Fast in silico protein folding by introduction of alternating hydrogen bond potentials

We accelerate protein folding in all-atom molecular dynamics simulations by introducing alternating hydrogen bond potentials as a supplement to the force field. The alternating hydrogen bond potentials result in accelerated hydrogen bond reordering, which leads to rapid formation of secondary structure elements. The method does not require knowledge of the native state but generates the potentials based on the development of the tertiary structure in the simulation. In protein folding, the formation of secondary structure elements, especially α-helix and β-sheet, is very important, and we show that our method can fold both efficiently and with great speed.     

K+/Na+ selectivity in K channels and valinomycin: over-coordination versus cavity-size constraints

Potassium channels and valinomycin molecules share the exquisite ability to select K(+) over Na(+). Highly selective K channels maintain a special local environment around their binding sites devoid of competing hydrogen bond donor groups, which enables spontaneous transfer of K(+) from states of low coordinations in water into states of over-coordination by eight carbonyl ligands. In such a phase-activated state, electrostatic interactions from these 8-fold binding sites, constrained to maintain high coordinations, result in K(+)/Na(+) selectivity with no need for a specific cavity size. Under such conditions, however, direct coordination from five or six carbonyl ligands does not result in selectivity. Yet, valinomycin molecules achieve selectivity by providing only six carbonyl ligands. Does valinomycin use additional coordinating ligands from the solvent or does it have special structural features not present in K channels? Quantum chemical investigations undertaken here demonstrate that valinomycin selectivity is due to cavity size constraints that physically prevent it from collapsing onto the smaller sodium ion. Valinomycin enforces these constraints by using a combination of intramolecular hydrogen bonds and other structural features, including its specific ring size and the spacing between its connected ligands. Results of these investigations provide a consistent explanation for the experimental data available for the ion-complexation properties of valinomycin in solvents of varying polarity. Together, investigations of these two systems reveal how nature, despite being popular for its parsimony in recycling functional motifs, can use different combinations of phase, coordination number, cavity size, and rigidity (constraints) to achieve K(+)/Na(+) selectivity.     

Low-mass molecular dynamics simulation: A simple and generic technique to enhance configurational sampling

CLN025 is one of the smallest fast-folding proteins. Until now it has not been reported that CLN025 can autonomously fold to its native conformation in a classical, all-atom, and isothermal–isobaric molecular dynamics (MD) simulation. This article reports the autonomous and repeated folding of CLN025 from a fully extended backbone conformation to its native conformation in explicit solvent in multiple 500-ns MD simulations at 277 K and 1 atm with the first folding event occurring as early as 66.1 ns. These simulations were accomplished by using AMBER forcefield derivatives with atomic masses reduced by 10-fold on Apple Mac Pros. By contrast, no folding event was observed when the simulations were repeated using the original AMBER forcefields of FF12SB and FF14SB. The results demonstrate that low-mass MD simulation is a simple and generic technique to enhance configurational sampling. This technique may propel autonomous folding of a wide range of miniature proteins in classical, all-atom, and isothermal–isobaric MD simulations performed on commodity computers—an important step forward in quantitative biology.     

Modelling the pH-dependent properties of Kv1 potassium channels

It is known that the pH dependence of conductance for the rat potassium channel Kv1.4 is susbstantially reduced upon mutation of either H508 or K532. These residues lie in the extracellular mouth of the channel pore. We have used continuum electrostatics to investigate their interactions with K(+) sites in the pore. The predicted scale of interactions between H508/K532 and potassium sites is sufficient to significantly alter potassium occupancy and thus channel function. We interpret the effect of K532 mutation as indicating that the pH-dependent effect requires not only an ionisable group with a suitable pK(a) value (i.e. histidine), but also that other charged groups set the potential profile at a threshold level. This hypothesis is examined in the context of pH dependence for other members of the Kv1 family, and may represent a general tool with which to study potassium channels.     

