Diffusional encounter of barnase and barstar

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.     

Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis and NMR

Phosphate is a competitive inhibitor of transesterification of GpC by the ribonuclease barnase. Barnase is significantly stabilized in the presence of phosphate against urea denaturation. The data are consistent with the existence of a single phosphate binding site in barnase with a dissociation constant, Kd, of 1.3 mM. The 2D 1H NMR spectrum of wild-type barnase with bound phosphate is assigned. Changes in chemical shifts and NOEs for wild type with bound phosphate compared with free wild type indicate that phosphate binds in the active site and that only small conformational changes occur on binding. Site-directed mutagenesis of the active site residues His-102, Lys-27, and Arg-87 to Ala increases the magnitude of Kd for phosphate by more than 20-fold. The 2D 1H NMR spectra of the mutants His-102----Ala, Lys-27----Ala, and Arg-87----Ala are assigned. Comparison with the spectra of wild-type barnase reveals that His-102----Ala and Lys-27----Ala have essentially the same structure as weild type, while some structural changes occur in Arg-87----Ala. It appears that phosphate binding by barnase is effected mainly by positively charge residues including His-102, Lys-27, and Arg-87. This may have applications for the design of phosphate binding sites in other proteins.     

The influence of intrinsic folding mechanism of an unfolded protein on the coupled folding-binding process during target recognition

Intrinsically disordered proteins (IDPs) are extensively involved in dynamic signaling processes which require a high association rate and a high dissociation rate for rapid binding/unbinding events and at the same time a sufficient high affinity for specific recognition. Although the coupled folding-binding processes of IDPs have been extensively studied, it is still impossible to predict whether an unfolded protein is suitable for molecular signaling via coupled folding-binding. In this work, we studied the interplay between intrinsic folding mechanisms and coupled folding-binding process for unfolded proteins through molecular dynamics simulations. We first studied the folding process of three representative IDPs with different folded structures, that is, c-Myb, AF9, and E3 rRNase. We found the folding free energy landscapes of IDPs are downhill or show low barriers. To further study the influence of intrinsic folding mechanism on the binding process, we modulated the folding mechanism of barnase via circular permutation and simulated the coupled folding-binding process between unfolded barnase permutant and folded barstar. Although folding of barnase was coupled to target binding, the binding kinetics was significantly affected by the intrinsic folding free energy barrier, where reducing the folding free energy barrier enhances binding rate up to two orders of magnitude. This accelerating effect is different from previous results which reflect the effect of structure flexibility on binding kinetics. Our results suggest that coupling the folding of an unfolded protein with no/low folding free energy barrier with its target binding may provide a way to achieve high specificity and rapid binding/unbinding kinetics simultaneously.     

Simulation of the diffusional association of barnase and barstar.

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.     

A comparative study of the interaction of Bacillus amyloliquefaciens ribonuclease (barnase) and Bacillus intermedius ribonuclease (binase) with barstar by brownian dynamics simulation

A comparative study of the interaction of two RNases (binase and barnase) with the polypeptide inhibitor barstar was performed by Brownian dynamics simulation. It was demonstrated that this method adequately reproduced the dependence of the association rate on the pH of solution as well as the effect of mutations at individual amino acid residues on the inhibition of barnase by barstar. Two types of energy-favorable binase-barstar encounter complexes were found. In type I complex, the amino acid residues of the binase active center are involved in formation of the complex; in type II complex, the active center remains free. It is suggested that temporary binding of free barstar into type II complex competes with the inhibition reaction. Presumably, this explains the decrease in the rate of binase inhibition by barstar as compared with the analogous reaction of barnase.     

How optimal are the binding energetics of barnase and barstar?

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.     

I87E Mutation Prevents Barstar Dimerization

The C40,82A;I87E mutant of barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, was obtained, and its physicochemical properties were studied. It was produced as a fusion protein with thioredoxin and then cleaved from this by EKmax enterokinase. The mutant was shown by NMR to retain the spatial structure of the wild-type protein but, in contrast to barstar, does not form the homodimers characteristic of barstar in aqueous solution. The mutant protein binds barnase with the dissociation constant (6.6 ± 1.1) × 10^–11 M and exhibits other physicochemical properties similar to those of the wild-type barstar. This allows the use of C40,82A;I87E mutant instead of wild-type barstar in the investigations where the protein dimerization is undesirable.     

