Structure-based Inhibitor Design for the Intrinsically Disordered Protein c-Myc.
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ABSTRACT:
SUBMITTER: Yu C
PROVIDER: S-EPMC4773988 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A growing body of research has demonstrated that targeting intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) is feasible and represents a new trending strategy in drug discovery. However, the number of inhibitors targeting IDPs/IDPRs is increasing slowly due to limitations of the methods that can be used to accelerate the discovery process. We have applied structure-based methods to successfully develop the first peptidic inhibitor (HIPe - Histone Inhibitory Peptide) that targets histone H4 that are released from NETs (Neutrophil Extracellular Traps). HIPe binds stably to the disordered N-terminal tail of histone H4, thereby preventing histone H4-induced cell death. Recently, by utilisation of the same state-of-the-art approaches, we have developed a novel peptidic inhibitor (CHIP - Cyclical Histone H2A Interference Peptide) that binds to NET-resident histone H2A, which results in a blockade of monocyte adhesion and consequently reduction in atheroprogression. Here, we present comprehensive details on the computational methods utilised to design and develop HIPe and CHIP. We have exploited protein-protein complexes as starting structures for rational peptide design and then applied binding free energy methods to predict and prioritise binding strength of the designed peptides with histone H4 and H2A. By doing this way, we have modelled only around 20 peptides and from these were able to select 4-5 peptides, from a total of more than a trillion candidate peptides, for functional characterisation in different experiments. The developed computational protocols are generic and can be widely used to design and develop novel inhibitors for other disordered proteins.
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Project description:Intrinsically disordaered proteins (IDPs) are a prevalent phenomenon with over 30% of human proteins estimated to have long disordered regions. Computational methods are widely used to study IDPs, however, nearly all treat disorder in a binary fashion, not accounting for the structural heterogeneity present in disordered regions. Here, we present a new de novo method, FRAGFOLD-IDP, which addresses this problem. Using 200 protein structural ensembles derived from NMR, we show that FRAGFOLD-IDP achieves superior results compared to methods which can predict related data (NMR order parameter, or crystallographic B-factor). FRAGFOLD-IDP produces very good predictions for 33.5% of cases and helps to get a better insight into the dynamics of the disordered ensembles. The results also show it is not necessary to predict the correct fold of the protein to reliably predict per-residue fluctuations. It implies that disorder is a local property and it does not depend on the fold. Our results are orthogonal to DynaMine, the only other method significantly better than the naïve prediction. We therefore combine these two using a neural network. FRAGFOLD-IDP enables better insight into backbone dynamics in IDPs and opens exciting possibilities for the design of disordered ensembles, disorder-to-order transitions, or design for protein dynamics.
| S-EPMC5539115 | biostudies-literature
Project description:Intrinsically disordered proteins (IDPs) carry out important biological functions and offer an instructive model system for folding and binding studies. However, their structural characterization in the absence of interactors is hindered by their highly dynamic conformation. The cyclin-dependent-kinase inhibitor (Cki) Sic1 from Saccharomyces cerevisiae is a key regulator of the yeast cell cycle, which controls entrance into S phase and coordination between cell growth and proliferation. Its last 70 out of 284 residues display functional and structural homology to the inhibitory domain of mammalian p21 and p27. Sic1 has escaped systematic structural characterization until now. Here, complementary biophysical methods are applied to the study of conformational properties of pure Sic1 in solution. Based on sequence analysis, gel filtration, circular dichroism (CD), electrospray-ionization mass spectrometry (ESI-MS), and limited proteolysis, it can be concluded that the whole molecule exists in a highly disordered state and can, therefore, be classified as an IDP. However, the results of these experiments indicate, at the same time, that the protein displays some content in secondary and tertiary structure, having properties similar to those of molten globules or premolten globules. Proteolysis-hypersensitive sites cluster at the N-terminus and in the middle of the molecule, whereas the most structured region resides at the C-terminus, including part of the inhibitory domain and the casein-kinase-2 (CK2) phosphorylation target S201. The mutations S201A and S201E, which are known to affect Sic1 function, do not have significant effects on the conformational properties of the pure protein.
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Project description:The hydrolysis of ATP by the ATP synthase in mitochondria is inhibited by a protein called IF1. Bovine IF1 has 84 amino acids, and its N-terminal inhibitory region is intrinsically disordered. In a known structure of bovine F1-ATPase inhibited with residues 1-60 of IF1, the inhibitory region from residues 1-50 is mainly ?-helical and buried deeply at the ?(DP)?(DP)-catalytic interface, where it forms extensive interactions with five of the nine subunits of F1-ATPase but mainly with the ?(DP)-subunit. As described here, on the basis of two structures of inhibited complexes formed in the presence of large molar excesses of residues 1-60 of IF1 and of a version of IF1 with the mutation K39A, it appears that the intrinsically disordered inhibitory region interacts first with the ?E?E-catalytic interface, the most open of the three catalytic interfaces, where the available interactions with the enzyme allow it to form an ?-helix from residues 31-49. Then, in response to the hydrolysis of an ATP molecule and the associated partial closure of the interface to the ?TP?TP state, the extent of the folded ?-helical region of IF1 increases to residues 23-50 as more interactions with the enzyme become possible. Finally, in response to the hydrolysis of a second ATP molecule and a concomitant 120° rotation of the ?-subunit, the interface closes further to the ?(DP)?(DP)-state, allowing more interactions to form between the enzyme and IF1. The structure of IF1 now extends to its maximally folded state found in the previously observed inhibited complex.