Kinetics and computational studies of ligand migration in nitrophorin 7 and its Δ1–3 mutant

Nitrophorins (NPs) are nitric oxide (NO)-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. Though NP7 exhibits a large sequence resemblance with other NPs, two major differential features are the ability to interact with negatively charged cell surfaces and the presence of a specific N-terminus composed of three extra residues (Leu1-Pro2-Gly3). The aim of this study is to examine the influence of the N-terminus on the ligand binding, and the topological features of inner cavities in closed and open states of NP7, which can be associated to the protein structure at low and high pH, respectively. Laser flash photolysis measurements of the CO rebinding kinetics to NP7 and its variant NP7(Δ1–3), which lacks the three extra residues at the N-terminus, exhibit a similar pattern and support the existence of a common kinetic mechanism for ligand migration and binding. This is supported by the existence of a common topology of inner cavities, which consists of two docking sites in the heme pocket and a secondary site at the back of the protein. The ligand exchange between these cavities is facilitated by an additional site, which can be transiently occupied by the ligand in NP7, although it is absent in NP4. These features provide a basis to explain the enhanced internal gas hosting capacity found experimentally in NP7 and the absence of ligand rebinding from secondary sites in NP4. The current data allow us to speculate that the processes of docking to cell surfaces and NO release may be interconnected in NP7, thereby efficiently releasing NO into a target cell. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Propagation of dynamic changes in barnase upon binding of barstar: an NMR and computational study

NMR spectroscopy and computer simulations were used to examine changes in chemical shifts and in dynamics of the ribonuclease barnase that result upon binding to its natural inhibitor barstar. Although the spatial structures of free and bound barnase are very similar, binding results in changes of the dynamics of both fast side-chains, as revealed by (2)H relaxation measurements, and NMR chemical shifts in an extended beta-sheet that is located far from the binding interface. Both side-chain dynamics and chemical shifts are sensitive to variations in the ensemble populations of the inter-converting molecular states, which can escape direct structural observation. Molecular dynamics simulations of free barnase and barnase in complex with barstar, as well as a normal mode analysis of barnase using a Gaussian network model, reveal relatively rigid domains that are separated by the extended beta-sheet mentioned above. The observed changes in NMR parameters upon ligation can thus be rationalized in terms of changes in inter-domain dynamics and in populations of exchanging states, without measurable structural changes. This provides an alternative model for the propagation of a molecular response to ligand binding across a protein that is based exclusively on changes in dynamics.     

The protonation state of the catalytic aspartates in plasmepsin II

Assigning the correct protonation state to the catalytic residues is essential for a realistic modelling of an enzyme's active site. Plasmepsins are pharmaceutically relevant aspartic proteases involved in haemoglobin degradation by Plasmodium spp. In aspartic proteases, one of the two catalytic aspartates is protonated, while the other is negatively charged. Here, multiple explicit-water molecular dynamics simulations of plasmepsin II, uncomplexed and with a hydroxypropylamine peptidomimetic inhibitor, indicate that protonation of Asp214 favours a stable active site structure. Moreover, the protonation state of the catalytic aspartate has a strong influence on a linear chain of hydrogen bonds with the adjacent side chains.     

Molecular dynamics simulations on SDF-1alpha: binding with CXCR4 receptor

Insights into the interacting mode of CXCR4 with SDF-1alpha are crucial in understanding the structural and functional characteristics of CXCR4 receptor. In this paper a computational pipeline, integrating protein structure prediction, molecular dynamics simulations, automated molecular docking, and Brownian dynamics simulations were employed to investigate the dynamic and energetic aspects of CXCR4 associating with SDF-1alpha. The entire simulation revealed the surface distribution feature of electrostatic potentials and conformational "open-close" process of the receptor. The possible binding conformation of CXCR4 was identified, and the CXCR4-SDF-1alpha binding complex was generated. Arg188-Glu277 salt bridge plays an important role for both the extracellular domain conformational change and SDF-1alpha binding. Two binding sites were mapped at the extracellular domain (Site 1) and inside the transmembrane domain (Site 2), which are composed of conserved residues. Sites 1 and 2 contribute approximately 60% and 40% to the binding affinity with SDF-1alpha, respectively. The binding model is in agreement with most of the experimental data. Transmembrane VI has more significant motion in the harmonious conformational transition of CXCR4 during SDF-1alpha binding, which may be possibly associated with signal transduction. Based on the modeling and simulation, a binding mechanism hypothesis between CXCR4 and SDF-1alpha and its relationship to the signal transduction has been proposed.     