Interaction energy decomposition in protein-protein association: a quantum mechanical study of barnase-barstar complex

Protein-protein interactions are very important in the function of a cell. Computational studies of these interactions have been of interest, but often they have utilized classical modelling techniques. In recent years, quantum mechanical (QM) treatment of entire proteins has emerged as a powerful approach to study biomolecular systems. Herein, we apply a semi-empirical divide and conquer (DC) methodology coupled with a dielectric continuum model for the solvent, to explore the contribution of electrostatics, polarization and charge transfer to the interaction energy between barnase and barstar in their complex form. Molecular dynamic (MD) simulation was performed to account for the dynamic behavior of the complex. The results show that electrostatics, charge transfer and polarization favor the formation of the complex. Our study shows that electrostatics dominates the interaction between barnase and barstar ( approximately 73%), while charge transfer and polarization are approximately 21% and approximately 6%, respectively. Close inspection of the polarization and charge-transfer effects on the charge distribution of the complex reveals the existence of two, well localized, regions in barstar. The first region includes the residues between P27 and Y47 and the second region is between N65 and D83. Since no such regions could be detected in barnase clearly suggests that barstar is well optimized for efficiently binding barnase. Furthermore, using our interaction energy decomposition scheme, we were able to identify all residues that have been experimentally determined to be important for the complex formation and to suggest other residues never have been investigated. This suggests that our approach will be useful as an aid in further understanding protein-protein contacts for the ultimate goal to produce successful inhibitors for protein complexes.     

The impact of protein flexibility on protein-protein docking

Accounting for protein flexibility in protein-protein docking algorithms is challenging, and most algorithms therefore treat proteins as rigid bodies or permit side-chain motion only. While the consequences are obvious when there are large conformational changes upon binding, the situation is less clear for the modest conformational changes that occur upon formation of most protein-protein complexes. We have therefore studied the impact of local protein flexibility on protein-protein association by means of rigid body and torsion angle dynamics simulation. The binding of barnase and barstar was chosen as a model system for this study, because the complexation of these 2 proteins is well-characterized experimentally, and the conformational changes accompanying binding are modest. On the side-chain level, we show that the orientation of particular residues at the interface (so-called hotspot residues) have a crucial influence on the way contacts are established during docking from short protein separations of approximately 5 A. However, side-chain torsion angle dynamics simulations did not result in satisfactory docking of the proteins when using the unbound protein structures. This can be explained by our observations that, on the backbone level, even small (2 A) local loop deformations affect the dynamics of contact formation upon docking. Complementary shape-based docking calculations confirm this result, which indicates that both side-chain and backbone levels of flexibility influence short-range protein-protein association and should be treated simultaneously for atomic-detail computational docking of proteins.     

The free energy landscape of small molecule unbinding

The spontaneous dissociation of six small ligands from the active site of FKBP (the FK506 binding protein) is investigated by explicit water molecular dynamics simulations and network analysis. The ligands have between four (dimethylsulphoxide) and eleven (5-diethylamino-2-pentanone) non-hydrogen atoms, and an affinity for FKBP ranging from 20 to 0.2 mM. The conformations of the FKBP/ligand complex saved along multiple trajectories (50 runs at 310 K for each ligand) are grouped according to a set of intermolecular distances into nodes of a network, and the direct transitions between them are the links. The network analysis reveals that the bound state consists of several subbasins, i.e., binding modes characterized by distinct intermolecular hydrogen bonds and hydrophobic contacts. The dissociation kinetics show a simple (i.e., single-exponential) time dependence because the unbinding barrier is much higher than the barriers between subbasins in the bound state. The unbinding transition state is made up of heterogeneous positions and orientations of the ligand in the FKBP active site, which correspond to multiple pathways of dissociation. For the six small ligands of FKBP, the weaker the binding affinity the closer to the bound state (along the intermolecular distance) are the transition state structures, which is a new manifestation of Hammond behavior. Experimental approaches to the study of fragment binding to proteins have limitations in temporal and spatial resolution. Our network analysis of the unbinding simulations of small inhibitors from an enzyme paints a clear picture of the free energy landscape (both thermodynamics and kinetics) of ligand unbinding.     

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.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Native and nonnative conformational preferences in the urea-unfolded state of barstar

The refolding of barstar from its urea-unfolded state has been studied extensively using various spectroscopic probes and real-time NMR, which provide global and residue-specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25 degrees C. Complete backbone resonance assignments of the urea-unfolded protein were obtained using the recently developed three-dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13C(alpha) and 13CO secondary shifts show positive deviations in the urea-unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to beta-strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the beta-strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.     

A New Method to Characterize Hydrophobic Organization of Proteins: Application to Rational Protein Engineering of Barnase

Abstract

We present a new algorithm for characterization of protein spatial structure basing on the molecular hydrophobicity potential approach. The method is illustrated by the analysis of three-dimensional structure of barnase and barnase-barstar complex. Current approach enables identification of amino acid residues situated in unfavorable environment (these residues may be “active” for binding), and to map quantitatively hydrophobic, hydrophilic and unfavorable hydrophobic-hydrophilic intra-and inter-molecular contacts involving backbone and side-chain segments of amino acid residues. Calculation of individual contributions of amino acid residues to such contacts permits identification of structurally-important residues. The contact plots obtained with molecular hydrophobicity potential calculations, provide easy rules to choose sites for mutations, which can increase a strength of intra- or inter-molecular hydrophobic interactions. The unfavorable hydrophobic-hydrophilic contact can be mutated to favorable hydrophobic, and already existing weak hydrophobic contact can be strengthen by increasing hydrophobicity of residues in contact. Basing on the analysis of the contact plots, we suggest several mutations of barnase which are supposed to increase intramolecular hydrophobic interactions, and thus might lead to increased stability of the protein. Part of these mutations was studied previously experimentally, and indeed stabilized barnase. The other of predicted mutations were not studied experimentally yet. Several new mutations of barnase and barstar are also proposed to enhance the hydrophobic interactions on their binding interface.     

