On the roles of substrate binding and hinge unfolding in conformational changes of adenylate kinase

We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier works of coarse-grained modeling. The trajectories of the all-atom molecular-dynamics simulations revealed the complexity of protein dynamics and the coupling between different domains during conformational change. Calculations of solvent density and density fluctuations surrounding AK did not show prominent variation during the transition between closed and open forms. Finally, we characterized the effects of local unfolding of an important hinge near Pro¹⁷⁷ on the closed-to-open transition of AK and identified a novel mechanism by which hinge unfolding modulates protein conformational change. The local unfolding of Pro¹⁷⁷ hinge induces alternative tertiary contacts that stabilize the closed structure and prevent the opening transition.     

A consistent empirical potential for water–protein interactions

A simple point-charge potential, developed earlier for the calculation of intermolecular forces in molecular-dynamics simulations of liquid water, has been extended to include interactions between water molecules and polar groups of proteins. A complete potential for use in the simulation of protein dynamics in water is reported.     

Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar Interactions in Configurational Transitions

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50–100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a “monkey-bar”. We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.     

Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar Interactions in Configurational Transitions

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50–100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a “monkey-bar”. We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.     

Defining the minimum size of a hydrophobic cluster in two-stranded alpha-helical coiled-coils: effects on protein stability

The alpha-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)(n) in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel alpha-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as [see text]) was investigated. Alanine residues (denoted as [see text]) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions ([see text]) was among the least stable coiled-coil (T(m) = 39.3 degrees C and Urea(1/2) = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette ([see text]), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette ([see text])([see text]) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes ([see text]). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette ([see text]) resulted in a further synergistic gain in stability of 1.7 kcal/mole (T(m) = 54.1 degrees C and Urea(1/2) = 3.3M). Therefore, the role of hydrophobicity in the hydrophobic core of coiled-coils is extremely context dependent and clustering is an important aspect of protein folding and stability.     

New approaches for predicting protein retention time in hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is an important technique for the purification of proteins. In this paper, we review three different approaches for predicting protein retention time in HIC, based either on a protein's structure or on its amino-acidic composition, and we have extended one of these approaches. The first approach correlates the protein retention time in HIC with the protein average surface hydrophobicity. This methodology is based on the protein three-dimensional structure data and considers the hydrophobic contribution of the exposed amino acid residues as a weighted average. The second approach, which we have extended, is based on the high correlation level between the average surface hydrophobicity of a protein's hydrophobic interacting zone and its retention time in HIC. Finally, a third approach carries out a prediction of the average surface hydrophobicity of a protein, using only its amino-acidic composition, without knowing its three-dimensional structure. These models would make it possible to test different operating conditions for the purification of a target protein by computer simulations, and thus make it easier to select the optimal conditions, contributing to the rational design and optimization of the process.     

Study of Thermomyces lanuginosa Lipase in the Presence of Tributyrylglycerol and Water

The Thermomyces lanuginosa lipase has been extensively studied in industrial and biotechnological research because of its potential for triacylglycerol transformation. This protein is known to catalyze both hydrolysis at high water contents and transesterification in quasi-anhydrous conditions. Here, we investigated the Thermomyces lanuginosa lipase structure in solution in the presence of a tributyrin aggregate using 30 ns molecular-dynamics simulations. The water content of the active-site groove was modified between the runs to focus on the protein-water molecule interactions and their implications for protein structure and protein-lipid interactions. The simulations confirmed the high plasticity of the lid fragment and showed that lipid molecules also bind to a secondary pocket beside the lid. Together, these results strongly suggest that the lid plays a role in the anchoring of the protein to the aggregate. The simulations also revealed the existence of a polar channel that connects the active-site groove to the outside solvent. At the inner extremity of this channel, a tyrosine makes hydrogen bonds with residues interacting with the catalytic triad. This system could function as a pipe (polar channel) controlled by a valve (the tyrosine) that could regulate the water content of the active site.     

