Conformational flexibility of aqueous monomeric and dimeric insulin: a molecular dynamics study

A series of molecular dynamics simulations have been used to investigate the nature of monomeric and dimeric insulin in aqueous solution. It is shown that in the absence of crystal contacts both monomeric and dimeric insulin have a high degree of intrinsic flexibility. Neither of the two monomer conformations of 2Zn crystalline insulin appears to be favored in solution nor is the asymmetry of the crystal dimer reduced in the absence of crystal contacts. A shift is observed in the relative positions of molecules 1 and 2 in the dimer compared with that found in the crystal, which may have consequences for the prediction of the effects of mutants in the monomer-monomer interface designed to alter the self-association properties of insulin.     

Structure of a variant of lac repressor with increased thermostability and decreased affinity for operator

A single amino acid substitution, K84L, in the Escherichia coli lac repressor produces a protein that has substantially increased stability compared to wild-type. However, despite the increased stability, this altered tetrameric repressor has a tenfold reduced affinity for operator and greatly decreased rate-constants of inducer binding as well as a reduced phenotypic response to inducer in vivo. To understand the dramatic increase in stability and altered functional properties, we have determined the X-ray crystal structures of a dimeric repressor with and without the K84L substitution at resolutions of 1.7 and 3.0 A, respectively. In the wild-type dimer, K84-11, Lys84 forms electrostatic interactions at the monomer-monomer interface and is partially exposed to solvent. In the K84L-11 substituted protein there is reorientation of the N-subdomains, which allows the leucine to become deeply buried at the monomer-monomer interface. This reorientation of the N-subdomains, in turn, results in an alteration of hydrogen bonding, ion pairing, and van der Waals interactions at the monomer-monomer interface. The lysine residue at position 84 appears to exert its key effects by destabilizing the "optimal" conformation of the repressor, effectively loosening the dimer interface and allowing the repressor to adopt the conformations necessary to function as a molecular switch.     

Evolutionary conservation of RecA genes in relation to protein structure and function.

Functional and structural regions inferred from the Escherichia coli R ecA protein crystal structure and mutation studies are evaluated in terms of evolutionary conservation across 63 RecA eubacterial sequences. Two paramount segments invariant in specific amino acids correspond to the ATP-binding A site and the functionally unassigned segment from residues 145 to 149 immediately carboxyl to the ATP hydrolysis B site. Not only are residues 145 to 149 conserved individually, but also all three-dimensional structural neighbors of these residues are invariant, strongly attesting to the functional or structural importance of this segment. The conservation of charged residues at the monomer-monomer interface, emphasizing basic residues on one surface and acidic residues on the other, suggests that RecA monomer polymerization is substantially mediated by electrostatic interactions. Different patterns of conservation also allow determination of regions proposed to interact with DNA, of LexA binding sites, and of filament-filament contact regions. Amino acid conservation is also compared with activities and properties of certain RecA protein mutants. Arginine 243 and its strongly cationic structural environment are proposed as the major site of competition for DNA and LexA binding to RecA. The conserved acidic and glycine residues of the disordered loop L1 and its proximity to the RecA acidic monomer interface suggest its involvement in monomer-monomer interactions rather than DNA binding. The conservation of various RecA positions and regions suggests a model for RecA-double-stranded DNA interaction and other functional and structural assignments.     

Envisioning the Loop Movements and Rotation of the Two Subdomains of Dihydrofolate Reductase by Elastic Normal Mode Analysis

Abstract

Normal mode analysis, using the elastic network model, has been employed to envision the low frequency normal mode motion trends in the structures of five intermediates and a transition state in the kinetic pathway of E. coli dihydrofolate reductase (DHFR). Five of the reaction pathway analog structures and a crystal structure resembling the transition state, using X-ray analyses determined by Kraut et al., have been adapted as structural models. The motions that poise pathways of the M20 loop transitions from closed to occluded conformations and sub domain rotation to close the substrate cleft, have been predicted and envisioned for the first time by this study. Pathway entries to the movement of the substrate binding cleft helices are also envisioned. These motions play roles in transition structure stabilization and in regulating the release of the product tetrahydrofolate (THF). The motions observed push the ground state conformation of each intermediate towards a higher energy sub state conformation. A set of conserved residues involved in the catalytic reactions and conformational changes, previously studied by kinetic, theoretical and NMR, have been analyzed. The importance of these motions in terms of protein dynamics are revealed and envisioned by the normal mode analysis. Additional residues are proposed as candidates for further study of their potential promotional function.     

Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The solution structure of the B9(Asp) mutant of human insulin has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy. Thirty structures were calculated by distance geometry from 451 interproton distance restraints based on intra-residue, sequential and long-range nuclear Overhauser enhancement data, 17 restraints on phi torsional angles obtained from 3JH alpha HN coupling constants, and the restraints from 17 hydrogen bonds, and the three disulphide bridges. The distance geometry structures were optimized using restrained molecular dynamics (RMD) and energy minimization. The average root-mean-square deviation for the best 20 RMD refined structures is 2.26 A for the backbone and 3.14 A for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are better defined, with root-mean-square deviation values of 1.11 A for the backbone and 2.03 A for all atoms. The data analysis and the calculations show that B9(Asp) insulin, in water solution at the applied pH (1.8 to 1.9), is a well-defined dimer with no detectable difference between the two monomers. The association of the two monomers in the solution dimer is relatively loose as compared with the crystal dimer. The overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer is found to be preserved. The conformation-averaged NMR structures obtained for the monomer is close to the structure of molecule 1 in the hexamer of the 2Zn insulin crystal. However, minor, but significant deviations from this structure, as well as from the structure of monomeric insulin in solution, exist and are ascribed to the absence of the hexamer and crystal packing forces, and to the presence of monomer-monomer interactions, respectively. Thus, the monomer in the solution dimer shows a conformation similar to that of the crystal monomer in molecular regions close to the monomer-monomer interface, whereas it assumes a conformation similar to that of the solution structure of monomeric insulin in other regions, suggesting that B9(Asp) insulin adopts a monomer-like conformation when this is not inconsistent with the monomer-monomer arrangement in the dimer.     

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.     

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.     

Tyr74 is essential for the formation, stability and function of Plasmodium falciparum triosephosphate isomerase dimer

Plasmodium falciparum triosephosphate isomerase (PfTIM) is known to be functional only as a homodimer. Although many studies have shown that the interface Cys13 plays a major role in the stability of the dimer, a few reports have demonstrated that structurally conserved Tyr74 may be essential for the stability of PfTIM dimer. To understand the role of Tyr74, we have performed molecular dynamics (MD) simulations of monomeric and dimeric PfTIM mutated to glycine and cysteine at position 74. Simulations of the monomer revealed that mutant Tyr74Gly does not produce changes in folding and stability of the monomer. Interestingly, comparison of the flexibility of Tyr74 in the monomer and dimer revealed that this residue possesses an intrinsic restricted mobility, indicating that Tyr74 is an anchor residue required for homodimerization. Tyr74 also appears to play an important role in binding by facilitating the disorder-to-order transitions of loops 1 and 3, which allows Cys13 to form favorable interactions with loop 3 and Lys12 to be locked in a favorable position for catalysis. High-temperature MD simulations of the wild-type and Tyr74Gly PfTIM dimers showed that the aromatic moiety of Tyr74 is necessary to preserve the geometry and native contacts between loops 1 and 3 at the interface of the dimer. Disulfide cross-linking between mutant Tyr74Cys and Cys13 further revealed that Tyr74 stabilizes the geometry of loop 1 (which contains the catalytic residue Lys12) and the interactions between loops 1 and 3 via aromatic-aromatic interactions with residues Phe69, Tyr101, and Phe102. Principal component analysis showed that Tyr74 is also necessary to preserve the collective motions in the dimer that contribute to the catalytic efficiency of PfTIM dimer. We conclude that Tyr74 not only plays a role in the stability of the dimer, but also participates in the dimerization process and collective motions via coupled disorder-to-order transitions of intrinsically disordered regions, necessary for efficiency in the catalytic function of PfTIM.     

Exploring the dihydrodipicolinate synthase tetramer: How resilient is the dimer-dimer interface?