A modular design of low‐background bioassays based on a high‐affinity molecular pair barstar:barnase

High‐affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (K_D) of the molecular pair, with avidin:biotin being the state‐of‐the‐art molecular pair (K_D ～ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high‐affinity protein pair, barstar:barnase (K_D ～ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non‐negligible background due to the non‐specific binding. A quantitative assessment of the non?specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin‐based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non‐specific binding, showing good prospects for high‐sensitivity rare biomolecular event nanoproteomic assays.     

New hypotheses about the structure–function of proprotein convertase subtilisin/kexin type 9: Analysis of the epidermal growth factor‐like repeat A docking site using WaterMap

LDL cholesterol (LDL‐C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low‐density lipoprotein cholesterol receptor (LDL‐R). LDL‐R is targeted for lysosomal degradation by association with proprotein convertase subtilisin‐kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor‐sparing phenomena, including loss‐of‐function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate‐determining steps for protein–ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild‐type versus gain‐of‐function mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins (“hydration sites”). We compared these results with binding kinetics data for PCSK9, full‐length LDL‐R ectodomain, and isolated EGF‐A repeat. We propose that the fast k_on and entropically driven thermodynamics observed for PCSK9‐EGF‐A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H‐bonds from water to polar EGF‐A groups). We further propose that the relatively fast k_off observed for EGF‐A unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower k_off observed for EGF‐A and LDL‐R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effect of DNA on the conformational dynamics of the endonucleases I‐DmoI as provided by molecular dynamics simulations

The conformational behavior of the wild‐type endonucleases I‐DmoI and two of its mutants has been studied in the presence and in the absence of DNA target sequences by means of extended molecular dynamics simulations. Our results show that in the absence of DNA, the three protein forms explore a similar essential conformational space, whereas when bound to the same DNA target sequence of 25 base pairs, they diversify and restrain the subspace explored. In addition, the differences in the essential subspaces explored by the residues near the catalytic site for both the bound and unbound forms are discussed in background of the experimental protein activity.     

Testing mechanisms of DNA sliding by architectural DNA-binding proteins: dynamics of single wild-type and mutant protein molecules in vitro and in vivo

Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.     

Characterization of in vitro oxidized barstar

The polypeptide inhibitor of the ribonuclease barnase, barstar, has two cysteine residues in positions 40 and 82. These have been proposed to form a disulfide bridge leading to an increase in stability without changing the inhibitory activity of the protein. Barstar and a mutant (E80A) were oxidized in vitro and the biochemical and physico-chemical properties of the oxidized monomers were analysed. The oxidized proteins show no inhibition of barnase using a plate assay and are significantly destabilized. CD spectra indicate a loss of secondary structure. The amino acid substitution E80 → A stabilizes the oxidized barstar to about the same extent as it does the reduced protein, indicating, however, that the helical region which it is in is intact.     

The interaction with the cytoplasmic loops of rhodopsin plays a crucial role in arrestin activation and binding

The binding of arrestin to rhodopsin is initiated by the interaction of arrestin with the phosphorylated rhodopsin C-terminus and/or the cytoplasmic loops, followed by conformational changes that expose an additional high-affinity site on arrestin. Here we use an arrestin mutant (R175E) that binds similarly to phosphorylated and unphosphorylated, wild-type rhodopsin to identify rhodopsin elements other than C-terminus important for arrestin interaction. R175E-arrestin demonstrated greatly reduced binding to unphosphorylated cytoplasmic loop mutants L72A, N73A, P142A and M143A, suggesting that these residues are crucial for high-affinity binding. Interestingly, when these rhodopsin mutants are phosphorylated, R175E-arrestin binding is less severely affected. This effect of phosphorylation on R175E-arrestin binding highlights the co-operative nature of the multi-site interaction between arrestin and the cytoplasmic loops and C-terminus of rhodopsin. However, a combination of any two mutations disrupts the ability of phosphorylation to enhance binding of R175E-arrestin. N73A, P142A and M143A exhibited accelerated rates of dissociation from wild-type arrestin. Using sensitivity to calpain II as an assay, these cytoplasmic loop mutants also demonstrated reduced ability to induce conformational changes in arrestin that correlated with their reduced ability to bind arrestin. These results suggest that arrestin bound to rhodopsin is in a distinct conformation that is co-ordinately regulated by association with the cytoplasmic loops and the C-terminus of rhodopsin.