Conformational dynamics of subtilisin-chymotrypsin inhibitor 2 complex by coarse-grained simulations

An off-lattice dynamic Monte Carlo (MC) method is used to investigate the conformational dynamics of chymotrypsin inhibitor 2 (CI2) and subtilisin in both free and complex forms over two time windows, referring to short and long time scales. The conformational dynamics of backbone bonds analysed from several independent trajectories reveal that: Both the inhibitor and the enzyme are restricted in their bond rotations, excluding a few bonds, upon binding; the effect being greatest for the loop regions, and for the inhibitor. A cooperativity in the near-neighbor bond rotations are observed on both time scales, whereas the cooperative rotations of the bonds far along the sequence appear only in the long time window, and the latter time window is where most of the interactions between the inhibitor and the enzyme are observed. Upon binding, the cooperatively rotating parts of the inhibitor and the enzyme are readjusted compared to their free forms, and new correlations appear. The binding loop, although it is the closest contact region, is not the only part of the inhibitor involved in the interactions with the enzyme. Loops 3 and 8 and the helices F and G in bound enzyme and the binding loop of the inhibitor contribute at the most to the collective motions of whole structure on the slow time scale and are apparently important for enzyme-inhibitor interactions and function. The results in general provide evidence for the contribution of the loops with cooperative motions to the extensive communication network of the complex.     

Conformational Dynamics of Subtilisin-Chymotrypsin Inhibitor 2 Complex by Coarse-Grained Simulations

Abstract

An off-lattice dynamic Monte Carlo (MC) method is used to investigate the conformational dynamics of chymotrypsin inhibitor 2 (CI2) and subtilisin in both free and complex forms over two time windows, referring to short and long time scales. The conformational dynamics of backbone bonds analysed from several independent trajectories reveal that: Both the inhibitor and the enzyme are restricted in their bond rotations, excluding a few bonds, upon binding; the effect being greatest for the loop regions, and for the inhibitor. A cooperativity in the near-neighbor bond rotations are observed on both time scales, whereas the cooperative rotations of the bonds far along the sequence appear only in the long time window, and the latter time window is where most of the interactions between the inhibitor and the enzyme are observed. Upon binding, the cooperatively rotating parts of the inhibitor and the enzyme are readjusted compared to their free forms, and new correlations appear. The binding loop, although it is the closest contact region, is not the only part of the inhibitor involved in the interactions with the enzyme. Loops 3 and 8 and the helices F and G in bound enzyme and the binding loop of the inhibitor contribute at the most to the collective motions of whole structure on the slow time scale and are apparently important for enzyme-inhibitor interactions and function. The results in general provide evidence for the contribution of the loops with cooperative motions to the extensive communication network of the complex.     

Correlated motions in the U1 snRNA stem/loop 2:U1A RBD1 complex

The complex formed by U1A RBD1 and the U1 snRNA stem/loop II is noted for its high affinity and exquisite specificity. Here, that complex is investigated by 5 ns molecular dynamics simulations and analyzed by reorientational eigenmode dynamics to determine the dynamic properties of the RNA:protein interface that could contribute to the binding mechanism. The analysis shows that there is extensive correlation between motions of the RNA and protein, involving 7 of the 10 RNA loop nucleotides, the protein beta-sheet surface, two of its loops, and its C-terminal tripeptide sequence. Order parameters of these regions of the complex are uniformly high, indicating restricted motion. However, several regions of both RNA and protein retain local flexibility, notably three nucleotides of the RNA loop and one loop of RBD1 that does not contact RNA. The highly correlated motions involving both molecules reflect the intricate network of interactions that characterize this complex and could account in part for the thermodynamic coupling observed for complex formation.     

