Elio A Cino

University of Waterloo, Waterloo, Ontario, Canada

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Publications (11)32.49 Total impact

  • Elio A. Cino · Wing-Yiu Choy · Mikko Karttunen
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    ABSTRACT: Intrinsically disordered proteins (IDPs) and regions are highly prevalent in eukaryotic proteomes, and like folded proteins, they perform essential biological functions. Interaction sites in folded proteins are generally formed by tertiary structures, whereas IDPs use short segments called Linear Motifs (LMs). Despite their short length and lack of stable structure, LMs may have considerable structural propensities, which often resemble bound state conformations with targets. Structural data is crucial for understanding the molecular basis of protein interactions and development of targeted pharmaceuticals, but IDPs present considerable challenges to experimental techniques. As a result, IDPs are largely underrepresented in the Protein Data Bank. In the face of experimental challenges, Molecular Dynamics (MD) simulations have proven to be a useful tool for structural characterization of IDPs. Here, the free state ensemble of the Nuclear Receptor Corepressor 1 (NCOR1) CoRNR box 3 motif, which is important for binding to nuclear receptors to control gene expression, is studied using MD simulations of a total of 8 µs. Transitions between disordered and α-helical conformations resembling a bound state structure were observed throughout the trajectory, indicating that the motif may have a natural conformational bias towards bound state structures. The data shows that the disordered and folded populations are separated by a low energy (4-6 kJ/mol) barrier, and the presence of off-pathway intermediates, leading to a Cterminally folded species that cannot efficiently transition into a completely folded conformation. Structural transitions and folding pathways within the free state ensemble were well described by Principal Component Analysis (PCA) of the peptide backbone dihedral angles, with the analysis providing insight for increasing structural homogeneity of the ensemble
    No preview · Article · Jan 2016 · The Journal of Physical Chemistry B
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    ABSTRACT: p53, p63, p73 family of proteins are transcription factors with crucial roles in regulating cellular processes such apoptosis, proliferation, differentiation, and DNA damage response. The three family members have both overlapping and unique biological functions. Sequence and structural homology are greatest in the DNA binding domains (DBD), which is the site of the majority of p53 mutations. Structurally unstable p53 DBD mutants can associate with themselves or p63 and p73 DBDs, impeding tumor suppressor functions. Evidence suggests that these proteins associate to form amyloid-like oligomers and fibrils through an aggregation-prone sequence within the DBDs. Despite having high sequence and structure similarities, p63 and p73 DBDs appear to have considerably lower tendencies to be incorporated into p53 aggregates, relative to p53. The backbone resonance assignments of p73 DBD reported here complement those previously reported for p53 and p63, allowing comparisons and providing molecular insights into their biological functions and roles in aggregation and tumor development.
    Full-text · Article · Aug 2015 · Biomolecular NMR Assignments
  • Elio Cino · Mikko Karttunen · Wing-Yiu Choy

    No preview · Chapter · Jan 2015
  • Elio A. Cino · Wing-Yiu Choy · Mikko Karttunen
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    ABSTRACT: Linear Motifs (LMs) are protein-protein interaction sites, typically consisting of ~4-20 amino acid residues that are often found in disordered proteins or regions, and function largely independent from other parts of the proteins they are found in. These short sequence patterns are involved in a wide spectrum of biological functions including cell cycle control, transcriptional regulation, enzymatic catalysis, cell signaling, protein trafficking, etc. Even though LMs may adopt defined structures in complexes with targets, which can be determined by conventional methods, their uncomplexed states can be highly dynamic and difficult to characterize. This hinders our understanding of the structure-function relationship of LMs. Here, the uncomplexed states of 6 different LMs are investigated using atomistic molecular dynamics (MD) simulations. The total simulation time was about 63 µs. The results show that LMs can have distinct conformational propensities, which often resemble their complexed state. As a result, the free state structure and dynamics of LMs may hold important clues regarding binding mechanisms, affinities and specificities. The findings should be helpful in advancing our understanding of the mechanisms whereby disordered amino acid sequences bind targets, modeling disordered proteins/regions, and computational prediction of binding affinities.
