Deoxymyoglobin studied by the conformational normal mode analysis. I. Dynamics of globin and the heme-globin interaction

Dynamic properties of deoxymyoglobin are studied theoretically by the analysis of conformational fluctuations. Root-mean-square atomic fluctuations and distance fluctuations between different segments reveal the mechanical construction of the molecule. Eight alpha-helices behave as relatively rigid bodies and corner regions are more flexible, showing larger fluctuations. More particularly, corner regions EF and GH are specific in that flanking alpha-helices extend their rigidity up to a point in the corner region and the two rigid segments are connected flexibly at that point. The FG corner is exceptional. A segment from the F helix to the beginning of the G helix, in which the FG corner is included, becomes relatively rigid by means of strong interactions with the heme group. The whole myoglobin molecule is divided into two large units of motion, one extending from the B to the E helix, and the other from the F to the H helix. These two units are connected covalently by the EF corner. However, dynamic interactions between these two units take place mainly through contacts between helices B and G and not through the EF corner. From correlation coefficients between fluctuational motions of residues and the heme group, 55 residues are identified as having strong dynamic interactions with the heme moiety. Among them, 18 residues in the three segments, one consisting of residues from the C helix to the CD corner, a second consisting of the E helix, and a third from the F helix to the beginning of the G helix, are in close contact with the heme group. Twenty-two of the 55 residues are within four residues of the 18 residues in their sequential residue number and are more than 3 A away from the heme group. The other 15 residues are located further in the sequential residue number and are all found in helices A and H. They are more than 6 A away from the heme group. By the use of correlation coefficients of fluctuations between residues, it is found that dynamic interaction with the heme group is transmitted to the A helix and the beginning of the H helix in the direction Leu(E15)----[Val(All) and Trp(A12)]. The transmission to the C-terminal end of the H helix is mediated by a long segment, from the end of the EF corner to the beginning of the G helix, that lies on the heme group and has close contacts over a wide range.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dynamical structure of transfer RNA studied by normal mode analysis

The internal motion of yeast phenylalanine transfer RNA is studied by normal mode analysis in extended dihedral angle space, in which the flexibility of five-membered ribose rings is treated faithfully by introducing a variable for its pseudo-rotational motion. Analysis of global molecular motions reveals that the molecule is very soft. We show that this softness comes not from the property of the “material” comprising the molecule but from its slender shape. Analysis of thermal distance fluctuations reveals that this molecule can be regarded as consisting dynamically of three blocks. Thermal fluctuations of the mainchain dihedral angles show rigidity of the anticodon region. They also show flexibility of regions around nonstacking bases. Base-stacking interactions cause suppression of the correlated functions of mainchain dihedral angles beyond a ribose ring. We analyze the thermal fluctuation of parameters describing the positions of two adjacent bases. Fluctuations of relative translational parameters in the anticodon and acceptor stem regions are found to be larger than those in other stem regions. The relative translational motions cause the two stem regions to undergo global twisting and bending motions. We show that the role of pseudo-rotational motion of sugars is important in regions around bases which are involved in nonregular interactions. Received: 29 October 1998 / Revised version: 8 February 1999 / Accepted: 11 February 1999     

Dynamic structure of subtilisin-eglin c complex studied by normal mode analysis

Normal mode analysis of subtilisin-eglin c complex was performed to investigate the dynamics at the interface between the enzyme and the inhibitor. The internal motions of the complex calculated from the normal modes were divided into three parts: the internal motions changing the shape of each molecule, the external rigid-body motions changing their mutual dispositions, and the coupling between the internal and external motions. From the results of the analysis, the following characteristic features were found in the dynamics at the interface regions: 1) negative correlation between the internal and external motions within each molecule, and 2) positive correlation between the external motions of the two molecules. The former decreases the apparent amplitudes of motions at the interface. The latter minimizes the interference between individual motions of the two molecules. These dynamic characteristics allow the enzyme and the inhibitor to move as freely as possible. This finding suggests that the experimental evidence of the large entropy gain on binding should be attributed not only to strong hydrophobic interactions, but also to the dynamic structure of the complex, which is found to minimize an unavoidable loss of the conformational entropy on binding. Proteins 32:324–333, 1998. © 1998 Wiley-Liss, Inc.     

