Molecular dynamics of the long neurotoxin LSIII

Long neurotoxins bind tightly and specifically to the nicotinic acetylcholine receptor (AChR) in postsynaptic membranes and are useful for exploring the biology of synapses. In crystallographic studies of long neurotoxins the principal binding loop appears disordered, but the NMR solution structure of the long neurotoxin LSIII revealed significant local order, even though the loop is disordered with respect to the globular core. A possible mechanism for conferring global disorder while preserving local order is rigid-body motion of the loop about a hinge region. Here we report investigations of LSIII dynamics based on (13)C(alpha) magnetic relaxation rates and molecular dynamics simulation. The relaxation rates and MD simulation both confirm the hypothesis of rigid-body motion of the loop and place bounds on the extent and time scale of the motion. The bending motion of the loop is slow compared to the rapid fluctuations of individual dihedral angles, reflecting the collective nature and largely entropic free energy profile for hinge bending. The dynamics of the central binding loop in LSIII illustrates two distinct mechanisms by which molecular dynamics directly impacts biological activity. The relative rigidity of key residues involved in recognition at the tip of the central binding loop lowers the otherwise substantial entropic cost of binding. Large excursions of the loop hinge angle may endow the protein with structural plasticity, allowing it to adapt to conformational changes induced in the receptor.     

Elbow‐ and hinge‐bending motions of IgG: Dielectric response and dynamic feature

Immunoglobulin G (IgG) is a Y‐shaped globular protein consisting of two Fab segments connecting to an Fc segment with a flexible hinge region, in which the Fab segments show secondary flexibility at an “elbow” region. In the present work, the hinge‐bending and elbow‐bending motions of aqueous solutions of IgG by microwave dielectric measurements below the freezing point of bulk water was observed. The presence of unfreezable water around the macromolecules reduced the effects of steric hindrance normally generated by ice and enabled the intramolecular motions of IgG. At the same time, the overall IgG molecule rotation was restricted by ice. Papain digestion and reduction of the disulfide linkage at the hinge region was used to generate Fab and Fc fragments. In solutions of these fragments, the dielectric relaxation process of the hinge‐bending motion was absent, although the elbow‐bending motion remained. Three relaxation processes were observed for papain‐digested IgG. The high, middle, and low frequency processes were attributed to unfrozen water, local peptide motions cooperating with bound water, and the elbow‐bending motion, respectively. In the case of the intact IgG, an additional relaxation process due to the hinge‐bending motion was observed at frequencies lower than that of the elbow‐bending motion. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 626–632, 2016.     

Dynamics of transfer RNAs analyzed by normal mode calculation.

Normal mode calculation is applied to tRNAPhe and tRNAAsp, and their structural and vibrational aspects are analyzed. Dihedral angles along the phosphate-ribose backbone (alpha, beta, gamma, epsilon, zeta) and dihedral angles of glycosyl bonds (chi) are selected as movable parameters. The calculated displacement of each atom agrees with experimental data. In modes with frequencies higher than 130 cm-1, the motions are localized around each stem and the elbow region of the L-shape. On the other hand, collective motions such as bending or twisting of arms are seen in modes with lower frequencies. Hinge axes and bend angles are calculated without prior knowledge. Movements in modes with very low frequencies are combinations of hinge bending motions with various hinge axes and bend angles. The thermal fluctuations of dihedral angles well reflect the structural characters of transfer RNAs. There are some dihedral angles of nucleotides located around the elbow region of L-shape, which fluctuate about five to six times more than the average value. Nucleotides in the position seem to be influential in the dynamics of the entire structure. The normal mode calculation seems to provide much information for the study of conformational changes of transfer RNAs induced by aminoacyl-tRNA synthetase or codon during molecular recognition.     

Domain motions and the open-to-closed conformational transition of an enzyme: a normal mode analysis of S-adenosyl-L-homocysteine hydrolase