    No preview · Article · Dec 2013 · The Journal of Physical Chemistry B
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    ABSTRACT: A small number of proteins, called hubs, have high connectivity and are essential for interactome functionality and integrity. Keap1 is a crucial hub in the oxidative stress response and apoptosis. The Kelch domain of Keap1 preferentially binds to disordered regions of its partners, which share similar binding motifs, but have a wide range of binding affinities. Isothermal titration calorimetry (ITC) and multi-microsecond molecular dynamics (MD) simulations were used to determine the factors that govern the affinity of all currently known disordered binding partners to Kelch. Our results show that the affinities to this hub are largely determined by the extent of preformed bound state-like conformation in the free state structures of these disordered targets. Based on our findings, we have designed a high-affinity peptide that can specifically disrupt the Keap1-NRF2 interaction and has the potential for therapeutic applications.
    Full-text · Article · Jul 2013 · Scientific Reports
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    ABSTRACT: Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin alpha (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, we have characterized the interaction between ProTα and the Kelch domain of Keap1 by using nuclear magnetic resonance spectroscopy (NMR), isothermal titration calorimetry (ITC), peptide array analysis, site-directed mutagenesis, and molecular dynamic (MD) simulations. The results of NMR chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTα retains a high level of flexibility, even in the bound state with Kelch. This finding is in agreement with the observations from the MD simulations of the ProTα-Kelch complex. Mutational analysis of ProTα, guided by peptide array data and ITC, further pinpointed that the region (38)NANEENGE(45) of ProTα is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding.
    No preview · Article · Jan 2013 · Journal of Molecular Biology
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    Elio A Cino · Mikko Karttunen · Wing-Yiu Choy
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    ABSTRACT: Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.
    Full-text · Article · Nov 2012 · PLoS ONE
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    Elio A Cino · Wing-Yiu Choy · Mikko Karttunen
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    ABSTRACT: We have compared molecular dynamics (MD) simulations of a β-hairpin forming peptide derived from the protein Nrf2 with 10 biomolecular force fields using trajectories of at least 1 μs. The total simulation time was 37.2 μs. Previous studies have shown that different force fields, water models, simulation methods, and parameters can affect simulation outcomes. The MD simulations were done in explicit solvent with a 16-mer Nrf2 β-hairpin forming peptide using Amber ff99SB-ILDN, Amber ff99SB*-ILDN, Amber ff99SB, Amber ff99SB*, Amber ff03, Amber ff03*, GROMOS96 43a1p, GROMOS96 53a6, CHARMM27, and OPLS-AA/L force fields. The effects of charge-groups, terminal capping, and phosphorylation on the peptide folding were also examined. Despite using identical starting structures and simulation parameters, we observed clear differences among the various force fields and even between replicates using the same force field. Our simulations show that the uncapped peptide folds into a native-like β-hairpin structure at 310 K when Amber ff99SB-ILDN, Amber ff99SB*-ILDN, Amber ff99SB, Amber ff99SB*, Amber ff03, Amber ff03*, GROMOS96 43a1p, or GROMOS96 53a6 were used. The CHARMM27 simulations were able to form native hairpins in some of the elevated temperature simulations, while the OPLS-AA/L simulations did not yield native hairpin structures at any temperatures tested. Simulations that used charge-groups or peptide capping groups were not largely different from their uncapped counterparts with single atom charge-groups. On the other hand, phosphorylation of the threonine residue located at the β-turn significantly affected the hairpin formation. To our knowledge, this is the first study comparing such a large set of force fields with respect to β-hairpin folding. Such a comprehensive comparison will offer useful guidance to others conducting similar types of simulations.
