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Linker histones (LHs) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed with 64% AT. The labeled gH consistently exhibited higher FRET efficiency with the labeled L-DNA containing the A-tract than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizing AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences., (Copyright © 2021. Published by Elsevier Inc.)

Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids, and xenobiotics. Microsomal CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal transmembrane (TM) helix that is connected to the globular catalytic domain by a flexible linker sequence. However, the structural and functional importance of the TM-helix is unclear because it has been shown that CYPs can still associate with the membrane and have enzymatic activity in reconstituted systems after truncation or modification of the N-terminal sequence. Here, we investigated the effect of mutations in the N-terminal TM-helix residues of two human steroidogenic enzymes, CYP 17A1 and CYP 19A1, that are major drug targets for cancer therapy. These mutations were originally introduced to increase the expression of the proteins in Escherichia coli. To investigate the effect of the mutations on protein-membrane interactions and function, we carried out coarse-grained and all-atom molecular dynamics simulations of the CYPs in a phospholipid bilayer. We confirmed the orientations of the globular domain in the membrane observed in the simulations by linear dichroism measurements in a Nanodisc. Whereas the behavior of CYP 19A1 was rather insensitive to truncation of the TM-helix, mutations in the TM-helix of CYP 17A1, especially W2A and E3L, led to a gradual drifting of the TM-helix out of the hydrophobic core of the membrane. This instability of the TM-helix could affect interactions with the allosteric redox partner, cytochrome b5, required for CYP 17A1's lyase activity. Furthermore, the simulations showed that the mutant TM-helix influenced the membrane interactions of the CYP 17A1 globular domain. In some simulations, the mutated TM-helix obstructed the substrate access tunnel from the membrane to the CYP active site, indicating a possible effect on enzyme function., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Linker histone (LH) proteins play a key role in higher-order structuring of chromatin for the packing of DNA in eukaryotic cells and in the regulation of genomic function. The common fruit fly (Drosophila melanogaster) has a single somatic isoform of the LH (H1). It is thus a useful model organism for investigating the effects of the LH on nucleosome compaction and the structure of the chromatosome, the complex formed by binding of an LH to a nucleosome. The structural and mechanistic details of how LH proteins bind to nucleosomes are debated. Here, we apply Brownian dynamics simulations to compare the nucleosome binding of the globular domain of D. melanogaster H1 (gH1) and the corresponding chicken (Gallus gallus) LH isoform, gH5, to identify residues in the LH that critically affect the structure of the chromatosome. Moreover, we investigate the effects of posttranslational modifications on the gH1 binding mode. We find that certain single-point mutations and posttranslational modifications of the LH proteins can significantly affect chromatosome structure. These findings indicate that even subtle differences in LH sequence can significantly shift the chromatosome structural ensemble and thus have implications for chromatin structure and transcriptional regulation., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

As a model for understanding how molecular crowding influences diffusion and transport of proteins in cellular environments, we combined experimental and theoretical approaches to study the diffusion of proteins in highly concentrated protein solutions. Bovine serum albumin and γ-Globulin were chosen as molecular crowders and as tracers. These two proteins are representatives of the main types of plasma protein and have different shapes and sizes. Solutions consisting of one or both proteins were studied. The self-diffusion coefficients of the fluorescently labeled tracer proteins were measured by means of fluorescence correlation spectroscopy at a total protein concentration of up to 400 g/L. γ-Globulin is found to have a stronger influence as a crowder on the tracer self-diffusion coefficient than Bovine serum albumin. Brownian dynamics simulations show that the excluded volume and the shape of the crowding protein have a significantly stronger influence on translational and rotational diffusion coefficients, as well as transient oligomerization, than hydrodynamic or direct interactions. Anomalous subdiffusion, which is not observed at the experimental fluorescence correlation spectroscopy timescales (>100 μs), appears only at very short timescales (<1 μs) in the simulations due to steric effects of the proteins. We envision that the combined experimental and computational approach employed here can be developed to unravel the different biophysical contributions to protein motion and interaction in cellular environments by systematically varying protein properties such as molecular weight, size, shape, and electrostatic interactions., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The study of solutions of biomacromolecules provides an important basis for understanding the behavior of many fundamental cellular processes, such as protein folding, self-assembly, biochemical reactions, and signal transduction. Here, we describe a Brownian dynamics simulation procedure and its validation for the study of the dynamic and structural properties of protein solutions. In the model used, the proteins are treated as atomically detailed rigid bodies moving in a continuum solvent. The protein-protein interaction forces are described by the sum of electrostatic interaction, electrostatic desolvation, nonpolar desolvation, and soft-core repulsion terms. The linearized Poisson-Boltzmann equation is solved to compute electrostatic terms. Simulations of homogeneous solutions of three different proteins with varying concentrations, pH, and ionic strength were performed. The results were compared to experimental data and theoretical values in terms of long-time self-diffusion coefficients, second virial coefficients, and structure factors. The results agree with the experimental trends and, in many cases, experimental values are reproduced quantitatively. There are no parameters specific to certain protein types in the interaction model, and hence the model should be applicable to the simulation of the behavior of mixtures of macromolecules in cell-like crowded environments., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Auxin Binding Protein 1 (ABP1) is ubiquitous in green plants. It binds the phytohormone auxin with high specificity and affinity, but its role in auxin-induced processes is unknown. To understand the proposed receptor function of ABP1 we carried out a detailed molecular modeling study. Molecular dynamics simulations showed that ABP1 can adopt two conformations differing primarily in the position of the C-terminus and that one of them is stabilized by auxin binding. This is in agreement with experimental evidence that auxin induces changes at the ABP1 C-terminus. Simulations of ligand egress from ABP1 revealed three main routes by which an auxin molecule can enter or leave the ABP1 binding site. Assuming the previously proposed orientation of ABP1 to plant cell membranes, one of the routes leads to the membrane and the other two to ABP1's aqueous surroundings. A network of hydrogen-bonded water molecules leading from the bulk water to the zinc-coordinated ligands in the ABP1 binding site was formed in all simulations. Water entrance into the zinc coordination sphere occurred simultaneously with auxin egress. These results suggest that the hydrogen-bonded water molecules may assist in protonation and deprotonation of auxin molecules and their egress from the ABP1 binding site.