Molecular dynamics study of water penetration in staphylococcal nuclease

The ionization properties of Lys and Glu residues buried in the hydrophobic core of staphylococcal nuclease (SN) suggest that the interior of this protein behaves as a highly polarizable medium with an apparent dielectric constant near 10. This has been rationalized previously in terms of localized conformational relaxation concomitant with the ionization of the internal residue, and with contributions by internal water molecules. Paradoxically, the crystal structure of the SN V66E variant shows internal water molecules and the structure of the V66K variant does not. To assess the structural and dynamical character of interior water molecules in SN, a series of 10-ns-long molecular dynamics (MD) simulations was performed with wild-type SN, and with the V66E and V66K variants with Glu66 and Lys66 in the neutral form. Internal water molecules were identified based on their coordination state and characterized in terms of their residence times, average location, dipole moment fluctuations, hydrogen bonding interactions, and interaction energies. The locations of the water molecules that have residence times of several nanoseconds and display small mean-square displacements agree well with the locations of crystallographically observed water molecules. Additional, relatively disordered water molecules that are not observed crystallographically were found in internal hydrophobic locations. All of the interior water molecules that were analyzed in detail displayed a distribution of interaction energies with higher mean value and narrower width than a bulk water molecule. This underscores the importance of protein dynamics for hydration of the protein interior. Further analysis of the MD trajectories revealed that the fluctuations in the protein structure (especially the loop elements) can strongly influence protein hydration by changing the patterns or strengths of hydrogen bonding interactions between water molecules and the protein. To investigate the dynamical response of the protein to burial of charged groups in the protein interior, MD simulations were performed with Glu66 and Lys66 in the charged state. Overall, the MD simulations suggest that a conformational change rather than internal water molecules is the dominant determinant of the high apparent polarizability of the protein interior.     

Backbone and side-chain contributions in protein denaturation by urea

Urea is a commonly used protein denaturant, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its effect on protein stability. Using the Trp-cage miniprotein as a model system, we report what we believe to be the first computation of changes in the preferential interaction coefficient of the protein upon urea denaturation from molecular-dynamics simulations and examine the contributions from the backbone and the side-chain groups. The preferential interaction is obtained from reversible folding/unfolding replica exchange molecular-dynamics simulations of Trp-cage in presence of urea, over a wide range of urea concentration. The increase in preferential interaction upon unfolding is dominated by the side-chain contribution, rather than the backbone. Similar trends are observed in simulations using two different force fields, Amber94 and Amber99sb, for the protein. The magnitudes of the side-chain and backbone contributions differ in the two force fields, despite containing identical protein-solvent interaction terms. The differences arise from the unfolded ensembles sampled, with Amber99sb favoring conformations with larger surface area and lower helical content. These results emphasize the importance of the side-chain interactions with urea in protein denaturation, and highlight the dependence of the computed driving forces on the unfolded ensemble sampled.     

Wetting characterisation of silicon (1,0,0) surface

Water droplets on bare silicon surfaces are studied to examine the wetting behaviour as a function of the surface energy and to parameterise water–silicon interactions in order to recover the hydrophobic behaviour measured by experiments. Two different wetting regimes characterised by a critical interaction strength value are observed. At a threshold value of the water–silicon interaction parameter, water molecules start penetrating into the first layer of silicon surface under thermally vibrating walls, resulting in two distinct wetting behaviours. Fixed (cold) silicon walls do not exhibit the two different wetting characteristics. Size effects are studied for nano-scale droplets, and line tension influence is observed depending on the surface wettability. Decrease in the droplet size increases the contact angle values for the low wetting cases, while contact angles decrease for smaller droplets on the high wetting surfaces. Considering the line tension effects and droplet size, ?_Si–O for water–silicon interactions to recover the hydrophobic behaviour of silicon surfaces is estimated to be 12.5% of the value predicted using the Lorentz–Berthelot mixing rule.     

Molecular-dynamics simulations of insertion of chemically modified DNA nanostructures into a water-chloroform interface

DNA-based two-dimensional and three-dimensional arrays have been used as templates for the synthesis of functional polymers and proteins. Hydrophobic or amphiphilic DNA arrays would be useful for the synthesis of hydrophobic molecules. The objective of this study was to design a modified amphiphilic double crossover DNA molecule that would insert into a water-chloroform interface, thus showing an amphiphilic character. Since experiments for such designs are tedious, we used molecular-dynamics simulations to identify and optimize the functional groups to modify the DNA backbone that would enable insertion into the water-chloroform interface before synthesis. By methylating the phosphates of the backbone to make phosphonates, in combination with placing a benzyl group at the 2′ position of the deoxyribose rings in the backbone, we observed that the simple B-DNA structure was able to insert into the water-chloroform interface. We find that the transfer free energy of methylated benzylated DNA is better than that of either just methylated or benzylated DNA. The driving force for this insertion comes from the entropic contribution to the free energy and the favorable van der Waals interaction of the chloroform molecules with the methyl and benzyl groups of the DNA.     