Rigid‐body motions of interacting proteins dominate multispecific binding of ubiquitin in a shape‐dependent manner

To understand the dynamic aspects of multispecificity of ubiquitin, we studied nine ubiquitin–ligand (partner protein) complexes by normal mode analysis based on an elastic network model. The coupling between ubiquitin and ligand motions was analyzed by decomposing it into rigid‐body (external) and vibrational (internal) motions of each subunit. We observed that in total the external motions in one of the subunits largely dominated the coupling. The combination of external motions of ubiquitin and the ligands showed general trends of rotations and translations. Moreover, we observed that the rotational motions of ubiquitin were correlated to the ligand orientations. We also identified ubiquitin atomic vibrations that differentiated the orientation of the ligand molecule. We observed that the extents of coupling were correlated to the shapes of the ligands, and this trend was more pronounced when the coupling involved vibrational motions of the ligand. In conclusion, an intricate interplay between internal and external motions of ubiquitin and the ligands help understand the dynamics of multispecificity, which is mostly guided by the shapes of the ligands and the complex. Proteins 2014; 82:77–89. © 2013 Wiley Periodicals, Inc.     

Retention of Conformational Entropy upon Calmodulin Binding to Target Peptides Is Driven by Transient Salt Bridges

Calmodulin (CaM) is a highly flexible calcium-binding protein that mediates signal transduction through an ability to differentially bind to highly variable binding sequences in target proteins. To identify how binding affects CaM motions, and its relationship to conformational entropy and target peptide sequence, we have employed fully atomistic, explicit solvent molecular dynamics simulations of unbound CaM and CaM bound to five different target peptides. The calculated CaM conformational binding entropies correlate with experimentally derived conformational entropies with a correlation coefficient R² of 0.95. Selected side-chain interactions with target peptides restrain interhelical loop motions, acting to tune the conformational entropy of the bound complex via widely distributed CaM motions. In the complex with the most conformational entropy retention (CaM in complex with the neuronal nitric oxide synthase binding sequence), Lys-148 at the C-terminus of CaM forms transient salt bridges alternating between Glu side chains in the N-domain, the central linker, and the binding target. Additional analyses of CaM structures, fluctuations, and CaM-target interactions illuminate the interplay between electrostatic, side chain, and backbone properties in the ability of CaM to recognize and discriminate against targets by tuning its conformational entropy, and suggest a need to consider conformational dynamics in optimizing binding affinities.     

Multi-timescale dynamics study of FKBP12 along the rapamycin-mTOR binding coordinate

Drugs can affect function in proteins by modulating their flexibility. Despite this possibility, there are very few studies on how drug binding affects the dynamics of target macromolecules. FKBP12 (FK506 binding protein 12) is a prolyl cis-trans isomerase and a drug target. The immunosuppressant drug rapamycin exerts its therapeutic effect by serving as an adaptor molecule between FKBP12 and the cell proliferation regulator mTOR (mammalian target of rapamycin). To understand the role of dynamics in rapamycin-based immunosuppression and to gain insight into the role of dynamics in the assembly of supramolecular complexes, we used ¹⁵N, ¹³C, and ²H NMR spin relaxation to characterize FKBP12 along the binding coordinate that leads to cell cycle arrest. We show that sequential addition of rapamycin and mTOR leads to incremental rigidification of the FKBP12 backbone on the picosecond-nanosecond timescale. Both binding events lead to perturbation of main-chain and side-chain dynamics at sites distal to the binding interfaces, suggesting tight coupling interactions dispersed throughout the FKBP12-rapamycin interface. Binding of the first molecule, rapamycin, quenches microsecond-millisecond motions of the FKBP12 80's loop. This loop provides much of the surface buried at the protein-protein interface of the ternary complex, leading us to assert that preorganization upon rapamycin binding facilitates binding of the second molecule, mTOR. Widespread microsecond-millisecond motions of the backbone persist in the drug-bound enzyme, and we provide evidence that these slow motions represent coupled dynamics of the enzyme and isomerization of the bound drug. Finally, the pattern of microsecond-millisecond dynamics reported here in the rapamycin complex is dramatically different from the pattern in the complex with the structurally related drug FK506. This raises the important question of how two complexes that are highly isomorphic based on high-resolution static models have such different flexibilities in solution.     