    Full-text · Article · Aug 2012 · Journal of Chemical Theory and Computation
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    ABSTRACT: Kelch-like ECH-associated Protein 1 (Keap1) is a multi-domain protein that functions as an inhibitor of the transcription factor nuclear factor E2-related factor 2 (Nrf2) in the cellular response to oxidative stress. Under normal conditions, Keap1 binds to Nrf2 via its C-terminal Kelch domain and the interaction ultimately leads to the ubiquitin-dependent degradation of Nrf2. It has been proposed that designing molecules to selectively disrupt the Keap1–Nrf2 interaction can be a potential therapeutic approach for enhancing the expression of cytoprotective genes. Here, we reported the 1H, 13C, and 15N backbone chemical shift assignments of the Kelch domain of mouse Keap1. Further, unlabeled Nrf2 peptide containing the Kelch-binding motif was added to the 15N-labeled Kelch sample. 1H–15N HSQC spectra of the protein in the absence and presence of an equimolar concentration of the Nrf2 peptide were presented. A significant number of resonance signals were shifted upon addition of the peptide, confirming the protein–peptide interaction. The results here will not just facilitate the further studies of the binding between Keap1 and Nrf2, it will also be valuable for probing interactions between the Kelch domain and small molecules, as well as a growing list of protein targets that have been identified recently.
    No preview · Article · Jun 2012 · Biomolecular NMR Assignments
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    ABSTRACT: PnTx3-4 is a toxin isolated from the venom of the spider Phoneutria nigriventer that blocks N-, P/Q-, and R-type voltage-gated calcium channels and has great potential for clinical applications. In this report we used the SUMO system to express large amounts of recombinant PnTx3-4 peptide, which was found in both soluble and insoluble fractions of bacterial extracts. We purified the recombinant toxin from both fractions and showed that the recombinant peptide showed biological activity similar to the native PnTx3-4. In silico analysis of the primary sequence of PnTx3-4 indicated that the peptide conforms to all the criteria of a knottin scaffold. Additionally, circular dichroism spectrum analysis of the recombinant PnTx3-4 predicted that the toxin structure is composed of approximately 53% turns/unordered, 31% α-helix and 16% β-strand, which is consistent with predicted model of the PnTx3-4 knottin scaffold available at the knottin database (http://knottin.cbs.cnrs.fr). These studies provide the basis for future large scale production and structure-function investigation of PnTx3-4.
    Full-text · Article · May 2012 · Toxicon
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    Dataset: Figure S6
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    ABSTRACT: All-atom RMSD values between the MD and crystal structures. The RMSD values were computed by subtracting the all-atom distance matrix at time t of the MD trajectories from the reference distance matrix determined from the crystal structures of the ProTα and Neh2 peptides bound to Keap1 (PDB ids: 2Z32 and 1X2R respectively) [50], [55]. The distance matrices consisted of residues i through i+3 of the β-turn regions of the ProTα and Neh2 peptides determined from the crystal structures [50], [55]. (TIF)
    Preview · Dataset · Nov 2011
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    ABSTRACT: Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProTα) and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2), with a common binding partner, Kelch-like ECH-associated protein 1(Keap1), are essential for regulating cellular response to oxidative stress. Misregulation of this pathway can lead to neurodegenerative diseases, premature aging and cancer. In order to understand the mechanisms these two disordered proteins employ to bind to Keap1, we performed extensive 0.5-1.0 microsecond atomistic molecular dynamics (MD) simulations and isothermal titration calorimetry experiments to investigate the structure/dynamics of free-state ProTα and Neh2 and their thermodynamics of bindings. The results show that in their free states, both ProTα and Neh2 have propensities to form bound-state-like β-turn structures but to different extents. We also found that, for both proteins, residues outside the Keap1-binding motifs may play important roles in stabilizing the bound-state-like structures. Based on our findings, we propose that the binding of disordered ProTα and Neh2 to Keap1 occurs synergistically via preformed structural elements (PSEs) and coupled folding and binding, with a heavy bias towards PSEs, particularly for Neh2. Our results provide insights into the molecular mechanisms Neh2 and ProTα bind to Keap1, information that is useful for developing therapeutics to enhance the oxidative stress response.
