Hamiltonian Replica Exchange Molecular Dynamics Research Articles

Extended Conformational Selection in the Antigen-Antibody Interaction of the PfAMA1 Protein.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a surface protein found in two stages of the malaria life cycle. This is a protein involved in a reorientation movement of the parasite so that cell invasion occurs in the so-called "moving junction", relevant when the membranes of the parasite and the host are in contact. The structure of a conformational epitope of domain III of PfAMA1 in complex with the monoclonal antibody Fab F8.12.19 is experimentally known. Here, we used molecular dynamics with enhanced sampling by Hamiltonian replica exchange molecular dynamics (HREMD) to understand the effect of intermolecular interactions, conformational variability, and intrinsically disordered regions on the mechanism of antigen-antibody interaction. Clustering methods and the analysis of conformational variability were used in order to understand the influence of the presence of the partner protein in the complex. The free-state epitope accesses a broader conformational pool, including disordered conformations not seen in the bound state. The simulations suggest an extended conformational selection mechanism in which the antibody stabilizes a conformational set of the epitope existing in the free state. The stabilization of the active conformation occurs mainly through hydrogen bonds: Tyr(H33)-Asp493, His(L94)-Val510, Ser(L93)-Glu511, Tyr(H56)-Asp485, and Tyr(H35)-Asp493. The antibody has a structure with few flexible regions, and only the complementarity determining region (CDR) H3 shows greater plasticity in the presence of the epitope.

Dissecting the geometric and hydrophobic constraints of stapled peptides.

Stapled peptides are a promising class of molecules with potential as highly specific probes of protein-protein interactions and as therapeutics. Hydrocarbon stapling affects the peptide properties through the interplay of two factors: enhancing the overall hydrophobicity and constraining the conformational flexibility. By constructing a series of virtual peptides, we study the role of each factor in modulating the structural properties of a hydrocarbon-stapled peptide PM2, which has been shown to enter cells, engage its target Mouse Double Minute 2 (MDM2), and activate p53. Hamiltonian replica exchange molecular dynamics (HREMD) simulations suggest that hydrocarbon stapling favors helical populations of PM2 through a combination of the geometric constraints and the enhanced hydrophobicity of the peptide. To further understand the conformational landscape of the stapled peptides along the binding pathway, we performed HREMD simulations by restraining the peptide at different distances from MDM2. When the peptide approaches MDM2, the binding pocket undergoes dehydration which appears to be greater in the presence of the stapled peptide compared with the linear peptide. In the binding pocket, the helicity of the stapled peptide is increased due to the favorable interactions between the peptide residues as well as the staple and the microenvironment of the binding pocket, contributing to enhanced affinity. The dissection of the multifaceted mechanism of hydrocarbon stapling into individual factors not only deepens fundamental understanding of peptide stapling, but also provides guidelines for the design of new stapled peptides.

Efficient and accurate calculation of proline cis/trans isomerization free energies from Hamiltonian replica exchange molecular dynamics simulations

Proline cis/trans isomerization plays an important role in many biological processes but occurs on time scales not accessible to brute-force molecular dynamics (MD) simulations. We have designed a new Hamiltonian replica exchange scheme, ω-bias potential replica exchange molecular dynamics (ωBP-REMD), to efficiently and accurately calculate proline cis/trans isomerization free energies. ωBP-REMD is applied to various proline-containing tripeptides and a biologically important proline residue in the N2-domain of the gene-3-protein of phage fd in the wildtype and mutant variants of the protein. Excellent cis/trans transition rates are obtained. Reweighting of the sampled probability distribution along the peptide bond dihedral angle allows construction of the corresponding free-energy profile and calculation of the cis/trans isomerization free energy with high statistical precision. Very good agreement with experimental data is obtained. ωBP-REMD outperforms standard umbrella sampling in terms of convergence and agreement with experiment and strongly reduces perturbation of the local structure near the proline residue.

De Novo Design of a β-Helix Tau Protein Scaffold: An Oligomer-Selective Vaccine Immunogen Candidate for Alzheimer's Disease.