Analysis of the relationship between enzyme activity and its internal motion using nuclear magnetic resonance: 15N relaxation studies of wild-type and mutant lysozyme

A mutant lysozyme where R14 and H15 are deleted together has higher activity and a similar binding ability to an inhibitor, trimer of N-acetylglucosamine ((NAG)3), compared with wild-type lysozyme. Since this has been attributed to intrinsic protein dynamic properties, we investigated the relationship between the activity and the internal motions of proteins. Backbone dynamics of the free and the complex forms with the (NAG)3 have been studied by measurement of the 15N T1 and T2 relaxation rates and NOE determinations at 600 MHz. Analysis of the data using the model-free formalism showed that the generalized order parameters (S2) were almost the same in wild-type and mutant lysozyme in unbound state, indicating that the mutation had little effect on the global internal motions. On the other hand, in the presence of (NAG)3, although some signals located around the active site were broadened or decreased in intensity because of strong perturbation by (NAG)3, there were several residues that showed increased or decreased backbone S2 in the complexed lysozymes. A comparison of the internal motions of the wild-type and mutant complexes showed a number of distinct dynamic differences between them. In particular, many residues located at or near active-site regions (turn 1, strand 2, turn 2 and long loop), displayed greater backbone dynamics reflecting the order parameter in mutant complex relative to mutant free. Furthermore, the Rex values at the loop C-D region, which was considered to be important for enzymatic activity, significantly increased. From these results, it was suggested that variations in the dynamics of these regions may play an important role in the enzyme activity.     

153 Wonderful roles of the entropy in protein dynamics,binding and folding

The entropy, which is central to the second law of thermodynamics, determines that the thermal energy always flows spontaneously from regions of higher temperature to regions of lower temperature. In the protein–solvent thermodynamic system, the entropy is defined as a measure of how evenly the thermal energy would distribute over the entire system (Liu et al., 2012). Such tendency to distribute energy as evenly as possible will reduce the state of order of the initial system, and hence, the entropy can be regarded as an expression of the disorder, or randomness of the system (Yang et al., 2012). For a protein–solvent system under a constant solvent condition, the origin of entropy is the thermal energy stored in atoms, which makes atoms jostle around and bump onto one another, thus leading to vibrations of the covalent bonds connecting two atoms (occurring on the fs timescale) and the rotational and translational motions of amino acid side chain groups (occurring on ps timescale) and water molecules. These motions break the noncovalent bonds around structural regions that are weakly constrained thereby triggering the competitive interactions among residues or between residues and water molecules leading ultimately to the loop motions (occurring on ns timescale) around the protein surface. The loop motions can further transmit either through the water network around the protein surface or via specific structural components (such as the hinge-bending regions) over the entire protein molecule leading to large concerted motions (occurring on μs to s timescales) that are most relevant to protein functions (Amadei, Linssen & Berendsen, 1993; Tao, Rao & Liu, 2010). Thus, the multiple hierarchies of the protein dynamics on distinct timescales (Henzler-Wildman & Kern, 2007) are a consequence of the cascade amplification of the microscopic motions of atoms and groups for which the entropy originating from atomic thermal energy is most fundamental. In the case of protein–ligand binding, the importance of the entropy is embodied in the following aspects. (i) The release of the water molecule kinetic energy (which is a process of the solvent entropy maximization) will cause Brownian motions of individual water molecules which result in strong Brownian bombardments to solute molecules causing molecule wanders/diffusions and subsequent accident contacts/collisions between proteins and ligands. (ii) Such collisions will inevitably cause water molecule displacement and, if the contact interfaces are properly complementary, the requirement to increase the solvent entropy would further displace the water network around the binding interfaces thus leading to the formation the initial protein-ligand complex. (iii) In the initial complex, the loose association of the two partners provide the opportunity for protein to increase conformational entropy, thus triggering the conformational adjustments through competitive interaction between protein residues and ligand, leading ultimately to the formation of tightly associated complex (Liu et al., 2012). In the protein folding process, the first stage, i.e. the rapid hydrophobic collapse (Agashe, Shastry & Udgaonkar, 1995; Dill, 1985), is in fact driven by the effect of the solvent entropy maximization. Specifically, the requirement to maintain as many as possible the dynamic hydrogen bonds among the water molecules will squeeze/sequestrate the hydrophobic amino acid side chains into the interior of the folding intermediates and expose the polar/charged side chains onto the intermediate surface. This will minimize the solvent accessible surface area of the folding intermediates and as thus maximize the entropy of the solvent. The resulting molten globule states (Ohgushi & Wada, 1983) may contain a few secondary structural components and native tertiary contacts, while many native contacts, or close residue–residue interactions present in the native state have not yet formed. However, the nature to increase the protein conformational entropy can trigger a further conformational adjustment process, i.e. the conformational entropy increase breaks the transient secondary or tertiary contacts and triggers the competitive interactions among protein residues and between residues and water. This process may repeat many rounds until the negative enthalpy change resulting from the noncovalent formations can overcompensate for protein conformational entropy loss. In summary, we consider that the tendency to maximize the entropy of the protein–solvent system, which originates from the atomic thermal energy, is the most fundamental driving factor for protein folding, binding, and dynamics, whereas the enthalpy reduction, an opposing factor that tends to make the system become ordered, can compensate for the effect of entropy loss to ultimately allow the system to reach equilibrium at the free energy minima, either global or local.     