We present an analysis of trajectories from Brownian dynamics simulations of diffusional protein-protein encounter for the well-studied system of barnase and barstar. This analysis reveals details about the optimal association pathways, the regions of the encounter complex, possible differences of the pathways for dissociation and association, the coupling of translational and rotation motion, and the effect of mutations on the trajectories. We found that a small free-energy barrier divides the energetically most favorable region into a region of the encounter complex above the barnase binding interface and a region around a second energy minimum near the RNA binding loop. When entering the region of the encounter complex from the region near the RNA binding loop, barstar has to change its orientation to increase the electrostatic attraction between the proteins. By concentrating the analysis on the successful binding trajectories, we found that the region of the second minimum is not essential for the binding of barstar to barnase. Nevertheless, this region may be helpful to steer barstar into the region of the encounter complex. When applying the same analysis to several barnase mutants, we found that single mutations may drastically change the free-energy landscape and may significantly alter the population of the two minima. Therefore, certain protein-protein pairs may require careful adaptation of the positions of encounter and transition states when interpreting mutation effects on kinetic rates of association and/or dissociation.

The extracellular ribonuclease barnase and its intracellular inhibitor barstar bind fast and with high affinity. Although extensive experimental and theoretical studies have been carried out on this system, it is unclear what the relative importance of different contributions to the high affinity is and whether binding can be improved through point mutations. In this work, we first applied Poisson-Boltzmann electrostatic calculations to 65 barnase-barstar complexes with mutations in both barnase and barstar. The continuum electrostatic calculations with a van der Waals surface dielectric boundary definition result in the electrostatic interaction free energy providing the dominant contribution favoring barnase-barstar binding. The results show that the computed electrostatic binding free energy can be improved through mutations at W44/barstar and E73/barnase. Furthermore, the determinants of binding affinity were quantified by applying COMparative BINding Energy (COMBINE) analysis to derive quantitative structure-activity relationships (QSARs) for the 65 complexes. The COMBINE QSAR model highlights approximately 20 interfacial residue pairs as responsible for most of the differences in binding affinity between the mutant complexes, mainly due to electrostatic interactions. Based on the COMBINE model, together with Brownian dynamics simulations to compute diffusional association rate constants, several mutants were designed to have higher binding affinities than the wild-type proteins.

A major problem in mathematical modeling of the dynamics of complex biological systems is the frequent lack of knowledge of kinetic parameters. Here, we apply Brownian dynamics simulations, based on protein three-dimensional structures, to estimate a previously undetermined kinetic parameter, which is then used in biochemical network simulations. The peroxidase-oxidase reaction involves many elementary steps and displays oscillatory dynamics important for immune response. Brownian dynamics simulations were performed for three different peroxidases to estimate the rate constant for one of the elementary steps crucial for oscillations in the peroxidase-oxidase reaction, the association of superoxide with peroxidase. Computed second-order rate constants agree well with available experimental data and permit prediction of rate constants at physiological conditions. The simulations show that electrostatic interactions depress the rate of superoxide association with myeloperoxidase, bringing it into the range necessary for oscillatory behavior in activated neutrophils. Such negative electrostatic steering of enzyme-substrate association presents a novel control mechanism and lies in sharp contrast to the electrostatically-steered fast association of superoxide and Cu/Zn superoxide dismutase, which is also simulated here. The results demonstrate the potential of an integrated and concerted application of structure-based simulations and biochemical network simulations in cellular systems biology.