The structure and fluctuations of the enzyme S-adenosyl-L-homocysteine hydrolase (SAHH) are analyzed in an effort to explain its biological function. Besides the previously identified open structure, characteristic of the substrate-free enzyme, we find two distinct structures in enzyme-inhibitor complexes, the closed and closed-twisted conformers. Both closed conformers differ from the open form by a hinge bending motion of two large domains within each subunit, which isolate the inhibitor bound in the active site from the bulk solvent. The closed-twisted form further differs from the closed form by a rigid body twist of the two-subunit dimers. The local structural fluctuations of SAHH are analyzed by performing block normal mode analysis of the tetrameric enzyme in its three forms. For the open form, we find that the four lowest-frequency normal modes, corresponding to the collective motions of the protein with the largest amplitudes, are essentially combinations of the hinge bending deformations of the individual subunits. Thus, the mechanical properties of the open structure of SAHH lead to the presence of structural fluctuations in the direction of the open-to-closed conformational transition. A candidate for such a motion has been observed in previous fluorescence depolarization studies of the enzyme. Both structural and normal mode analyses indicate that residues 180-190 and 350-356 form hinge regions, connecting large domains which tend to move as rigid bodies in response to interactions with substrate, intermediates, and the product of the enzymatic reactions. We propose that these hinge regions play a crucial role in the enzymatic mechanism of SAHH. In contrast to the open form, normal mode calculations for the closed conformations show strong coupling of the hinge bending motions of the individual subunits to each other and to other low-frequency vibrations. Thus, information about structural changes related to reaction progress in one active site may be mechanically transmitted to other subunits of the protein, explaining the cooperativity found in the enzyme kinetics.     

Chromophore conformation and the evolution of tertiary structural changes in photoactive yellow protein

We use time-resolved crystallography to observe the structural progression of a bacterial blue light photoreceptor throughout its photocycle. Data were collected from 10 ns to 100 ms after photoactivation of the E46Q mutant of photoactive yellow protein. Refinement of transient chromophore conformations shows that the spectroscopically distinct intermediates are formed via progressive disruption of the hydrogen bond network to the chromophore. Although structural change occurs within a few nanoseconds on and around the chromophore, it takes milliseconds for a distinct pattern of tertiary structural change to fully progress through the entire molecule, thus generating the putative signaling state. Remarkably, the coupling between the chromophore conformation and the tertiary structure of this small protein is not tight: there are leads and lags between changes in the conformation of the chromophore and the protein tertiary structure.     

Normal mode analysis of human lysozyme: study of the relative motion of the two domains and characterization of the harmonic motion

A normal mode analysis of human lysozyme has been carried out at room temperature. Human lysozyme is an enzyme constituted of two domains separated by an active site cleft, the motion of which is thought to be relevant for biological function. This motion has been described as a hinge bending motion. McCammon et al. have determined the characteristics of the hinge bending motion but they assumed a prior knowledge of the hinge axis. In this work we propose a method which is free from this assumption and determines the hinge axis and root mean square (rms) rotation angle which give the best agreement with the pattern of changes in all the distances between nonhydrogen atoms in the two domains, obtained by the normal mode analysis. The hinge axis we found is notably different from the one previously determined and goes, roughly, through the C alpha 55 and C alpha 76, i.e., it is located at the base of the beta-sheet of the second domain. The rms value for the rotation angle is also twice as large as the previous one: 3.37 degrees. It is shown that this hinge bending motion provides a fairly good approximation of the dynamics of human lysozyme and that the normal mode with the lowest frequency has a dominating contribution to this hinge bending motion. A study of the accessible surface area of the residues within the cleft reveals that the motion does not result in a better exposure to the solvent of these residues. A characterization of the thermally excited state (under the hypothesis of the harmonicity of the potential energy surface) has been done using the concept of topology of atom packing. Under this hypothesis the thermal fluctuations result only in a small change of the topology of atom packing, leading therefore to nearly elastic deformations of the protein.     

Fine-tuning function: correlation of hinge domain interactions with functional distinctions between LacI and PurR

LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function.     

Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 Å resolution

The structure of a triclinic complex between liver alcohol dehydrogenase, reduced coenzyme NADH, and the inhibitor dimethylsulfoxide has been determined to 2.9 Å resolution using isomorphous replacement methods. The heavy-atom positions were derived by molecular replacement methods using phase angles derived from a model of the orthorhombic apoenzyme structure previously determined to 2.4 Å resolution. A model of the present holoenzyme molecule was built on a Vector General 3400 display system using the RING system of programs. This model gave a crystallographic R-value of 37.9%.There are extensive conformational differences between the protein molecules in the two forms. The conformational change involves a rotation of 7.5 ° of the catalytic domains relative to the coenzyme binding domains. A hinge region for this rotation is defined within a hydrophobic core between two helices. The internal structures of the domains are preserved with the exception of a movement of a small loop in the coenzyme binding domain. A cleft between the domains is closed by this coenzyme-induced conformational change, making the active site less accessible from solution and thus more hydrophobic.The two crystallographically independent subunits are very similar and bind both coenzyme and inhibitor in an identical way within the present limits of error. The coenzyme molecule is bound in an extended conformation with the two ends in hydrophobic crevices on opposite sides of the central pleated sheet of the coenzyme binding domain. There are hydrogen bonds to oxygen atoms of the ribose moities from Asp223, Lys228 and His51. The pyrophosphate group is in contact with the side-chains of Arg47 and Arg369.No new residues are brought into the active site compared to the apoenzyme structure. The active site zinc atom is close to the hinge region, where the smallest structural changes occur. Small differences in the co-ordination geometry of the ligands Cys46, His67 and Cysl74 are not excluded and may account for the ordered mechanism. The oxygen atom of the inhibitor dimethylsulfoxide is bound directly to zinc confirming the structural basis for the suggested mechanism of action based on studies of the apoenzyme structure.     