Backbone dynamics of a winged helix protein and its DNA complex at different temperatures: changes of internal motions in genesis upon binding to DNA.

The dynamic properties of a winged helix protein, Genesis, and its DNA complex at different temperatures were studied. Due to the complexity of motions, the commonly used model-free formalism could not be used to reflect the dynamic properties. The reduced spectral density function mapping approach was proven to be a useful tool to describe the overall and internal motion of molecules on the picosecond to nanosecond time-scale, and conformational exchanges on the microsecond to millisecond time-scale. The local motions in DNA-free Genesis showed strong temperature dependence and the backbone dynamics of each secondary structural element responds to the temperature change differently, while the Genesis-DNA complex showed more stability with changing the temperatures. Furthermore, each DNA contact sequence of Genesis showed distinct dynamic perturbation after Genesis binds to DNA.     

Quasi‐harmonic fluctuations of two bound peptides

Binding of two short peptides of sequences ASN‐ASP‐MET‐PHE‐ARG‐LEU and LEU‐LEU‐PHE‐MET‐GLN‐HIS and their bound complex structures is studied. Molecular dynamic simulations of the three structures around their respective minimum energy conformations are performed and a quasi‐harmonic analysis is performed over the trajectories generated. The fluctuation correlation matrix is constructed for all C^α‐atoms of the peptides for the full trajectory. The spring constant matrix between peptide C^α‐atoms is obtained from the correlation matrix. Statistical thermodynamics of fluctuations, the energies, entropies, and the free energies of binding are discussed in terms of the quasi‐harmonic model. Sites contributing to the stability of the system and presenting high affinity for binding are determined. Contribution of hydrophobic forces to binding is discussed. Quasi‐harmonic approximation identifies the essential subspace of motions, the important interactions, and binding sites, gives the energetic contribution of each individual interaction, and filters out noise observed in molecular dynamics owing to uncorrelated motions. Comparison of the molecular dynamics results with those of the quasi‐harmonic model shows the importance of entropy change, resulting from water molecules being liberated from the surfaces of the two peptides upon binding. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Evaluation of the Accuracy of Calculation of the Standard Binding Entropy of Molecules from their Average Mobility in Molecular Crystals