| S-EPMC4128166 | biostudies-literature
Project description:IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.
| S-EPMC3149264 | biostudies-literature
Project description:In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI's most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme's binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.
| S-EPMC7234811 | biostudies-literature
Project description:The small neuroendocrine protein 7B2 has been shown to be required for the productive maturation of proprotein convertase 2 (proPC2) to an active enzyme form; this action is accomplished via its ability to block aggregation of proPC2 into nonactivatable forms. Recent data show that 7B2 can also act as a postfolding chaperone to block the aggregation of a number of other proteins, for example, α-synuclein. To gain insight into the mechanism of action of 7B2 in blocking protein aggregation, we performed structural studies of this protein using gel filtration chromatography, intrinsic tryptophan fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Gel filtration studies indicated that 7B2 exists as an extended monomer, eluting at a molecular mass higher than that expected for a globular protein of similar size. However, chemical cross-linking showed that 7B2 exhibits concentration-dependent oligomerization. CD experiments showed that both full-length 27 kDa 7B2 and the C-terminally truncated 21 kDa form lack appreciable secondary structure, although the longer protein exhibited more structural content than the latter, as demonstrated by intrinsic and ANS fluorescence studies. NMR spectra confirmed the lack of structure in native 7B2, but a disorder-to-order transition was observed upon incubation with one of its client proteins, α-synuclein. We conclude that 7B2 is a natively disordered protein whose function as an antiaggregant chaperone is likely facilitated by its lack of appreciable secondary structure and tendency to form oligomers.
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Project description:Intrinsically disordered proteins are attractive therapeutic targets owing to their prevalence in several diseases. Yet their lack of well-defined structure renders ligand discovery a challenging task. An intriguing example is provided by the oncoprotein c-Myc, a transcription factor that is over expressed in a broad range of cancers. Transcriptional activity of c-Myc is dependent on heterodimerization with partner protein Max. This protein-protein interaction is disrupted by the small molecule 10058-F4 (1), that binds to monomeric and disordered c-Myc. To rationalize the mechanism of inhibition, structural ensembles for the segment of the c-Myc domain that binds to 1 were computed in the absence and presence of the ligand using classical force fields and explicit solvent metadynamics molecular simulations. The accuracy of the computed structural ensembles was assessed by comparison of predicted and measured NMR chemical shifts. The small molecule 1 was found to perturb the composition of the apo equilibrium ensemble and to bind weakly to multiple distinct c-Myc conformations. Comparison of the apo and holo equilibrium ensembles reveals that the c-Myc conformations binding 1 are already partially formed in the apo ensemble, suggesting that 1 binds to c-Myc through an extended conformational selection mechanism. The present results have important implications for rational ligand design efforts targeting intrinsically disordered proteins.
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Project description:Phosphorylation is a major post-translational modification that plays a central role in signaling pathways. Protein kinases phosphorylate substrates (phosphoproteins) by adding phosphate at Ser/Thr or Tyr residues (phosphosites). A large amount of data identifying and describing phosphosites in phosphoproteins has been reported but the specificity of phosphorylation is not fully resolved. In this report, data of kinase-substrate pairs identified by the Kinase-Interacting Substrate Screening (KISS) method were used to analyze phosphosites in intrinsically disordered regions (IDRs) of intrinsically disordered proteins. We compared phosphorylated and nonphosphorylated IDRs and found that the phosphorylated IDRs were significantly longer than nonphosphorylated IDRs. The phosphorylated IDR is often the longest IDR (71%) in a phosphoprotein when only a single phosphosite exists in the IDR, and when the phosphoprotein has multiple phosphosites in an IDR(s), the phosphosites are primarily localized in a single IDR (78%) and this IDR is usually the longest one (81%). We constructed a stochastic model of phosphorylation to estimate the effect of IDR length. The model that accounted for IDR length produced more realistic results when compared with a model that excluded the IDR length. We propose that the IDR length is a significant determinant for locating kinase phosphorylation sites in phosphoproteins.
| S-EPMC6954741 | biostudies-literature
Project description:Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine ?-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric ?-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ?4.6 nm to ?1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed.
| S-EPMC3183800 | biostudies-other