Characterizing a Histidine Switch Controlling pH-Dependent Conformational Changes of the Influenza Virus Hemagglutinin

During the fusion of the influenza virus to the host cell, bending of the HA2 chain of hemagglutinin into a hairpin-shaped structure in a pH-dependent manner facilitates the fusion of the viral envelope and the endosomal membrane. To characterize the structural and dynamical responses of the hinge region of HA2 to pH changes and examine the role of a conserved histidine in this region (the hinge histidine), we have performed an extensive set of molecular dynamics (MD) simulations of 26-residue peptides encompassing the hinge regions of several hemagglutinin subtypes under both neutral and low pH conditions, modeled by the change of the protonation state of the hinge histidine. More than 70 sets of MD simulations (collectively amounting to 25.1 μs) were performed in both implicit and explicit solvents to study the effect of histidine protonation on structural dynamics of the hinge region. In both explicit and implicit solvent simulations, hinge bending was consistently observed upon the protonation of the histidine in all the simulations starting with an initial straight helical conformation, whereas the systems with a neutral histidine retained their primarily straight conformation throughout the simulations. Conversely, the MD simulations starting from an initially bent conformation resulted in the formation of a straight helical structure upon the neutralization of the hinge histidine, whereas the bent structure was maintained when the hinge histidine remained protonated. Finally, mutation of the hinge histidine to alanine abolishes the bending response of the peptide altogether. A molecular mechanism based on the interaction of the hinge histidine with neighboring acidic residues is proposed to be responsible for its role in controlling the conformation of the hinge. We propose that this might present a common mechanism for pH-controlled structural changes in helical structures when histidines act as the pH sensor.     

Characterizing a Histidine Switch Controlling pH-Dependent Conformational Changes of the Influenza Virus Hemagglutinin

During the fusion of the influenza virus to the host cell, bending of the HA2 chain of hemagglutinin into a hairpin-shaped structure in a pH-dependent manner facilitates the fusion of the viral envelope and the endosomal membrane. To characterize the structural and dynamical responses of the hinge region of HA2 to pH changes and examine the role of a conserved histidine in this region (the hinge histidine), we have performed an extensive set of molecular dynamics (MD) simulations of 26-residue peptides encompassing the hinge regions of several hemagglutinin subtypes under both neutral and low pH conditions, modeled by the change of the protonation state of the hinge histidine. More than 70 sets of MD simulations (collectively amounting to 25.1 μs) were performed in both implicit and explicit solvents to study the effect of histidine protonation on structural dynamics of the hinge region. In both explicit and implicit solvent simulations, hinge bending was consistently observed upon the protonation of the histidine in all the simulations starting with an initial straight helical conformation, whereas the systems with a neutral histidine retained their primarily straight conformation throughout the simulations. Conversely, the MD simulations starting from an initially bent conformation resulted in the formation of a straight helical structure upon the neutralization of the hinge histidine, whereas the bent structure was maintained when the hinge histidine remained protonated. Finally, mutation of the hinge histidine to alanine abolishes the bending response of the peptide altogether. A molecular mechanism based on the interaction of the hinge histidine with neighboring acidic residues is proposed to be responsible for its role in controlling the conformation of the hinge. We propose that this might present a common mechanism for pH-controlled structural changes in helical structures when histidines act as the pH sensor.     

Hinge sequences as signaling agents?

We report an unexpected finding of common structural principles in two unrelated signaling systems: the FAS death domain transformation that initializes the extrinsic apoptotic pathway and signaling by calmodulin bending. The location and design of the hinge is postulated to be a general principle for creating potential signaling event. We suggest that already existing tool can predict the existence of such a hinge and formulate the hypothesis that the internal instabilities designed into the hinge sequences are necessary devices in effective signaling events.     