The oxidation of cytochrome f by the soluble cupredoxin plastocyanin is a central reaction in the photosynthetic electron transfer chain of all oxygenic organisms. Here, two different computational approaches are used to gain new insights into the role of molecular recognition and protein-protein association processes in this redox reaction. First, a comparative analysis of the computed molecular electrostatic potentials of seven single and multiple point mutants of spinach plastocyanin (D42N, E43K, E43N, E43Q/D44N, E59K/E60Q, E59K/E60Q/E43N, Q88E) and the wt protein was carried out. The experimentally determined relative rates (k(2)) for the set of plastocyanin mutants are found to correlate well (r(2) = 0.90 - 0.97) with the computed measure of the similarity of the plastocyanin electrostatic potentials. Second, the effects on the plastocyanin/cytochrome f association rate of these mutations in the plastocyanin "eastern site" were evaluated by simulating the association of the wild type and mutant plastocyanins with cytochrome f by Brownian dynamics. Good agreement between the computed and experimental relative rates (k(2)) (r(2) = 0.89 - 0.92) was achieved for the plastocyanin mutants. The results obtained by applying both computational techniques provide support for the fundamental role of the acidic residues at the plastocyanin eastern site in the association with cytochrome f and in the overall electron-transfer process.

The WW domain consists of approximately 40 residues, has no disulfide bridges, and forms a three-stranded antiparallel beta-sheet that is monomeric in solution. It thus provides a model system for studying beta-sheet stability in native proteins. We performed molecular dynamics simulations of two WW domains, YAP65 and FBP28, with very different stability characteristics, in order to explore the initial unfolding of the beta-sheet. The less stable YAP domain is much more sensitive to simulation conditions than the FBP domain. Under standard simulation conditions in water (with or without charge-balancing counterions) at 300 K, the beta-sheet of the YAP WW domain disintegrated at early stages of the simulations. Disintegration commenced with the breakage of a hydrogen bond between the second and third strands of the beta-sheet due to an anticorrelated transition of the Tyr-28 psi and Phe-29 phi angles. Electrostatic interactions play a role in this event, and the YAP WW domain structure is more stable when simulated with a complete explicit model of the surrounding ionic strength. Other factors affecting stability of the beta-sheet are side-chain packing, the conformational entropy of the flexible chain termini, and the binding of cognate peptide.

The accurate and efficient treatment of electrostatic interactions is one of the challenging problems of molecular dynamics simulation. Truncation procedures such as switching or shifting energies or forces lead to artifacts and significantly reduced accuracy. The particle mesh Ewald (PME) method is one approach to overcome these problems by providing a computationally efficient means of calculating all long-range electrostatic interactions in a periodic simulation box by use of fast Fourier transformation techniques. For the application of the PME method to the simulation of a protein with a net charge in aqueous solution, counterions are added to neutralize the system. The usual procedure is to add charge-balancing counterions close to charged residues to neutralize the protein surface. In the present article, we show that for MD simulation of a small protein of marginal stability, the YAP-WW domain, explicit modeling of 0.2 M ionic strength (in addition to the charge-balancing counterions) is necessary to maintain a stable protein structure. Without explicit ions throughout the periodic simulation box, the charge-balancing counterions on the protein surface diffuse away from the protein, resulting in destruction of the beta-sheet secondary structure of the WW domain.

Beta-lactamases are responsible for resistance to penicillins and related beta-lactam compounds. Despite numerous studies, the identity of the general base involved in the acylation step is still unclear. It has been proposed, on the basis of a previous pKa calculation and analysis of structural data, that the unprotonated Lys73 in the active site could act as the general base. Using a continuum electrostatic model with an improved treatment of the multiple titration site problem, we calculated the pKa values of all titratable residues in the substrate-free TEM-1 and Bacillus licheniformis class A beta-lactamases. The pKa of Lys73 in both enzymes was computed to be above 10, in good agreement with recent experimental data on the TEM-1 beta-lactamase, but inconsistent with the proposal that Lys73 acts as the general base. Even when the closest titratable residue, Glu166, is mutated to a neutral residue, the predicted downward shift of the pKa of Lys73 shows that it is unlikely to act as a proton abstractor in either enzyme. These results support a mechanism in which the proton of the active Ser70 is transferred to the carboxylate group of Glu166.