Tau pathology is associated with many neurodegenerative disorders, including Alzheimer's disease (AD), where the spatio-temporal pattern of tau neurofibrillary tangles strongly correlates with disease progression, which motivates therapeutics selective for misfolded tau. Here, we introduce a new avidity-enhanced, multi-epitope approach for protein-misfolding immunogen design, which is predicted to mimic the conformational state of an exposed epitope in toxic tau oligomers. A predicted oligomer-selective tau epitope 343KLDFK347 was scaffolded by designing a β-helix structure that incorporated multiple instances of the 16-residue tau fragment 339VKSEKLDFKDRVQSKI354. Large-scale conformational ensemble analyses involving Jensen-Shannon Divergence and the embedding depth showed that the multi-epitope scaffolding approach, employed in designing the β-helix scaffold, was predicted to better discriminate toxic tau oligomers than other "monovalent" strategies utilizing a single instance of an epitope for vaccine immunogen design. Using Rosetta, 10,000 sequences were designed and screened for the linker portions of the β-helix scaffold, along with a C-terminal stabilizing α-helix that interacts with the linkers, to optimize the folded structure and stability of the scaffold. Structures were ranked by energy, and the lowest 1% (82 unique sequences) were verified using AlphaFold. Several selection criteria involving AlphaFold are implemented to obtain a lead-designed sequence. The structure was further predicted to have free energetic stability by using Hamiltonian replica exchange molecular dynamics (MD) simulations. The synthesized β-helix scaffold showed direct binding in surface plasmon resonance (SPR) experiments to several antibodies that were raised to the structured epitope using a designed cyclic peptide. Moreover, the strength of binding of these antibodies to in vitro tau oligomers correlated with the strength of binding to the β-helix construct, suggesting that the construct presents an oligomer-like conformation and may thus constitute an effective oligomer-selective immunogen.

Alchemical Free Energy and Hamiltonian Replica Exchange Molecular Dynamics to Compute Hydrofluorocarbon Isotherms in Imidazolium-Based Ionic Liquids.

Ionic liquids (ILs) have shown promise for applications that leverage differential gas solubility in an IL solvent, e.g., gas separations. Although most available literature provides Henry's law constants, the ability to efficiently estimate full isotherms is important for engineering design calculations. Molecular simulation can be used as a tool to predict full isotherms of gas in ILs. However, particle insertions or deletions in a charge-dense IL medium and the sluggish conformational dynamics of ILs present two sampling challenges for these systems. We therefore devised a method that uses Hamiltonian replica exchange (HREX) molecular dynamics (MD) combined with alchemical free energy calculations to compute full solubility isotherms of two different hydrofluorocarbons (HFCs) in imidazolium-based IL binary mixtures. This workflow is significantly faster than the Gibbs ensemble Monte Carlo (GEMC) simulations which fail to deal with the slow conformational relaxation caused by the sluggish dynamics of ILs. Multiple free energy estimators, including thermodynamic integration, free energy perturbation, and multistate Bennett acceptance ratio method, provided consistent results. Overall, the simulated Henry's law constant, isotherm curvature, and solubility trends match experimental results reasonably well. We close by calculating the full solubility isotherms of two HFCs in IL mixtures that have not been reported in the literature, demonstrating the potential of this method to be used for solubility prediction and setting the stage for future computational screening studies that search for the "best" IL to separate azeotropic HFC mixtures.

Disordered Domain Shifts the Conformational Ensemble of the Folded Regulatory Domain of the Multidomain Oncoprotein c-Src.

c-Src kinase is a multidomain non-receptor tyrosine kinase that aberrantly phosphorylates several signaling proteins in cancers. Although the structural properties of the regulatory domains (SH3-SH2) and the catalytic kinase domain have been extensively characterized, there is less knowledge about the N-terminal disordered region (SH4UD) and its interactions with the other c-Src domains. Here, we used domain-selective isotopic labeling combined with the small-angle neutron scattering contrast matching technique to study SH4UD interactions with SH3-SH2. Our results show that in the presence of SH4UD, the radius of gyration (Rg) of SH3-SH2 increases, indicating that it has a more extended conformation. Hamiltonian replica exchange molecular dynamics simulations provide a detailed molecular description of the structural changes in SH4UD-SH3-SH2 and show that the regulatory loops of SH3 undergo significant conformational changes in the presence of SH4UD, while SH2 remains largely unchanged. Overall, this study highlights how a disordered region can drive a folded region of a multidomain protein to become flexible, which may be important for allosteric interactions with binding partners. This may help in the design of therapeutic interventions that target the regulatory domains of this important family of kinases.

