-of-the-art methods, the simulation results show that our method is competitive.

scholarly journals Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

2017 ◽  
Vol 114 (31) ◽  
pp. E6332-E6341 ◽  
Author(s):  
Patricia Santofimia-Castaño ◽  
Bruno Rizzuti ◽  
Ángel L. Pey ◽  
Philippe Soubeyran ◽  
Miguel Vidal ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
In Silico ◽  
Intrinsically Disordered Proteins ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Disordered Proteins ◽  
Wild Type ◽  
Intrinsically Disordered ◽  
Polycomb Proteins

Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 μM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins.

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