Structure, dynamics, and topological orientation of the polyether, ionophore antibiotic monensin, in a micellar environment

The structure and dynamics of the ionophoric antibiotic monensin in the presence of micelles have been determined. The conformation of monensin was derived from 50 nuclear Overhauser enhancement (NOE) derived distance restraints and metric-matrix based distance geometry calculations. The conformation was further refined with extensive NOE restrained molecular dynamics simulations carried out in a biphasic simulation cell. From the addition of doxylstearate and monitoring of the induced relaxation of the nmr signals, the relative topological orientation of the molecule within the micelle was ascertained. The results indicate two dihedral angles that act as hinge regions allowing the molecule to adopt a wide range of conformations. Considering the biological activity of monensin, i.e., the capture and transport of cations across cell membranes, an open and closed form of monensin have been postulated. The identification of these hinge regions, which are only observed in the membrane-like environment of the detergent micelles, provides insight into the mechanism of action and can serve as targets for modification to alter the biological profile of monensin.     

Identification of Allosteric Disulfides from Prestress Analysis

Disulfide bonds serve to form physical cross-links between residues in protein structures, thereby stabilizing the protein fold. Apart from this purely structural role, they can also be chemically active, participating in redox reactions, and they may even potentially act as allosteric switches controlling protein functions. Specific types of disulfide bonds have been identified in static protein structures from their distinctive pattern of dihedral bond angles, and the allosteric function of such bonds is purported to be related to the torsional strain they store. Using all-atom molecular-dynamics simulations for ∼700 disulfide bonded proteins, we analyzed the intramolecular mechanical forces in 20 classes of disulfide bonds. We found that two particular classes, the −RHStaple and the −/+RHHook disulfides, are indeed more stressed than other disulfide bonds, but the stress is carried primarily by stretching of the S-S bond and bending of the neighboring bond angles, rather than by dihedral torsion. This stress corresponds to a tension force of magnitude ∼200 pN, which is balanced by repulsive van der Waals interactions between the cysteine Cα atoms. We confirm stretching of the S-S bond to be a general feature of the −RHStaples and the −/+RHHooks by analyzing ∼20,000 static protein structures. Given that forced stretching of S-S bonds is known to accelerate their cleavage, we propose that prestress of allosteric disulfide bonds has the potential to alter the reactivity of a disulfide, thereby allowing us to readily switch between functional states.     

Buried waters and internal cavities in monomeric proteins

We have analyzed the buried water molecules and internal cavities in a set of 75 high-resolution, nonhomologous, monomeric protein structures. The number of hydrogen bonds formed between each water molecule and the protein varies from 0 to 4, with 3 being most common. Nearly half of the water molecules are found in pairs or larger clusters. Approximately 90% are shown to be associated with large cavities within the protein, as determined by a novel program, PRO_ACT. The total volume of a protein's large cavities is proportional to its molecular weight and is not dependent on structural class. The largest cavities in proteins are generally elongated rather than globular. There are many more empty cavities than hydrated cavities. The likelihood of a cavity being occupied by a water molecule increases with cavity size and the number of available hydrogen bond partners, with each additional partner typically stabilizing the occupied state by 0.6 kcal/mol.     

What is the relationship between the global structures of apo and holo proteins?

It is well known that ligand binding and release may induce a wide range of structural changes in a receptor protein, varying from small movements of loops or side chains in the binding pocket to large‐scale domain hinge‐bending and shear motions or even partial unfolding that facilitates the capture and release of a ligand. An interesting question is what in general are the conformational changes triggered by ligand binding? The aim of this work is analyze the magnitude of structural changes in a protein resulting from ligand binding to assess if the state of ligand binding needs to be included in template‐based protein structure prediction algorithms. To address this issue, a nonredundant dataset of 521 paired protein structures in the ligand‐free and ligand‐bound form was created and used to estimate the degree of both local and global structure similarity between the apo and holo forms. In most cases, the proteins undergo relatively small conformational rearrangements of their tertiary structure upon ligand binding/release (most root‐mean‐square‐deviations from native, RMSD, are <1 Å). However, a clear difference was observed between single‐ and multiple‐domain proteins. For the latter, RMSD changes greater than 1 Å and sometimes larger were found for almost 1/3 of the cases; these are mainly associated with large‐scale hinge‐bending movements of entire domains. The changes in the mutual orientation of individual domains in multiple‐domain proteins upon ligand binding were investigated using a mechanistic model based on mass‐weighted principal axes as well as interface buried surface calculations. Some preferences toward the anticipated mechanism of protein domain movements are predictable based on the examination of just the ligand‐free structural form. These results have applications to protein structure prediction, particularly in the context of protein domain assembly, if additional information concerning ligand binding is exploited. Proteins 2008. © 2007 Wiley‐Liss, Inc.     

