Elio A. Cino

Comparison of Secondary Structure Formation Using 10 Different Force Fields in Microsecond Molecular Dynamics Simulations

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.

Effects of Molecular Crowding on the Dynamics of Intrinsically Disordered Proteins

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.

Microsecond molecular dynamics simulations of intrinsically disordered proteins involved in the oxidative stress response

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.

Fuzzy complex formation between the intrinsically disordered prothymosin α and the Kelch domain of Keap1 involved in the oxidative stress response.

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 α (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, isothermal titration calorimetry, peptide array analysis, site-directed mutagenesis, and molecular dynamic simulations. The results of nuclear magnetic resonance 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 molecular dynamic simulations of the ProTα-Kelch complex. Mutational analysis of ProTα, guided by peptide array data and isothermal titration calorimetry, 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.

Binding of disordered proteins to a protein hub

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.

Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds.

The p53 family of proteins is comprised of p53, p63 and p73. Because the p53 DNA binding domain (DBD) is naturally unstable and possesses an amyloidogenic sequence, it is prone to form amyloid fibrils, causing loss of functions. To develop p53 therapies, it is necessary to understand the molecular basis of p53 instability and aggregation. Light scattering, thioflavin T (ThT) and high hydrostatic pressure (HHP) assays showed that p53 DBD aggregates faster and to a greater extent than p63 and p73 DBDs, and was more susceptible to denaturation. The aggregation tendencies of p53, p63, and p73 DBDs were strongly correlated with their thermal stabilities. Molecular Dynamics (MD) simulations indicated specific regions of structural heterogeneity unique to p53, which may be promoted by elevated incidence of exposed backbone hydrogen bonds (BHBs). The results indicate regions of structural vulnerability in the p53 DBD, suggesting new targetable sites for modulating p53 stability and aggregation, a potential approach to cancer therapy.

Conformational Biases of Linear Motifs

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.

Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer

Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimers, Parkinsons, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53-MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73. Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor-acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and combined efforts, the outlook is positive for the development of strategies for regulating p53 amyloid transformation.

Characterization of the Free State Ensemble of the CoRNR Box Motif by Molecular Dynamics Simulations

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 toward 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 C-terminally 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.

(1)H, (15)N and (13)C backbone resonance assignments of the Kelch domain of mouse Keap1.

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.