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) is a tetrameric enzyme that catalyses the first committed step of the lysine biosynthetic pathway. Dimeric variants of DHDPS have impaired catalytic activity due to aberrant protein motions within the dimer unit. Thus, it is thought that the tetrameric structure functions to restrict these motions and optimise enzyme dynamics for catalysis. Despite the importance of dimer-dimer association, the interface between subunits of each dimer is small, accounting for only 4.3% of the total monomer surface area, and the structure of the interface is not conserved across species. We have probed the tolerance of dimer-dimer association to mutation by introducing amino acid substitutions within the interface. All point mutations resulted in destabilisation of the ‘dimer of dimers’ tetrameric structure. Both the position of the mutation in the interface and the physico-chemical nature of the substitution appeared to effect tetramerisation. Despite only weak destabilisation of the tetramer by some mutations, catalytic activity was reduced to ∼10-15% of the wild-type in all cases, suggesting that the dimer-dimer interface is finely tuned to optimise function.     

Dramatic differences in the motions of the mouth of open and closed cytochrome P450BM-3 by molecular dynamics simulations

Molecular dynamics trajectories were calculated separately for each of the two molecules in the asymmetric unit of the crystal structure of the hemoprotein domain of cytochrome P450BM-3. Each simulation was 200 ps in length and included a 10 Å layer of explicit solvent. The simulated time-average structure of each P450BM-3 molecule is closer to its crystal structure than the two molecular dynamics time-averaged structures are to each other. In the crystal structure, molecule 2 has a more accessible substrate binding pocket than molecule 1, and this difference is maintained throughout the simulations presented here. In particular, the substrate docking regions of molecule 1 and molecule 2 diverge in the solution state simulations. The mouth of the substrate binding pocket is significantly more mobile in the simulation of molecule 2 than in the simulation of molecule 1. For molecule 1, the width of the mouth is only slightly larger than its X-ray value of 8.7 Å and undergoes fluctuations of about 1 Å. However, in molecule 2, the mouth of the substrate binding pocket is dramatically more open in the time-average molecular dynamics structure (14.7 Å) than in the X-ray structure (10.9 Å). Furthermore, this region of the protein undergoes large amplitude motions during the trajectory that are not seen in the trajectory of molecule 1, repeatedly opening and closing up to 7 Å. Presumably, the binding of different substrates will induce the mouth region to adopt different conformations from within the wide range of structures that are accessible. © 1995 Wiley-Liss, Inc.     

Thermodynamic analysis of unfolding and dissociation in lactose repressor protein.

Lactose repressor protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low protein concentrations (<10(-)12 M). To examine the unfolding and dissociation of this tetrameric protein, structural transitions in the presence of varying concentrations of urea were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation, and functional activities. The spectroscopic data demonstrated a single cooperative transition with no evidence of folded dimeric or monomeric species of this protein. These spectroscopic transitions were reversible provided a long incubation step was employed in the refolding reaction at approximately 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-type repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M4 <--> 4U) in the spectral transitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M4 <--> U4) that may occur prior to the dissociation of the dimer-dimer interface. Thus, we propose that the unfolded monomers remain associated at the C-terminus by the 4-helical coiled-coil structure that forms the dimer-dimer interface and that this intermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibited by the aggregation of this intermediate. Based upon these observations, the wild-type fluorescence and CD data were fit to a model, M4 <--> U4, which resulted in an overall DeltaG degrees for unfolding of 40 kcal/mol. Using a mutant protein, K84L, in which the monomer-monomer interface is stabilized, sedimentation equilibrium results demonstrated that the dimer-dimer interface of lac repressor could persist at higher levels of urea than the monomer-monomer interface. The tetramer-dimer transition monitored using this mutant repressor yields a DeltaG degrees of 20.4 kcal/mol. Using this free energy value for the dissociation process of U4 <--> 4U, an overall free energy change of approximately 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting the exceptional stability of this protein.     

His74 conservation in the bilin reductase PcyA family reflects an important role in protein-substrate structure and dynamics

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the proton-coupled four-electron reduction of biliverdin IXα’s two vinyl groups to produce phycocyanobilin, an essential chromophore for phytochromes, cyanobacteriochromes and phycobiliproteins. Previous site directed mutagenesis studies indicated that the fully conserved residue His74 plays a critical role in the H-bonding network that permits proton transfer. Here, we exploit X-ray crystallography, enzymology and molecular dynamics simulations to understand the functional role of this invariant histidine. The structures of the H74A, H74E and H74Q variants of PcyA reveal that a “conserved” buried water molecule that bridges His74 and catalytically essential His88 is not required for activity. Despite distinct conformations of Glu74 and Gln74 in the H74E and H74Q variants, both retain reasonable activity while the H74A variant is inactive, suggesting smaller residues may generate cavities that increase flexibility, thereby reducing enzymatic activity. Molecular dynamic simulations further reveal that the crucial active site residue Asp105 is more dynamic in H74A compared to wild-type PcyA and the two other His74 variants, supporting the conclusion that the Ala74 mutation has increased the flexibility of the active site.     