One of the main problems in attempts to predict the binding constants of molecules (or free energies of their binding) is the correct evaluation of configurational binding entropy. This evaluation is possible by methods of molecular dynamics simulation, but these simulations require a lot of computational time. Earlier, we have developed an alternative approach which allows the fast calculation of the binding entropy from summarizing the available data on sublimation of crystals. Our method is based on evaluating the mean amplitude of the movements that are restricted in the bound molecule, e.g., in a crystal, but are not restricted in the free state, e.g., in vapor. In this work, it is shown that the standard entropy of binding of molecules by crystals under standard conditions (1 atm, 25°C) can be assessed rather accurately from geometric and physical parameters of the molecule and the average amplitude of the molecule motions in crystals estimated in our previous work.     

NMR studies of Fusarium solani pisi cutinase in complex with phosphonate inhibitors.

The backbone dynamics of Fusarium solani pisi cutinase in complex with a phosphonate inhibitor has been studied by a variety of nuclear magnetic resonance experiments to probe internal motions on different time scales. The results have been compared with dynamical studies performed on free cutinase. In solution, the enzyme adopts its active conformation only upon binding the inhibitor. While the active site Ser120 is rigidly attached to the stable alpha/beta core of the protein, the remainder of the binding site is very flexible in the free enzyme. The other two active site residues Asp175 and His188 as well as the oxyanion hole residues Ser42 and Gln121 are only restrained into their proper positions upon binding of the substrate-like inhibitor. The flap helix, which opens and closes the binding site in the free molecule, is also fixed in the cutinase-inhibitor complex. Our results are in contrast with the X-ray analysis results, namely that in the protein crystal, free cutinase has a well-defined active site and a preformed oxyanion hole and that it does not need any rearrangements to bind its substrate. Our solution studies show that cutinase does need conformational rearrangements to bind its substrate, which may form the rate-limiting step in catalysis.     

The role of slow and fast protein motions in allosteric interactions

Allostery is fundamentally thermodynamic in nature. Long-range communication in proteins may be mediated not only by changes in the mean conformation with enthalpic contribution but also by changes in dynamic fluctuations with entropic contribution. The important role of protein motions in mediating allosteric interactions has been established by NMR spectroscopy. By using CAP as a model system, we have shown how changes in protein structure and internal dynamics can allosterically regulate protein function and activity. The results indicate that changes in conformational entropy can give rise to binding enhancement, binding inhibition, or have no effect in the expected affinity, depending on the magnitude and sign of enthalpy–entropy compensation. Moreover, allosteric interactions can be regulated by the modulation a low-populated conformation states that serve as on-pathway intermediates for ligand binding. Taken together, the interplay between fast internal motions, which are intimately related to conformational entropy, and slow internal motions, which are related to poorly populated conformational states, can regulate protein activity in a way that cannot be predicted on the basis of the protein’s ground-state structure.     

Exploring the role of structure and dynamics in the function of chymotrypsin inhibitor 2

Increasing awareness of the possible role of internal dynamics in protein function has led to the development of new methods for experimentally characterizing protein dynamics across multiple time scales, especially using NMR spectroscopy. A few analyses of the conformational dynamics of proteins ranging from nonallosteric single domains to multidomain allosteric enzymes are now available; however, demonstrating a connection between dynamics and function remains difficult on account of the comparative lack of studies examining both changes in dynamics and changes in function in response to the same perturbations. In previous work, we characterized changes in structure and dynamics on the ps–ns time scale resulting from hydrophobic core mutations in chymotrypsin inhibitor 2 and found that there are moderate, persistent global changes in dynamics in the absence of gross structural changes (Whitley et al., Biochemistry 2008;47:8566–8576). Here, we assay those and additional mutants for inhibitory ability toward the serine proteases elastase and chymotrypsin to determine the effects of mutation on function. Results indicate that core mutation has only a subtle effect on CI2 function. Using chemical shifts, we also studied the effect of complex formation on CI2 structure and found that perturbations are greatest at the complex interface but also propagate toward CI2's hydrophobic core. The structure–dynamics–function data set completed here suggests that dynamics plays a limited role in the function of this small model system, although we do observe a correlation between nanosecond-scale reactive loop motions and inhibitory ability for mutations at one key position in the hydrophobic core.     