Folding free-energy landscape of a 10-residue mini-protein, chignolin

Chignolin is an artificial mini-protein composed of 10 residues (GYDPETGTWG) that has been shown to cooperatively fold into a beta-hairpin structure in water. We extensively explored the conformational space of chignolin using a 180-ns multicanonical molecular dynamics (MD) simulation and analyzed its folding free-energy landscape. In the MD trajectory, we found structures that satisfy 99% of the experimental restraints and are quite close to the experimentally determined structures with C(alpha) root-mean-square-deviations of less than 0.5 Angstroms. These structures formed a large cluster in the conformational space with the largest probability of existence, agreeing well with the experiment.     

Union of Geometric Constraint-Based Simulations with Molecular Dynamics for Protein Structure Prediction

Although proteins are a fundamental unit in biology, the mechanism by which proteins fold into their native state is not well understood. In this work, we explore the assembly of secondary structure units via geometric constraint-based simulations and the effect of refinement of assembled structures using reservoir replica exchange molecular dynamics. Our approach uses two crucial features of these methods: i), geometric simulations speed up the search for nativelike topologies as there are no energy barriers to overcome; and ii), molecular dynamics identifies the low free energy structures and further refines these structures toward the actual native conformation. We use eight α-, β-, and α/β-proteins to test our method. The geometric simulations of our test set result in an average RMSD from native of 3.7 Å and this further reduces to 2.7 Å after refinement. We also explore the question of robustness of assembly for inaccurate (shifted and shortened) secondary structure. We find that the RMSD from native is highly dependent on the accuracy of secondary structure input, and even slightly shifting the location of secondary structure along the amino acid sequence can lead to a rapid decrease in RMSD to native due to incorrect packing.     

Molecular dynamics simulation of highly charged proteins: comparison of the particle-particle particle-mesh and reaction field methods for the calculation of electrostatic interactions

Molecular dynamics (MD) simulations of the activation domain of porcine procarboxypeptidase B (ADBp) were performed to examine the effect of using the particle-particle particle-mesh (P3M) or the reaction field (RF) method for calculating electrostatic interactions in simulations of highly charged proteins. Several structural, thermodynamic, and dynamic observables were derived from the MD trajectories, including estimated entropies and solvation free energies and essential dynamics (ED). The P3M method leads to slightly higher atomic positional fluctuations and deviations from the crystallographic structure, along with somewhat lower values of the total energy and solvation free energy. However, the ED analysis of the system leads to nearly identical results for both simulations. Because of the strong similarity between the results, both methods appear well suited for the simulation of highly charged globular proteins in explicit solvent. However, the lower computational demand of the RF method in the present implementation represents a clear advantage over the P3M method.     

Ensemble docking and molecular dynamics identify knoevenagel curcumin derivatives with potent anti-EGFR activity

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) is an attractive target for cancer therapy. Despite a number of effective EGFR inhibitors that are constantly expanding and different methods being employed to obtain novel compounds, the search for newer EGFR inhibitors is still a major scientific challenge. In the present study, a molecular docking and molecular dynamics investigation has been carried out with an ensemble of EGFR-TK structures against a synthetically feasible library of curcumin analogs to discover potent EGFR inhibitors. To resolve protein flexibility issue we have utilized 5 EGFR wild type crystal structures during docking as this gives improved possibility of identifying an active compound as compared to using a single crystal structure. We then identified five curcumin analogs representing different scaffolds that can serve as lead molecules. Finally, the 5 ns molecular dynamics simulation shows that knoevenagel condensate of curcumin specifically C29 and C30 can be used as starting blocks for developing effective leads capable of inhibiting EGFR.     