    Full-text · Article · Nov 2011 · PLoS ONE
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    Dataset: Table S1
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    ABSTRACT: Frequencies of intra-turn hydrogen bond formations in full-length human ProTα and human 32-mer Neh2 trajectories. (DOCX)
    Preview · Dataset · Nov 2011
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    Dataset: Figure S3
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    ABSTRACT: χ1 and χ2 angles from the MD and bound-state structures. Plots of the sidechain χ1 and χ2 angles for residues i to i+3 of the β-turns are shown. Red dots indicate the angles from the last 0.1 µs of the full-length ProTα and 32-mer Neh2 trajectories. Black dots indicate the angles from the crystal structures (PDB ids: 2Z32 and 1X2R) for ProTα and Neh2 respectively) [50], [55]. (TIF)
    Preview · Dataset · Nov 2011
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    Dataset: Figure S1
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    ABSTRACT: Overlays of the starting structure (grey) and crystal structure (pink) β-turns. Residues i through i+3 of the β-turns from the starting structures, generated in CNS [54], were superimposed onto the corresponding residues from their bound state crystal structures. The RMSD values were computed by subtracting the all-atom distance matrix of the starting structures from the reference distance matrix determined from the crystal structures of the ProTα and Neh2 peptides bound to Keap1 (PDB ids: 2Z32 and 1X2R respectively) [50], [55]. The distance matrices consisted of residues i through i+3 of the β-turn regions of the ProTα and Neh2 peptides determined from the crystal structures [50], [55]. The starting structures for human ProTα and Neh2 were compared to the mouse structures (PDB ids: 2Z32 and 1X2R) [50], [55] as their bound-state references. Hydrogen atoms were added for clarity. (TIF)
    Preview · Dataset · Nov 2011
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    Dataset: Figure S4
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    ABSTRACT: Isothermal titration calorimetry (ITC) measurements. Panels A and B correspond to titrations of 16-mer ProTα and 9-mer Neh2 peptide to the mouse Kelch domain of Keap1, respectively. (Upper) The raw data of two ITC experiments each performed at 25°C. (Lower) The integrated heat changes, corrected for the heat of dilution, and the fitted curve assuming single-site binding. (TIF)
    Preview · Dataset · Nov 2011
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    Dataset: Video S1
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    ABSTRACT: Transition of the 9-mer mouse Neh2 peptide from an extended to a bound-state-like β-turn conformation. (MPG)
    Preview · Dataset · Nov 2011
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    Dataset: Video S2
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    ABSTRACT: Convergence of the 32-mer mouse Neh2 peptide to a bound-state-like β-turn conformation. (MPG)
    Preview · Dataset · Nov 2011
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    Dataset: Figure S7
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    ABSTRACT: C αi−C αi+3 distances and their deviations from their crystal structure distances. Panels A and B show the C αi−C αi+3 distances and the deviations from the corresponding distances in the crystal structures respectively. Data was collected over the last 0.1 µs of the full-length human ProTα and human 32-mer Neh2 trajectories. Deviations were calculated for C αi−C αi+3 pairs from the β-turns, determined from the mouse crystal structures [50], [55], by subtraction of the i to i+3 distance at time t of the trajectory from the fixed distance of the corresponding atom pair from the crystal structures (PDB ids: 2Z32 and 1X2R) for ProTα and Neh2 respectively) [50], [55]. (TIF)
    Preview · Dataset · Nov 2011
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    Dataset: Figure S2
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    ABSTRACT: Overlays of the β-turn structures from the 16-mer ProTα and 9-mer Neh2 MD simulations (white) with those from the longer sequence simulations (pink). The RMSD values were computed by subtracting the all-atom distance matrices. The distance matrices consisted of residues i through i+3 of the β-turn regions of the ProTα and Neh2 peptides determined from the crystal structures [50], [55]. Centroid structures from the shorter peptide simulations with lowest RMSDs to the bound state (820–830 ns and 630–640 ns from the ProTα and Neh2 simulations, respectively) were superimposed onto the corresponding centroid structures from the last 100 ns of the longer sequence simulations. (TIF)
    Preview · Dataset · Nov 2011