Structural aspects of the inhibition of DNase and rRNase colicins by their immunity proteins

Nuclease E colicins that exert their cytotoxic activity through either non-specific DNase or specific rRNase action are inhibited by immunity proteins in a high affinity interaction that gives complete protection to the producing host cell from the deleterious effects of the toxin. Previous X-ray crystallographic analysis of these systems has revealed that in both cases, the immunity protein inhibitor forms its highly stable complex with the enzyme by binding as an exosite inhibitor-adjacent to, but not obscuring, the enzyme active site. The structures of the free E9 DNase domain and its complex with an ssDNA substrate now show that inhibition is achieved without deformation of the enzyme and by occlusion of a limited number of residues of the enzyme critical in recognition and binding of the substrate that are 3' to the cleaved scissile phosphodiester. No sequence or structural similarity is evident between the two classes of cytotoxic domain, and the heterodimer interfaces are also dissimilar. Thus, whilst these structures suggest the basis for specificity in each case, they give few indications as to the basis for the remarkably strong binding that is observed. Structural analyses of complexes bearing single site mutations in the immunity protein at the heterodimer interface reveal further differences. For the DNases, a largely plastic interface is suggested, where optimal binding may be achieved in part by rigid body adjustment in the relative positions of inhibitor and enzyme. For the rRNases, a large solvent-filled cavity is found at the immunity-enzyme interface, suggesting that other considerations, such as that arising from the entropy contribution from bound water molecules, may have greater significance in the determination of rRNase complex affinity than for the DNases.     

Dynamic polymorphism of actin as activation mechanism for cell motility

Actin filament dynamics are crucial in cell motility. Actin filaments, and their bundles, networks, and gels assemble and disassemble spontaneously according to thermodynamic rules. These dynamically changing structures of actin are harnessed for some of its functions in cells. The actin systems respond to external signals, forces, or environments by biasing the fluctuation of actin assembly structures. In this study, dynamic conformation of actin molecules was studied by monitoring conformational dynamics of actin molecules at the single molecule level in real time. Actin conformation spontaneously fluctuates between multiple conformational states. Regarding myosin motility, the dynamic equilibrium of actin conformation was interpreted as between states that activates and inhibits the motility. The binding of myosin to actin filaments activates myosin motility by shifting the conformational fluctuation of actin towards the state that activates the motility. Thus, the activation mechanism based on thermal fluctuation is suggested at molecular level as well as at cellular level.     

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

Chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL-BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo- and holo-cL-BABP but are lost immediately after the start of molecular dynamics. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Substrate binding modifies the hinge bending characteristics of human 3‐phosphoglycerate kinase: A molecular dynamics study

3‐Phosphogycerate kinase (PGK) is a two domain enzyme, with a binding site of the 1,3‐bisphosphoglycerate on the N‐domain and of the ADP on the C‐domain. To transfer a phosphate group the enzyme has to undergo a hinge bending motion from open to closed conformation to bring the substrates to close proximity. Molecular dynamics simulation was used to elucidate the effect of ligand binding onto the domain motions of this enzyme. The simulation results of the apo form indicate a hinge bending motion in the ns timescale while the time period of the hinge bending motion of the complex form is clearly over the 20 ns simulation time. The apo form exhibits several hinge points that contribute to the hinge bending motion while upon binding the ligands, the hinge bending becomes strictly restrained with one dominant hinge point in the vicinity of the substrates. At the same time, ligand binding results in an enhanced correlation of internal domain motions. Proteins 2009. © 2009 Wiley‐Liss, Inc.