Ligand-induced conformational change of a protein reproduced by a linear combination of displacement vectors obtained from normal mode analysis

The conformational change of a protein upon ligand binding was examined by normal mode analysis (NMA) based on an elastic-network model (ENM) for a full-atom system using dihedral angles as independent variables. Specifically, we investigated the extent to which conformational change vectors of atoms from an apo form to a holo form of a protein can be represented by a linear combination of the displacement vectors of atoms in the apo form calculated for the lowest-frequency m normal modes (m=1, 2,…, 20). In this analysis, the latter vectors were best fitted to the former ones by the least-squares method. Twenty-two paired proteins in the holo and apo forms, including three dimer pairs, were examined. The results showed that, in most cases, the conformational change vectors were reproduced well by a linear combination of the displacement vectors of a small number of low-frequency normal modes. The conformational change around an active site was reproduced as well as the entire conformational change, except for some proteins that only undergo significant conformational changes around active sites. The weighting factors for 20 normal modes optimized by the least-squares fitting characterize the conformational changes upon ligand binding for these proteins. The conformational changes sampled around the apo form of a protein by the linear combination of the displacement vectors obtained by ENM-based NMA may help solve the flexible-docking problem of a protein with another molecule because the results presented herein suggest that they have a relatively high probability of being involved in an actual conformational change.     

Fluorescence-monitored conformational change on the 3'-end of tRNA upon aminoacylation.

Fluorescent tRNAs species with formycine in the 3'-terminal position (tRNA-CCF) were derived from Escherichia coli tRNA(Val). Thermus thermophilus tRNA(Aap) and Thermus thermophilus tRNA(Phe). The fluorescence of formycine was used to monitor the conformational changes at the 3'-terminus of tRNA caused by aminoacylation and hydrolysis of aminoacyl residue from aminoacyl-tRNAs. An increase of about 15% in the fluorescence intensity was observed after aminoacylation of the three tRNA-CCF. This change in fluorescence amplitude that is reversed by hydrolysis of the aminoacyl residue, does not depend on the structure of the amino acid or tRNA sequence. A local conformational change at the 3'-terminal formycine probably involving a partial destacking of the base moiety in the ACCF end takes place as a consequence of aminoacylation. A structural change at the 3'-terminus of tRNA induced by attachment and detachment of the acyl residue may be important in controlling the substrate/product relationship in reactions in which tRNA participates during protein biosynthesis.     

Modeling protein conformational transitions by a combination of coarse-grained normal mode analysis and robotics-inspired methods

Background

Obtaining atomic-scale information about large-amplitude conformational transitions in proteins is a challenging problem for both experimental and computational methods. Such information is, however, important for understanding the mechanisms of interaction of many proteins.

Methods

This paper presents a computationally efficient approach, combining methods originating from robotics and computational biophysics, to model protein conformational transitions. The ability of normal mode analysis to predict directions of collective, large-amplitude motions is applied to bias the conformational exploration performed by a motion planning algorithm. To reduce the dimension of the problem, normal modes are computed for a coarse-grained elastic network model built on short fragments of three residues. Nevertheless, the validity of intermediate conformations is checked using the all-atom model, which is accurately reconstructed from the coarse-grained one using closed-form inverse kinematics.

Results

Tests on a set of ten proteins demonstrate the ability of the method to model conformational transitions of proteins within a few hours of computing time on a single processor. These results also show that the computing time scales linearly with the protein size, independently of the protein topology. Further experiments on adenylate kinase show that main features of the transition between the open and closed conformations of this protein are well captured in the computed path.

Conclusions

The proposed method enables the simulation of large-amplitude conformational transitions in proteins using very few computational resources. The resulting paths are a first approximation that can directly provide important information on the molecular mechanisms involved in the conformational transition. This approximation can be subsequently refined and analyzed using state-of-the-art energy models and molecular modeling methods.