The rate of protein association places an upper limit on the response time due to protein interactions, which, under certain circumstances, can be diffusion-controlled. Simulations of model proteins show that diffusion-limited association rates are approximately 10(6)-10(7) M-1 s-1 in the absence of long-range forces (Northrup, S. H., and H. P. Erickson. 1992. Kinetics of protein-protein association explained by Brownian dynamics computer simulations. Proc. Natl. Acad. Sci. U.S.A. 89:3338-3342). The measured association rates of barnase and barstar are 10(8)-10(9) M-1 s-1 at 50 mM ionic strength, and depend on ionic strength (Schreiber, G., and A. R. Fersht. 1996. Rapid, electrostatically assisted association of proteins. Nat. Struct. Biol. 3:427-431), implying that their association is electrostatically facilitated. We report Brownian dynamics simulations of the diffusional association of barnase and barstar to compute association rates and their dependence on ionic strength and protein mutation. Crucial to the ability to reproduce experimental rates is the definition of encounter complex formation at the endpoint of diffusional motion. Simple definitions, such as a required root mean square (RMS) distance to the fully bound position, fail to explain the large influence of some mutations on association rates. Good agreement with experiments could be obtained if satisfaction of two intermolecular residue contacts was required for encounter complex formation. In the encounter complexes, barstar tends to be shifted from its position in the bound complex toward the guanine-binding loop on barnase.

Interfacial activation of Rhizomucor miehei lipase is accompanied by a hinge-type motion of a single helix (residues 83-94) that acts as a lid over the active site. Activation of the enzyme involves the displacement of the lid to expose the active site, suggesting that the dynamics of the lid could be of mechanistic and kinetic importance. To investigate possible activation pathways and to elucidate the effect of a hydrophobic environment (as would be provided by a lipid membrane) on the lid opening, we have applied molecular dynamics and Brownian dynamics techniques. Our results indicate that the lipase activation is enhanced in a hydrophobic environment. In nonpolar low-dielectric surroundings, the lid opens in approximately 100 ns in the BD simulations. In polar high-dielectric (aqueous) surroundings, the lid does not always open up in simulations of up to 900 ns duration, but it does exhibit some gating motion, suggesting that the enzyme molecule may exist in a partially active form before the catalytic reaction. The activation is controlled by the charged residues ARG86 and ASP91. In the inactive conformation, ASP91 experiences repulsive forces and pushes the lid toward the open conformation. Upon activation ARG86 approaches ASP61, and in the active conformation, these residues form a salt bridge that stabilizes the open conformation.

Ordered water molecules are observed by crystallography and nuclear magnetic resonance to mediate protein-ligand interactions. Here, we examine the energetics of hydrating cavities formed at protein-ligand interfaces using molecular dynamics simulations. The free energies of hydrating two cavities in the active site of two liganded complexes of cytochrome P450cam were calculated by multiconfigurational thermodynamic integration. The complex of cytochrome P450cam with 2-phenyl-imidazole contains a crystallographically well defined water molecule mediating hydrogen bonds between the protein and the inhibitor. We calculate that this water molecule is stabilized by a binding free energy of -11.6 +/- kJ/mol. The complex of cytochrome P450cam with its natural substrate, camphor, contains a cavity that is empty in the crystal structure although a water molecule in it could make a hydrogen bond to camphor. Here, solvation of this cavity is calculated to be unfavorable by +15.8 +/- 5.0 kJ/mol. The molecular dynamics simulations can thus distinguish a hydrated interfacial cavity from an empty one. They also provide support for the notion that protein-ligand complexes can accommodate empty interfacial cavities and that such cavities are likely to be unhydrated unless more than one hydrogen bond can be made to a water molecule in the cavity.

The enzyme triose phosphate isomerase has flexible peptide loops at its active sites. The loops close over these sites upon substrate binding, suggesting that the dynamics of the loops could be of mechanistic and kinetic importance. To investigate these issues, the loop motions in the dimeric enzyme were simulated by Brownian dynamics. The two loops, one on each monomer, were represented by linear chains of appropriately parameterized spheres, each sphere corresponding to an amino acid residue. The loops moved in the electrostatic field of the rest of the enzyme, which was held rigid in its crystallographically observed conformation. In the absence of substrate, the loops exhibited gating of the active site with a period of about 1 ns and occupied "closed" conformations for about half of the time. As the period of gating is much shorter than the enzyme-substrate relaxation time, the motion of the loops does not reduce the rate constant for the approach of substrate from its simple diffusion-controlled value. This suggests that the flexible loops may have evolved to create the appropriate environment for catalysis while, at the same time, minimizing the kinetic penalty for gating the active site.