In silico chaperonin-like cycle helps folding of proteins for structure prediction

Currently, one of the most serious problems in protein-folding simulations for de novo structure prediction is conformational sampling of medium-to-large proteins. In vivo, folding of these proteins is mediated by molecular chaperones. Inspired by the functions of chaperonins, we designed a simple chaperonin-like simulation protocol within the framework of the standard fragment assembly method: in our protocol, the strength of the hydrophobic interaction is periodically modulated to help the protein escape from misfolded structures. We tested this protocol for 38 proteins and found that, using a certain defined criterion of success, our method could successfully predict the native structures of 14 targets, whereas only those of 10 targets were successfully predicted using the standard protocol. In particular, for non-α-helical proteins, our method yielded significantly better predictions than the standard approach. This chaperonin-inspired protocol that enhanced de novo structure prediction using folding simulations may, in turn, provide new insights into the working principles underlying the chaperonin system.     

Structural Features of Transiently Modified Beta-Lactoglobulin Relevant to the Stable Binding of Large Hydrophobic Molecules

Binding sites for hydrophobic molecules on bovine β-lactoglobulin, and their susceptibility to temperature, were studied by using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual probes was found to be required for binding to β-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible denaturation. Binding became extremely tight and stable upon cooling of the protein–probe mixture. Comparison among the various probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially—and reversibly—modified form of β-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes binding to β-lactoglobulin may be modulated by simple physical treatment of the protein.     

Counteraction of Urea by Trimethylamine N-Oxide Is Due to Direct Interaction

Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins, induces folding, and counteracts the denaturing effects of urea, pressure, and ice. To establish the mechanism behind these effects, isotopic substitution neutron-scattering measurements were performed on aqueous solutions of TMAO and 1:1 TMAO-urea at a solute mole fraction of 0.05. The partial pair distribution functions were extracted using the empirical potential structure refinement method. The results were compared with previous results obtained with isosteric tert-butanol, as well as the available data from spectroscopy and molecular-dynamics simulations. In solution, the oxygen atom of TMAO is strongly hydrogen-bonded to, on average, between two and three water molecules, and the hydrogen-bond network is tighter in water than in pure water. In TMAO-urea solutions, the oxygen atom in TMAO preferentially forms hydrogen bonds with urea. This explains why the counteraction is completed at a 2:1 urea/TMAO concentration ratio, independently of urea concentration. These results strongly support models for the effect of TMAO on the stability of proteins based on a modification of the simultaneous equilibria that control hydrogen bonding between the peptide backbone and water or intramolecular sites, without any need for direct interaction between TMAO and the protein.     

Taste of Sugar at the Membrane: Thermodynamics and Kinetics of the Interaction of a Disaccharide with Lipid Bilayers

Sugar recognition at the membrane is critical in various physiological processes. Many aspects of sugar-membrane interaction are still unknown. We take an integrated approach by combining conventional molecular-dynamics simulations with enhanced sampling methods and analytical models to understand the thermodynamics and kinetics of a di-mannose molecule in a phospholipid bilayer system. We observe that di-mannose has a slight preference to localize at the water-phospholipid interface. Using umbrella sampling, we show the free energy bias for this preferred location to be just −0.42 kcal/mol, which explains the coexistence of attraction and exclusion mechanisms of sugar-membrane interaction. Accurate estimation of absolute entropy change of water molecules with a two-phase model indicates that the small energy bias is the result of a favorable entropy change of water molecules. Then, we incorporate results from molecular-dynamics simulation in two different ways to an analytical diffusion-reaction model to obtain association and dissociation constants for di-mannose interaction with membrane. Finally, we verify our approach by predicting concentration dependence of di-mannose recognition at the membrane that is consistent with experiment. In conclusion, we provide a combined approach for the thermodynamics and kinetics of a weak ligand-binding system, which has broad implications across many different fields.     

Membrane contact probability: An essential and predictive character for the structural and functional studies of membrane proteins

One of the unique traits of membrane proteins is that a significant fraction of their hydrophobic amino acids is exposed to the hydrophobic core of lipid bilayers rather than being embedded in the protein interior, which is often not explicitly considered in the protein structure and function predictions. Here, we propose a characteristic and predictive quantity, the membrane contact probability (MCP), to describe the likelihood of the amino acids of a given sequence being in direct contact with the acyl chains of lipid molecules. We show that MCP is complementary to solvent accessibility in characterizing the outer surface of membrane proteins, and it can be predicted for any given sequence with a machine learning-based method by utilizing a training dataset extracted from MemProtMD, a database generated from molecular dynamics simulations for the membrane proteins with a known structure. As the first of many potential applications, we demonstrate that MCP can be used to systematically improve the prediction precision of the protein contact maps and structures.     