Molecular dynamics simulations of the calmodulin-induced α-helix in the SK2 calcium-gated potassium ion channel

The family of small-conductance Ca2+-activated potassium ion channels (SK channels) is composed of four members (SK1, SK2, SK3, and SK4) involved in neuron-firing regulation. The gating of these channels depends on the intracellular Ca2+ concentration, and their sensitivity to this ion is provided by calmodulin (CaM). This protein binds to a specific region in SK channels known as the calmodulin-binding domain (CaMBD), an event which is essential for their gating. While CaMBDs are typically disordered in the absence of CaM, the SK2 channel subtype displays a small prefolded α-helical region in its CaMBD even if CaM is not present. This small helix is known to turn into a full α-helix upon CaM binding, although the molecular-level details for this conversion are not fully understood yet. In this work, we offer new insights on this physiologically relevant process by means of enhanced sampling, atomistic Hamiltonian replica exchange molecular dynamics simulations, providing a more detailed understanding of CaM binding to this target. Our results show that CaM is necessary for inducing a full α-helix along the SK2 CaMBD through hydrophobic interactions with V426 and L427. However, it is also necessary that W431 does not compete for these interactions; the role of the small prefolded α-helix in the SK2 CaMBD would be to stabilize W431 so that this is the case. In conclusion, our findings provide further insight into a key interaction between CaM and SK channels that is important for channel sensitivity to Ca2+.

A coarse-grained approach to NMR-data-assisted modeling of protein structures.

The ESCASA algorithm for analytical estimation of proton positions from coarse-grained geometry developed in our recent work has been implemented in modeling protein structures with the highly coarse-grained UNRES model of polypeptide chains (two sites per residue) and nuclear magnetic resonance (NMR) data. A penalty function with the shape of intersecting gorges was applied to treat ambiguous distance restraints, which automatically selects consistent restraints. Hamiltonian replica exchange molecular dynamics was used to carry out the conformational search. The method was tested with both unambiguous and ambiguous restraints producing good-quality models with GDT_TS from 7.4units higher to 14.4units lower than those obtained with the CYANA or MELD software for protein-structure determination from NMR data at the all-atom resolution. The method can thus be applied in modeling the structures of flexible proteins, for which extensive conformational search enabled by coarse-graining is more important than high modeling accuracy.

Computer-aided comprehensive explorations of RNA structural polymorphism through complementary simulation methods.

While RNA folding was originally seen as a simple problem to solve, it has been shown that the promiscuous interactions of the nucleobases result in structural polymorphism, with several competing structures generally observed for non-coding RNA. This inherent complexity limits our understanding of these molecules from experiments alone, and computational methods are commonly used to study RNA. Here, we discuss three advanced sampling schemes, namely Hamiltonian-replica exchange molecular dynamics (MD), ratchet-and-pawl MD and discrete path sampling, as well as the HiRE-RNA coarse-graining scheme, and highlight how these approaches are complementary with reference to recent case studies. While all computational methods have their shortcomings, the plurality of simulation methods leads to a better understanding of experimental findings and can inform and guide experimental work on RNA polymorphism.

Advanced Molecular Dynamics Approaches to Model a Tertiary Complex APRIL/TACI with Long Glycosaminoglycans.

Glycosaminoglycans (GAGs) are linear anionic periodic polysaccharides participating in a number of biologically relevant processes in the extracellular matrix via interactions with their protein targets. Due to their periodicity, conformational flexibility, pseudo-symmetry of the sulfation pattern, and the key role of electrostatics, these molecules are challenging for both experimental and theoretical approaches. In particular, conventional molecular docking applied for GAGs longer than 10-mer experiences severe difficulties. In this work, for the first time, 24- and 48-meric GAGs were docked using all-atomic repulsive-scaling Hamiltonian replica exchange molecular dynamics (RS-REMD), a novel methodology based on replicas with van der Waals radii of interacting molecules being scaled. This approach performed well for proteins complexed with oligomeric GAGs and is independent of their length, which distinguishes it from other molecular docking approaches. We built a model of long GAGs in complex with a proliferation-inducing ligand (APRIL) prebound to its receptors, the B cell maturation antigen and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). Furthermore, the prediction power of the RS-REMD for this tertiary complex was evaluated. We conclude that the TACI–GAG interaction could be potentially amplified by TACI’s binding to APRIL. RS-REMD outperformed Autodock3, the docking program previously proven the best for short GAGs.