Molecular recognition of Cullin3 by KCTDs: Insights from experimental and computational investigations

Recent investigations have highlighted a key role of the proteins of the KCTD (K-potassium channel tetramerization domain containing proteins) family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptor in E3 ligases during protein ubiquitination. Here we present a characterization of the KCTD5-Cullin3 interactions which are mediated by the KCTD5 BTB domain. Isothermal titration calorimetry experiments reveal that KCTD5 avidly binds the Cullin3 (Cul3). The complex presents a 5:5 stoichiometry and a dissociation constant of 59 nM. Molecular modeling and molecular dynamics simulations clearly indicate that the two proteins form a stable (KCTD5–Cul3)₅ pinwheel-shaped heterodecamer in which two distinct KCTD5 subunits cooperate in the binding of each cullin chain. Molecular dynamics simulations indicate that different types of interactions contribute to the stability of the assembly. Interestingly, residues involved in Cul3 recognitions are conserved in the KCTD5 orthologs and paralogs implicated in important biological processes. These residues are also rather well preserved in most of the other KCTD proteins. By using molecular modeling techniques, the entire ubiquitination system including the E3 ligase, the E2 conjugating enzyme and ubiquitin was generated. The analysis of the molecular architecture of this complex machinery provides insights into the ubiquitination processes which involve E3 ligases with a high structural complexity.     

The DNA structure responds differently to physiological concentrations of K(+) or Na(+)

The influence of monovalent cations on DNA conformation and readout is an open question. This NMR study of DNA with either Na(+) or K(+) at physiological concentrations shows that the nature of the cation affects the (31)P chemical shifts (deltaP) and the sequential distances H2'(i)-H6/8(i+1), H2"(i)-H6/8(i+1), and H6/8(i)-H6/8(i+1). The deltaP and distance variations ascertain that the nature of the cation affects the DNA overall structure, i.e. both the conformational equilibria between the backbone BI (epsilon-zeta <0 degrees ) and BII (epsilon-zeta >0 degrees ) states and the helical parameters, via their strong mechanical coupling. These results reveal that Na(+) and K(+) interactions with DNA are different and sequence-dependent. These ions modulate the overall intrinsic properties of DNA, and possibly its packaging and readout.     

Analyzing the flexibility of RNA structures by constraint counting

RNA requires conformational dynamics to undergo its diverse functional roles. Here, a new topological network representation of RNA structures is presented that allows analyzing RNA flexibility/rigidity based on constraint counting. The method extends the FIRST approach, which identifies flexible and rigid regions in atomic detail in a single, static, three-dimensional molecular framework. Initially, the network rigidity of a canonical A-form RNA is analyzed by counting on constraints of network elements of increasing size. These considerations demonstrate that it is the inclusion of hydrophobic contacts into the RNA topological network that is crucial for an accurate flexibility prediction. The counting also explains why a protein-based parameterization results in overly rigid RNA structures. The new network representation is then validated on a tRNA^ASP structure and all NMR-derived ensembles of RNA structures currently available in the Protein Data Bank (with chain length ≥40). The flexibility predictions demonstrate good agreement with experimental mobility data, and the results are superior compared to predictions based on two previously used network representations. Encouragingly, this holds for flexibility predictions as well as mobility predictions obtained by constrained geometric simulations on these networks. Potential applications of the approach to analyzing the flexibility of DNA and RNA/protein complexes are discussed.     

The structure of the CD3 ζζ transmembrane dimer in POPC and raft-like lipid bilayer: A molecular dynamics study

Plasma membrane lipids significantly affect assembly and activity of many signaling networks. The present work is aimed at analyzing, by molecular dynamics simulations, the structure and dynamics of the CD3 ζζ dimer in palmitoyl-oleoyl-phosphatidylcholine bilayer (POPC) and in POPC/cholesterol/sphingomyelin bilayer, which resembles the raft membrane microdomain supposed to be the site of the signal transducing machinery. Both POPC and raft-like environment produce significant alterations in structure and flexibility of the CD3 ζζ with respect to nuclear magnetic resonance (NMR) model: the dimer is more compact, its secondary structure is slightly less ordered, the arrangement of the Asp6 pair, which is important for binding to the Arg residue in the alpha chain of the T cell receptor (TCR), is stabilized by water molecules. Different interactions of charged residues with lipids at the lipid–cytoplasm boundary occur when the two environments are compared. Furthermore, in contrast to what is observed in POPC, in the raft-like environment correlated motions between transmembrane and cytoplasmic regions are observed. Altogether the data suggest that when the TCR complex resides in the raft domains, the CD3 ζζ dimer assumes a specific conformation probably necessary to the correct signal transduction.     