Ligand-induced conformational changes in lactose repressor: a phosphorescence and ODMR study of single-tryptophan mutants.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on four single-tryptophan mutants of lac repressor protein from Escherichia coli: H74W/Wless, W201Y, Y273W/Wless, and F293W/Wless, where Wless represents a protein background containing the double mutation W201Y/W220Y. The single-tryptophan residues are located in the protein core region, either in the monomer-monomer interface of the tetrameric protein or in the region of the inducer binding cleft. Inducer binding elicits large changes in the energy (0,0-band wavelength shifts) and zero-field splitting energies (ZFS) of the triplet states for each of the mutant proteins except W201Y which exhibits more modest effects. F293W/Wless exists in two distinguishable conformations, only one of which appears to be sensitive to the presence of inducer. These effects of inducer binding can be attributed to a conformational change that alters specific polar interactions that occur at each affected tryptophan site. Changes in the tryptophan triplet state indicator depend on the existence of specific polar interactions that are altered by local atomic relocations.     

Utilizing a Dynamical Description of IspH to Aid in the Development of Novel Antimicrobial Drugs

The nonmevalonate pathway is responsible for isoprenoid production in microbes, including H. pylori, M. tuberculosis and P. falciparum, but is nonexistent in humans, thus providing a desirable route for antibacterial and antimalarial drug discovery. We coordinate a structural study of IspH, a [4Fe-4S] protein responsible for converting HMBPP to IPP and DMAPP in the ultimate step in the nonmevalonate pathway. By performing accelerated molecular dynamics simulations on both substrate-free and HMBPP-bound [Fe₄S₄]²⁺ IspH, we elucidate how substrate binding alters the dynamics of the protein. Using principal component analysis, we note that while substrate-free IspH samples various open and closed conformations, the closed conformation observed experimentally for HMBPP-bound IspH is inaccessible in the absence of HMBPP. In contrast, simulations with HMBPP bound are restricted from accessing the open states sampled by the substrate-free simulations. Further investigation of the substrate-free simulations reveals large fluctuations in the HMBPP binding pocket, as well as allosteric pocket openings – both of which are achieved through the hinge motions of the individual domains in IspH. Coupling these findings with solvent mapping and various structural analyses reveals alternative druggable sites that may be exploited in future drug design efforts.     

Slowly interchanging conformers of bovine neurophysin-I in the unliganded dimeric state.

The effect of neurophysin dimerization on Tyr-49, a residue adjacent to the hormone-binding site, was investigated by proton NMR in order to analyze the basis of the dimerization-induced increase in neurophysin hormone affinity. Dimerization-induced changes in Tyr-49 resonances, in two unliganded bovine neurophysins, suggested that Tyr-49 perturbation is an intrinsic consequence of dimerization, although Tyr-49 is distant from the monomer-monomer interface in the crystalline liganded state. To determine whether this perturbation reflects a conformational difference between liganded and unliganded states that places Tyr-49 at the interface in the unliganded state, or a dimerization-induced change in secondary (2 degrees) or tertiary (3 degrees) structure, the more general structural consequences of dimerization were further analyzed. No change in 2 degrees structure upon dimerization was demonstrable by CD. On the other hand, a general similarity of regions involved in dimerization in unliganded and liganded states was indicated by NMR evidence of participation of His-80 and Phe-35 in dimerization in the unliganded state; both residues are at the interface in the crystal structure and distant from Tyr-49. Consistent with a lack of direct participation of Tyr-49 at the monomer-monomer interface, dimerization induced at least two distinct slowly exchanging environmental states for the 3.5 ring protons of Tyr-49 without significantly increased dipolar broadening relative to the monomer. Two environments were also found in the dimer of des-1-8 neurophysin-I for the methyl protons of Thr-9, another residue distant from the monomer-monomer interface and close to the binding site in the liganded state.(ABSTRACT TRUNCATED AT 250 WORDS)     