PH-Dependent Conformations of an Antimicrobial Spider Venom Peptide, Cupiennin 1a, from Unbiased HREMD Simulations.

Cupiennin 1a is an antimicrobial peptide found in the venom of the spider Cupiennius salei. A highly cationic peptide, its cell lysis activity has been found to vary between neutral and charged membranes. In this study, Hamiltonian replica-exchange molecular dynamics (HREMD) was used to determine the conformational ensemble of the peptide in both charged (pH 3) and neutral (pH 11) states. The obtained free energy landscapes demonstrated the conformational diversity of the neutral peptide. At high pH, the peptide was found to adopt helix–hinge–helix and disordered structures. At pH 3, the peptide is structured with a high propensity toward α-helices. The presence of these α-helices seems to assist the peptide in recognizing membrane surfaces. These results highlight the importance of the charged residues in the stabilization of the peptide structure and the subsequent effects of pH on the peptide’s conformational diversity and membrane activity. These findings may provide insights into the antimicrobial activity of Cupiennin 1a and other amphipathic linear peptides toward different cell membranes.

Full structural ensembles of intrinsically disordered proteins from unbiased molecular dynamics simulations

Molecular dynamics (MD) simulation is widely used to complement ensemble-averaged experiments of intrinsically disordered proteins (IDPs). However, MD often suffers from limitations of inaccuracy. Here, we show that enhancing the sampling using Hamiltonian replica-exchange MD (HREMD) led to unbiased and accurate ensembles, reproducing small-angle scattering and NMR chemical shift experiments, for three IDPs of varying sequence properties using two recently optimized force fields, indicating the general applicability of HREMD for IDPs. We further demonstrate that, unlike HREMD, standard MD can reproduce experimental NMR chemical shifts, but not small-angle scattering data, suggesting chemical shifts are insufficient for testing the validity of IDP ensembles. Surprisingly, we reveal that despite differences in their sequence, the inter-chain statistics of all three IDPs are similar for short contour lengths (< 10 residues). The results suggest that the major hurdle of generating an accurate unbiased ensemble for IDPs has now been largely overcome.

Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin B.

Ligand‐based NMR techniques to study protein–ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi‐frequency STD NMR approach, differential epitope mapping (DEEP)‐STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so‐called DEEP‐STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP‐STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi‐subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.

Further Optimization and Validation of the Classical Drude Polarizable Protein Force Field.

The CHARMM Drude-2013 polarizable force field (FF) was developed to include the explicit treatment of induced electronic polarizability, resulting in a more accurate description of the electrostatic interactions in molecular dynamics (MD) simulations. While the Drude-2013 protein FF has shown success in improving the folding properties of α-helical peptides and to reproduce experimental observables in simulations up to 1 μs, some limitations were noted regarding the stability of β-sheet structures in simulations longer than 100 ns as well as larger deviations from crystal structures in simulations of a number of proteins compared to the additive CHARMM36 protein FF. The origin of the instability has been identified and appears to be primarily due to overestimated atomic polarizabilities and induced dipole-dipole interactions on the Cβ, Cγ, and Cδ side chain atoms. To resolve this and other issues, a number of aspects of the model were revisited, resulting in Drude-2019 protein FF. Backbone parameters were optimized targeting the conformational properties of the (Ala)5 peptide in solution along with gas phase properties of the alanine dipeptide. Dipeptides that contain N-acetylated and N'-methylamidated termini, excluding Gly, Pro, and Ala, were used as models to optimize the atomic polarizabilities and Thole screening factors on selected Cβ, Cγ, and Cδ carbons by targeting quantum mechanical (QM) dipole moments and molecular polarizabilities. In addition, to obtain better conformational properties, side chain χ1 and χ2 dihedral parameters were optimized targeting QM data for the respective side chain dipeptide conformations as well as Protein Data Bank survey data based on the χ1, χ2 sampling from Hamiltonian replica-exchange MD simulations of (Ala)4-X-(Ala)4 in solution, where X is the amino acid of interest. Further improvements include optimizing nonbonded interactions between charged residues to reproduce QM interaction energies of the charged-protein model compounds and experimental osmotic pressures. Validation of the optimized Drude protein FF includes MD simulations of a collection of peptides and proteins including β-sheet structures, as well as transmembrane ion channels. Results showed that the updated Drude-2019 protein FF yields smaller overall root-mean-square differences of proteins as compared to the additive CHARMM36m and Drude-2013 FFs as well as similar or improved agreement with experimental NMR properties, allowing for long time scale simulation studies of proteins and more complex biomolecular systems in conjunction with the remainder of the Drude polarizable FF.