Structural insights into the molecular mechanism of H‐NOX activation

Nitric oxide (NO) signaling in mammals controls important processes such as smooth muscle relaxation and neurotransmission by the activation of soluble guanylate cyclase (sGC). NO binding to the heme domain of sGC leads to dissociation of the iron–histidine (Fe–His) bond, which is required for enzyme activity. The heme domain of sGC belongs to a larger class of proteins called H‐NOX (Heme‐Nitric oxide/OXygen) binding domains. Previous crystallographic studies on H‐NOX domains demonstrate a correlation between heme bending and protein conformation. It was unclear, however, whether these structural changes were important for signal transduction. Subsequent NMR solution structures of H‐NOX proteins show a conformational change upon disconnection of the heme and proximal helix, similar to those observed in the crystallographic studies. The atomic details of these conformational changes, however, are lacking in the NMR structures especially at the heme pocket. Here, a high‐resolution crystal structure of an H‐NOX mutant mimicking a broken Fe–His bond is reported. This mutant exhibits specific changes in heme conformation and major N‐terminal displacements relative to the wild‐type H‐NOX protein. Fe–His ligation is ubiquitous in all H‐NOX domains, and therefore, the heme and protein conformational changes observed in this study are likely to occur throughout the H‐NOX family when NO binding leads to rupture of the Fe–His bond.     

Some Thoughts about Bond Energies,Bond Lengths,and Force Constants

The bond energy (BE) of a polyatomic molecule cannot be measured and, therefore, determination of BEs can only be done within a model using a set of assumptions. The bond strength is reflected by the intrinsic BE (IBE), which is related to the intrinsic atomization energy (IAE) and which represents the energy of dissociation under the provision that the degree of hybridization is maintained for all atoms of the molecule. IBE and BE differ in the case of CC and CH bonds by the promotion, the hybridization, and the charge reorganization energy of carbon. Since the latter terms differ from molecule to molecule, IBE and BE are not necessarily parallel and the use of BEs from thermochemical models can be misleading. The stretching force constant is a dynamical quantity and, therefore, it is related to the bond dissociation energy (BDE). Calculation and interpretation of stretching force constants for local internal coordinate modes are discussed and it is demonstrated that the best relationship between BDEs and stretching force constants is obtained within the model of adiabatic internal modes. The valence stretching force constants are less suitable since they are related to an artificial bond dissociation process with geometrical relaxation effects suppressed, which leads to an intrinsic BDE (IBDE). In the case of AX_n molecules, symmetric coordinates can be used to get an appropriate stretching force constant that is related to the BE. However, in general stretching force constants determined for symmetry coordinates do not reflect the strength of a particular bond since the related dissociation processes are strongly influenced by the stability of the products formed.     

Refined crystal structure of carboxypeptidase A at 1.54 A resolution

The crystal structure of bovine carboxypeptidase A (Cox) has been refined at 1.54 A resolution using the restrained least-squares algorithm of Hendrickson & Konnert (1981). The crystallographic R factor (formula; see text) for structure factors calculated from the final model is 0.190. Bond lengths and bond angles in the carboxypeptidase A model have root-mean-square deviations from ideal values of 0.025 A and 3.6 degrees, respectively. Four examples of a reverse turn like structure (the "Asx" turn) requiring an aspartic acid or asparagine residue are observed in this structure. The Asx turn has the same number of atoms as a reverse turn, but only one peptide bond, and the hydrogen bond that closes the turn is between the Asx side-chain CO group and a main-chain NH group. The distributions of CO-N and NH-O hydrogen bond angles in the alpha-helices and beta-sheet structures of carboxypeptidase A are centered about 156 degrees. A total of 192 water molecules per molecule of enzyme are included in the final model. Unlike the hydrogen bonding geometry observed in the secondary structure of the enzyme, the CO-O(wat) hydrogen bond angle is distributed about 131 degrees, indicating the role of the lone pair electrons of the carbonyl oxygen in the hydrogen bond interaction. Twenty four solvent molecules are observed buried within the protein. Several of these waters are organized into hydrogen-bonded chains containing up to five waters. The average temperature factor for atoms in carboxypeptidase A is 8 A2, and varies from 5 A2 in the center of the protein, to over 30 A2 at the surface.