     

Grb2 adaptor undergoes conformational change upon dimerization

Grb2 is an adaptor protein that couples activated receptor tyrosine kinases to downstream effector molecules such as Ras and Akt. Despite being a central player in mitogenic signaling and a target for therapeutic intervention, the role of Grb2 oligomerization in cellular signaling is not well understood. Here, using the techniques of size-exclusion chromatography, mass spectrometry, analytical ultra-centrifugation and isothermal titration calorimetry, we demonstrate that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven without an unfavorable enthalpic change at physiological temperatures. Our data indicate that enthalpy and entropy of dimer dissociation are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the overall free energy. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change derived is much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon Grb2 dimerization, implying that Grb2 monomers undergo conformational rearrangement upon dimerization. 3D structural models of Grb2 dimer and monomers suggest strongly that such conformational rearrangement upon dimerization may arise from domain swapping. Taken together, our study provides novel insights into the role of Grb2 as an adaptor in cellular signaling circuitry and how Grb2 dimerization may impart high fidelity in signal transduction as well as lead to rapid signal amplification upon receptor stimulation.     

Experimental verification of conformational variation of human fatty acid synthase as predicted by normal mode analysis

Fatty acid synthase (FAS) is a 550 kDa homodimeric enzyme with multiple functional and structural domains. Normal mode analysis of a previously determined 19 A structure of FAS suggested that this enzyme might assume different conformational states with several distinct hinge movements. We have used a simultaneous multiple-model refinement method to search for the presence of the structural conformers from the electron images of FAS. We have demonstrated that the resulting models observed in the electron images are consistent with the predicted conformational changes. This technique demonstrates the potential of the combination of normal mode analysis with multiple model refinement to elucidate the multiple conformations of flexible proteins. Since each of these structures is based on a more homogeneous particle set, this technique has the potential, provided that sufficient references are used, to improve the resolution of the final reconstructions of single particles from electron cryomicroscopy.     

Simplified normal mode analysis of conformational transitions in DNA-dependent polymerases: the elastic network model

The Elastic Network Model is used to investigate the open/closed transition in all DNA-dependent polymerases whose structure is known in both forms. For each structure the model accounts well for experimental crystallographic B-factors. It is found in all cases that the transition can be well described with just a handful of the normal modes. Usually, only the lowest and/or the second lowest frequency normal modes deduced from the open form give rise to calculated displacement vectors that have a correlation coefficient larger than 0.50 with the observed difference vectors between the two forms. This is true for every structural class of DNA-dependent polymerases where a direct comparison with experimental structural data is available. In cases where only one form has been observed by X-ray crystallography, it is possible to make predictions concerning the possible existence of another form in solution by carefully examining the vector displacements predicted for the lowest frequency normal modes. This simple model, which has the advantage to be computationally inexpensive, could be used to design novel kind of drugs directed against polymerases, namely drugs preventing the open/closed transition from occurring in bacterial or viral DNA-dependent polymerases.     

Electric field-induced conformational change of poly(L-lysine) studied by transient electric birefringence

Transient electric birefringence measurements on poly(L -lysine hydrobromide) in methanol–water mixtures have been carried out at various solvent compositions in the vicinity of the helix–coil transition region (from 87 to 98 vol % methanol). Anomalous birefringence transients were observed between 90 and 95 vol % methanol above a threshold field strength. A distinct difference between the responses to weak and strong electric fields was noticed over a narrow range of the solvent composition. The effects of polymer concentration and temperature on the field-strength dependence of the birefringence were studied at a solvent composition of 90 vol % methanol where the anomalous transients appeared most clearly. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different concentrations and temperatures could be superimposed by shifting them horizontally along the abscissa. The threshold field strength which was determined from the shift factor decreased with decreasing concentration. The results provide further evidence that strong electric fields can cause a helix–coil transition in this system under favorable conditions.     

Elastic network model-based normal mode analysis reveals the conformational couplings in the tripartite AcrAB-TolC multidrug efflux complex

The AcrAB-TolC drug efflux system, energized by proton movement down the transmembrane electrochemical gradient, is responsible for the resistance of the organism to a wide range of drugs. Experimental data suggest functional roles of each part of the assembly, but the detailed working mechanism of this machinery remains elusive. We used elastic network-based normal mode analysis (NMA) to explore the conformational dynamics of the AcrAB-TolC complex. The intrinsic flexibilities of the pore domain in AcrB monomer conform to the previously proposed three-step functionally rotating mechanism for asymmetric AcrB trimer. Conformational couplings across monomers in the AcrB trimer were observed, and the coupling between the transmembrane domain and the other parts of AcrB are strengthened through trimeric assembly. In the tripartite AcrAB-TolC assembly obtained through molecular docking, concerted motions were observed not only at the direct contact interfaces between various components but also between distant parts of the whole complex. The presence of AcrA was shown to significantly strengthen the motional couplings between AcrB and TolC. Overall, NMA revealed an allosteric network in the AcAB-TolC efflux system, which provides hints to our understanding of its detailed working mechanism.     