Role of Oxidized Gly25, Gly29, and Gly33 Residues on the Interactions of Aβ1-42 with Lipid Membranes.

Oxidative stress is known to play an important role in the pathogenesis of Alzheimer's disease. Moreover, it is becoming increasingly evident that the plasma membrane of neurons plays a role in modulating the aggregation and toxicity of Alzheimer's amyloid-β peptide (Aβ). In this study, the combined and interdependent effects of oxidation and membrane interactions on the 42 residues long Aβ isoform are investigated using molecular simulations. Hamiltonian replica exchange molecular dynamics simulations are utilized to elucidate the impact of selected oxidized glycine residues of Aβ42 on the interactions of the peptide with a model membrane comprised of 70% POPC, 25% cholesterol, and 5% of the ganglioside GM1. The main findings are that, independent of the oxidation state, Aβ prefers binding to GM1 over POPC, which is further enhanced by the oxidation of Gly29 and Gly33 and reduced the formation of β-sheet. Our results suggest that the differences observed in Aβ42 conformations and its interaction with a lipid bilayer upon oxidation originate from the position of the oxidized Gly residue with respect to the hydrophobic sequence of Aβ42 involving the Gly29-XXX-Gly33-XXX-Gly37 motif and from specific interactions between the peptide and the terminal sugar groups of GM1.

The dissociation mechanism of processive cellulases.

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

Inhibition Mechanisms of Rhodococcus Erythropolis 2'-Hydroxybiphenyl-2-sulfinate Desulfinase (DszB).

Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2'-Hydroxybiphenyl-2-sulfinate desulfinase (DszB), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, understanding the molecular mechanisms of DszB activity may enable development of the cascade as industrial biotechnology. Based on crystallographic evidence, we hypothesized that DszB undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipated this conformational change is responsible, in part, for enhancing product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced and purified via Escherichia coli BL21 to test these hypotheses. Activity and the resulting conformational change of DszB in the presence of HBP were evaluated. The activity of recombinant DszB was comparable to the natively expressed enzyme and was inhibited via competitive binding of the product, HBP. Using circular dichroism, global changes in DszB conformation were monitored in response to HBP concentration, which indicated that both product and substrate produced similar structural changes. Molecular dynamics (MD) simulations and free energy perturbation with Hamiltonian replica exchange molecular dynamics (FEP/λ-REMD) calculations were used to investigate the molecular-level phenomena underlying the connection between conformation change and kinetic inhibition. In addition to the HBP, MD simulations of DszB bound to common, yet structurally diverse, crude oil contaminants 2',2-biphenol (BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA), and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation trajectories, including root-mean-square fluctuation (RMSF), center of mass (COM) distances, and strength of nonbonded interactions, when compared with FEP/λ-REMD calculations of ligand binding free energy, showed excellent agreement with experimentally determined inhibition constants. Together, the results show that the combination of a molecule's hydrophobicity and nonspecific interactions with nearby functional groups contributes to a competitive inhibition mechanism that locks DszB in a closed conformation and precludes substrate access to the active site.