Mechanism of DNA Recognition by the Restriction Enzyme EcoRV

EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV-cognate DNA crystallographic complex, a sharp kink of 50° has been found at the center base-pair step (TA). Here, we examine the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme by performing all-atom molecular dynamics simulations of EcoRV unbound and interacting with three DNA sequences: the cognate sequence, GATATC (TA); the non-cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GA_CH3TATC (TA-CH₃). In the unbound EcoRV, the cleft between the two C-terminal subdomains is found to be open. Binding to AT narrows the cleft and forms a partially bound state. However, the intrinsic bending propensity of AT is insufficient to allow tight binding. In contrast, the cognate TA sequence is easier to bend, allowing specific, high-occupancy hydrogen bonds to form in the complex. The absence of cleavage for this methylated sequence is found to arise from the loss of specific hydrogen bonds between the first adenine of the recognition sequence and Asn185. On the basis of the results, we suggest a three-step recognition mechanism. In the first step, EcoRV, in an open conformation, binds to the DNA at a random sequence and slides along it. In the second step, when the two outer base pairs, GAxxTC, are recognized, the R loops of the protein become more ordered, forming strong hydrogen-bonding interactions, resulting in a partially bound EcoRV-DNA complex. In the third step, the flexibility of the center base pair is probed, and in the case of the full cognate sequence the DNA bends, the complex strengthens and the protein and DNA interact more closely, allowing cleavage.     

Structural insights into the regulation and the recognition of histone marks by the SET domain of NSD1

The development of epigenetic therapies fuels cancer hope. DNA-methylation inhibitors, histone-deacetylase and histone-methyltransferase (HMTase) inhibitors are being developed as the utilization of epigenetic targets is emerging as an effective and valuable approach to chemotherapy as well as chemoprevention of cancer. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. A growing number of studies have reported alterations or amplifications of NSD1, NSD2, or NSD3 in numerous carcinogenic events. Reducing NSDs activity through specific lysine-HMTase inhibitors appears promising to help suppressing cancer growth. However, little is known about the NSD pathways and our understanding of the histone lysine-HMTase mechanism is partial. To shed some light on both the recognition and the regulation of epigenetic marks by the SET domain of the NSD family, we investigate the structural mechanisms of the docking of the histone-H4 tail on the SET domain of NSD1. Our finding exposes a key regulatory and recognition mechanism driven by the flexibility of a loop at the interface of the SET and postSET region. Finally, we prospect the special value of this regulatory region for developing specific and selective NSD inhibitors for the epigenetic therapy of cancers.     

Selectivity and permeation of alkali metal ions in K+-channels

Ion conduction in K⁺-channels is usually described in terms of concerted movements of K⁺ progressing in a single file through a narrow pore. Permeation is driven by an incoming ion knocking on those ions already inside the protein. A fine-tuned balance between high-affinity binding and electrostatic repulsive forces between permeant ions is needed to achieve efficient conduction. While K⁺-channels are known to be highly selective for K⁺ over Na⁺, some K⁺ channels conduct Na⁺ in the absence of K⁺. Other ions are known to permeate K⁺-channels with a more moderate preference and unusual conduction features. We describe an extensive computational study on ion conduction in K⁺-channels rendering free energy profiles for the translocation of three different alkali ions and some of their mixtures. The free energy maps for Rb⁺ translocation show at atomic level why experimental Rb⁺ conductance is slightly lower than that of K⁺. In contrast to K⁺ or Rb⁺, external Na⁺ block K⁺ currents, and the sites where Na⁺ transport is hindered are characterized. Translocation of K⁺/Na⁺ mixtures is energetically unfavorable owing to the absence of equally spaced ion-binding sites for Na⁺, excluding Na⁺ from a channel already loaded with K⁺.