A plausible sequence of the conformational changes of hemoglobin induced by oxygenation.

Recent experimental data of oxygen equilibrium constants of human adult hemoglobin, which are measured over a wide range of oxygen pressures, are analyzed successfully from the viewpoint that the change in the molecular structure of hemoglobin induced by oxygenation is considered individually at each stage of oxygenation. Then, a simple phenomenological rule, which explains quantitatively the values of the four Adair constants with only three parameters, is found for hemoglobin under normal physiological conditions. The temperature dependence of these parameters suggests a sequence of the conformational changes such that until the third stage of oxygenation the conformational changes occur within the deoxy quaternary structure and at the fourth stage of oxygenation the deoxy quaternary structure is altered to the oxy one. The effects of pH and phosphate compounds on the Adair constants are discussed, and a possible modification and extension of the rule is suggested. The connection between the rule and the molecular structures of deoxy- and oxyhemoglobin is also discussed.     

The active ring-like structure of SecA revealed by electron crystallography: conformational change upon interaction with SecB

SecA is a multifunctional protein involved in protein translocation in bacteria. The structure of SecA on membrane is dramatically altered compared with that in solution, accompanying with functional changes. We previously reported the formation of a novel ring-like structure of SecA on lipid layers, which may constitute part of the preprotein translocation channel. In the present work, two-dimensional crystallization of Escherichia coli SecA on lipid monolayers was performed to reveal the structural details of SecA on lipid layers and to investigate its function. The 2D crystals composed of ring-like structures were obtained by specific interaction between SecA and negatively charged lipid. The 2D projection map and 3D reconstruction from negative stained 2D crystals exhibited a distinct open channel-like structure of SecA, with an outer diameter of 7 nm and an inner diameter of 2 nm, providing the structural evidence for SecA importance in forming the part of the translocation channel. This pore structure is altered after transferring crystals to the SecB solution, indicating that the lipid-specific SecA structure has the SecB binding activity. The strategy developed here provides a promising technique for studying structure of SecA complex with its ligand on membrane.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

10-Formyltetrahydrofolate synthetase. Evidence for a conformational change in the enzyme upon binding of tetrahydropteroylpolyglutamates

The 10-formyltetrahydrofolate synthetase domain of the trifunctional enzyme C1-tetrahydrofolate synthase appears to undergo a conformational change in the presence of tetrahydropteroylpolyglutamates, MgATP, and ammonium ion. The binding of these ligands increases the denaturation temperature of the enzyme by 12 degrees C, abolishes the cold lability of the enzyme, and alters its susceptibility to digestion by chymotrypsin. The results suggest that a conformational change is dependent upon binding of the third glutamate residue of tetrahydropteroylpolyglutamates and the beta-phosphoryl group of MgATP. The Km values for MgATP and formate are lowered 3.6- and 520-fold, respectively, when tetrahydropteroyltriglutamate is used as the substrate in place of tetrahydropteroylmonoglutamate. A sensitive coupled assay involving C1-tetrahydrofolate synthase and serine hydroxymethyltransferase was developed to determine the activity of 10-formyltetrahydrofolate synthetase. The assay gives linear rates with the tetrahydropteroylpolyglutamates as substrates but not with the monoglutamate form.     

Evidence for a phosphorylation-induced conformational change in phospholamban cytoplasmic domain by CD analysis.

Phospholamban (PLB), an integral membrane protein of cardiac sarcoplasmic reticulum (SR), is described as the regulator of the Ca(2+)-ATPase pump, via its phosphorylation-dephosphorylation of Ser-16. Recently it has been shown that a direct interaction between the N-terminal hydrophilic domain of PLB and Ca(2+)-ATPase may be one of the mechanisms of regulation. In order to show that this interaction could be modulated by a phosphorylation-induced conformational change in PLB, we ran CD studies on the synthetic peptide PLB(2-33) in its phosphorylated and non-phosphorylated forms, at various pHs, concentrations and in the absence or presence of trifluoroethanol. The results show a clear difference in structure of the phosphorylated and non-phosphorylated peptide.