The backrub motion: how protein backbone shrugs when a sidechain dances

Surprisingly, the frozen structures from ultra-high-resolution protein crystallography reveal a prevalent, but subtle, mode of local backbone motion coupled to much larger, two-state changes of sidechain conformation. This "backrub" motion provides an influential and common type of local plasticity in protein backbone. Concerted reorientation of two adjacent peptides swings the central sidechain perpendicular to the chain direction, changing accessible sidechain conformations while leaving flanking structure undisturbed. Alternate conformations in sub-1 angstroms crystal structures show backrub motions for two-thirds of the significant Cbeta shifts and 3% of the total residues in these proteins (126/3882), accompanied by two-state changes in sidechain rotamer. The Backrub modeling tool is effective in crystallographic rebuilding. For homology modeling or protein redesign, backrubs can provide realistic, small perturbations to rigid backbones. For large sidechain changes in protein dynamics or for single mutations, backrubs allow backbone accommodation while maintaining H bonds and ideal geometry.     

Crystal structures of Escherichia coli RecA in a compressed helical filament

The X-ray crystal structure of uncomplexed Escherichia coli RecA protein has been determined in three new crystal forms at resolutions of 1.9 A, 2.0 A, and 2.6 A. The RecA protein used for this study contains the extra residues Gly-Ser-His-Met at the N terminus, but retains normal ssDNA-dependent ATPase and coprotease activities. In all three crystals, RecA is packed in a right-handed helical filament with a pitch of approximately 74 A. These RecA filaments are compressed relative to the original crystal structure of RecA, which has a helical pitch of 82.7 A. In the structures of the compressed RecA filament, the monomer-monomer interface and the core domain are essentially the same as in the RecA structure with the 83 A pitch. The change in helical pitch is accommodated by a small movement of the N-terminal domain, which is reoriented to preserve the contacts it makes at the monomer-monomer interface. The new crystal structures show significant variation in the orientation and conformation of the C-terminal domain, as well as in the inter-filament packing interactions. In crystal form 2, a calcium ion is bound closely to a beta-hairpin of the C-terminal domain and to Asp38 of a neighboring filament, and residues 329-331 of the C-terminal tail become ordered to contact a neighboring filament. In crystal forms 3 and 4, a sulfate ion or a phosphate anion is bound to the same site on RecA as the beta-phosphate group of ADP, causing an opening of the P-loop. Altogether, the structures show the conformational variability of RecA protein in the crystalline state, providing insight into many aspects of RecA function.     

Molecular dynamics simulations of hemoglobin A in different states and bound to DPG: effector-linked perturbation of tertiary conformations and HbA concerted dynamics

Recent functional studies reported on human adult hemoglobin (HbA) show that heterotropic effector-linked tertiary structural changes are primarily responsible for modulating the oxygen affinity of hemoglobin. We present the results of 6-ns molecular dynamics simulations performed to gain insights into the dynamical and structural details of these effector-linked tertiary changes. All-atom simulations were carried out on a series of models generated for T- and R-state HbA, and for 2,3-diphosphoglycerate-bound models. Cross-correlation analyses identify both intra- and intersubunit correlated motions that are perturbed by the presence of the effector. Principal components analysis was used to decompose the covariance matrix extracted from the simulations and reconstruct the trajectories along the principal coordinates representative of functionally important collective motions. It is found that HbA in both quaternary states exists as ensembles of tertiary conformations that introduce dynamic heterogeneity in the protein. 2,3-Diphosphoglycerate induces significant perturbations in the fluctuations of both HbA states that translate into the protein visiting different tertiary conformations within each quaternary state. The analysis reveals that the presence of the effector affects the most important components of HbA motions and that heterotropic effectors modify the overall dynamics of the quaternary equilibrium via tertiary changes occurring in regions where conserved functionally significant residues are located, namely in the loop regions between helices C and E, E and F, and F and G, and in concerted helix motions. The changes are not apparent when comparing the available x-ray crystal structures in the presence and absence of effector, but are striking when comparing the respective dynamic tertiary conformations of the R and T tetramers.