Thermodynamics of Antimicrobial Lipopeptide Binding to Membranes: Origins of Affinity and Selectivity

The development of novel antibiotic drugs is one of the most pressing biomedical problems due to the increasing number of antibiotic-resistant pathogens. Antimicrobial peptides and lipopeptides are a promising category of candidates, but the molecular origins of their antimembrane activity is unclear. Here we explore a series of recently developed antimicrobial lipopeptides, using coarse-grained molecular-dynamics simulations and free energy methods to uncover the thermodynamics governing their binding to membranes. Specifically, we quantify C16-KGGK’s binding affinity to the two types of membrane by umbrella sampling. We also examined the origin of C16-KGGK’s selectivity for bacterial versus mammalian membranes by systematically varying the peptide sequence and salt concentration. Our data showed that the C16 hydrophobic tail is the main contributor to its affinity to lipid membrane, whereas the peptide portion is mainly responsible for its selectivity. Furthermore, the electrostatic interaction between the cationic peptide and anionic bacterial membrane plays a significant role in the selectivity.     

Computer assisted simulations and molecular graphics methods in molecular design 2. Proteinases and receptor and transport proteins

A survey is presented of model building techniques and computer-assisted quantum-chemical and molecular-dynamics simulations, as applied to the study of protease and receptor design, to the determination of the properties of related effectors, agonist and antagonist molecules, and to the design of fatty-acid transport proteins and molecular carriers. Studies covering integral membrane protein design have been reviewed: they include porins, ion channels and G protein coupled receptors. In the transport molecule class, hydrophobic ligand transporters, such as serum and cellular retinoid binding proteins, have been reviewed.     

Prediction of the binding mode between GSK3β and a peptide derived from GSKIP using molecular dynamics simulation

GSK3β plays an important role in many physiological functions; dysregulated GSK3β is involved in human diseases such as diabetes, cancer, and Alzheimer's disease. This study uses MD simulations to determine the interaction between GSK3β and a peptide derived from GSKIP, a novel GSK3β interacting protein. Results show that GSKIPtide is inlaid in a binding pocket consisting of an α-helix and an extended loop near the carboxy-terminal end. This binding pocket is hydrophobic, and is responsible for the protein-protein interaction of two other GSK3β interacting proteins: FRAT and Axin. The GSKIPtide binding mode is closer to that of AxinGID (in the Axin-GSK3-interacting domain). The single-point mutations of V267G and Y288F in GSK3β differentiate the binding modes between GSK3 and GSKIPtide, AxinGID, and FRATide. The V2677G mutation of GSK3β reduces the GSKIPtide binding affinity by 70% and abolishes the binding affinity with AxinGID, but has no effect on FRATide. However, GSK3β Y288F completely abolishes the FRATide binding without affecting GSKIPtide or AxinGID binding. An analysis of the GSK3β-GSKIPtide complex structure and the X-ray crystal structures of GSK3β-FRATide and GSK3β-AxinGID complexes suggests that the hydroxyl group of Y288 is crucial to maintaining a hydrogen bond network in GSK3β-FRATide. The hydrophobic side chain of V267 maintains the integrity of helix-helix ridge-groove hydrophobic interaction for GSK3β-GSKIPtide and GSK3β-AxinGID. This study simulates these two mutant systems to provide atomic-level evidence of the aforementioned experimental results and validate the wild-type complex structure prediction.     

Long-Timescale Molecular-Dynamics Simulations of the Major Urinary Protein Provide Atomistic Interpretations of the Unusual Thermodynamics of Ligand Binding

The mouse major urinary protein (MUP) has proved to be an intriguing test bed for detailed studies on protein-ligand recognition. NMR, calorimetric, and modeling investigations have revealed that the thermodynamics of ligand binding involve a complex interplay between competing enthalpic and entropic terms. We performed six independent, 1.2 μs molecular-dynamics simulations on MUP—three replicates on the apo-protein, and three on the complex with the pheromone isobutylmethoxypyrazine. Our findings provide the most comprehensive picture to date of the structure and dynamics of MUP, and how they are modulated by ligand binding. The mechanical pathways by which amino acid side chains can transmit information regarding ligand binding to surface loops and either increase or decrease their flexibility (entropy-entropy compensation) are identified. Dewetting of the highly hydrophobic binding cavity is confirmed, and the results reveal an aspect of ligand binding that was not observed in earlier, shorter simulations: bound ligand retains extensive rotational freedom. Both of these features have significant implications for interpretations of the entropic component of binding. More generally, these simulations test the ability of current molecular simulation methods to produce a reliable and reproducible picture of protein dynamics on the microsecond timescale.