The conformational ensemble of an intrinsically disordered protein

It is becoming increasingly realized that conformational flexibility, manifested as transitions among multiple states accessible to a macromolecular system, is also of critical importance to function. Many examples now exist of functional assemblies where flexibility is of paramount biological importance including intrinsically disordered proteins and multidomain kinases. Understanding this solution conformational flexibility remains a critical challenge in structural biology. We determined the conformational ensemble of the disordered N terminal of the c-Src kinase by combining small-angle X-ray (SAXS) scattering and molecular dynamics (MD) simulation. To interpret the ensemble averaged structural information obtained by SAXS, we conducted Hamiltonian replica exchange MD (HREXM), which enhance considerably the sampling of configurational states. We calculated theoretical SAXS intensities from the atomic coordinates of the simulations and compared to the experiments. We found that HERMD reproduced, without any fitting, the experimental SAXS intensities. The combined experimental and computational approach provides a detailed physical model of the conformational ensemble of c-Src. Simulation structure of the intrinsically disordered c-Src This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellulose-specific Type B carbohydrate binding modules: understanding oligomeric and non-crystalline substrate recognition mechanisms

BackgroundEffective enzymatic degradation of crystalline polysaccharides requires a synergistic cocktail of hydrolytic enzymes tailored to the wide-ranging degree of substrate crystallinity. To accomplish this type of targeted carbohydrate recognition, nature produces multi-modular enzymes, having at least one catalytic domain appended to one or more carbohydrate binding modules (CBMs). The Type B CBM categorization encompasses several families (i.e., protein folds) of CBMs that are generally thought to selectively bind oligomeric polysaccharides; however, a subset of cellulose-specific CBM families (17 and 28) appear to bind non-crystalline cellulose more tightly than oligomers and in a manner that discriminates between surface topology.ResultsTo provide insight into this unexplained phenomenon, we investigated the molecular-level origins of oligomeric and non-crystalline carbohydrate recognition in cellulose-specific Type B CBMs using molecular dynamics (MD) simulation and free energy calculations. Examining two CBMs from three different families (4, 17, and 28), we describe how protein–ligand dynamics contribute to observed variations in binding affinity of oligomers within the same CBM family. Comparisons across the three CBM families identified factors leading to modified functionality prohibiting competitive binding, despite similarity in sequence and specificity. Using free energy perturbation with Hamiltonian replica exchange MD, we also examined the hypothesis that the open topology of the binding grooves in families 17 and 28 necessitates tight binding of an oligomer, while the more confined family 4 binding groove does not require the same degree of tight binding. Finally, we elucidated the mechanisms of non-crystalline carbohydrate recognition by modeling CBMs complexed with a partially decrystallized cellulose substrate. Molecular simulation provided structural and dynamic data for direct comparison to oligomeric modes of carbohydrate recognition, and umbrella sampling MD was used to determine ligand binding free energy. Comparing both protein–carbohydrate interactions and ligand binding free energies, which were in good agreement with experimental values, we confirmed the hypothesis that family 17 and 28 CBMs bind non-crystalline cellulose and oligomers with different affinities (i.e., high and low).ConclusionsOur study provides an unprecedented level of insight into the complex solid and soluble carbohydrate substrate recognition mechanisms of Type B CBMs, the findings of which hold considerable promise for enhancing lignocellulosic biomass conversion technology and development of plant cell wall probes.

Characterizing the conformational landscape of MDM2-binding p53 peptides using Molecular Dynamics simulations

The conformational landscapes of p53 peptide variants and phage derived peptide (12/1) variants, all known to bind to MDM2, are studied using hamiltonian replica exchange molecular dynamics simulations. Complementing earlier observations, the current study suggests that the p53 peptides largely follow the ‘conformational selection’ paradigm in their recognition of and complexation by MDM2 while the 12/1 peptides likely undergo some element of conformational selection but are mostly driven by ‘binding induced folding’. This hypothesis is further supported by pulling simulations that pull the peptides away from their bound states with MDM2. This data extends the earlier mechanisms proposed to rationalize the entropically driven binding of the p53 set and the enthalpically driven binding of the 12/1 set. Using our hypothesis, we suggest mutations to the 12/1 peptide that increase its helicity in simulations and may, in turn, shift the binding towards conformational selection. In summary, understanding the conformational landscapes of the MDM2-binding peptides may suggest new peptide designs with bespoke binding mechanisms.