Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.     

A toxic monomeric conformer of the polyglutamine protein

Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a beta-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into beta-sheet-rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble beta-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic beta-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.     

Conformational change of the chloroplast ATP synthase on the enzyme activation process detected by the trypsin sensitivity of the gamma subunit

Delta mu H(+) is known to stimulate the enzyme activity of chloroplast ATP synthase in addition to its important role as energy supply for ATP synthesis. In the present study, we focused on the relationship between the proton translocation via the membrane sector of ATP synthase, F(o), and the conformational change of the central stalk subunit gamma. The conformational change of CF(1) mainly at the gamma subunit was induced by the proton flow via F(o) in the absence of substrates. The effects of inhibitors on CF(o) or CF(1) for this conformational change were also examined. The observed conformational change was partially suppressed by ADP binding. From these results, we propose the Delta mu H(+)-dependent conformational change of CF(1) on the enzyme activation process, which is affected by both ADP binding to the catalytic sites and proton flow via F(o) portion.     

Sequence dependence of the B-A conformational transition of DNA

We have studied, by conformational analysis, the sequence dependence of DNA conformational transition between B- and A-forms. We have considered intramolecular interactions between base pairs, without backbone, to examine their role in the conformational transition between B- and A-forms, and found that base pairs themselves usually have intrinsic conformational preferences for the B- or A-form. Calculation of all ten possible base steps shows that the base combinations, CC (or GG), GC, AT, and TA, have tendencies to assume the A-conformation. Results show that it is particularly easy to slide along the long axis of the base pair for these steps, with AT and CC showing especially flat energies. These calculations show that a preference for the B- or A-conformation depends on the electrostatic energy parameters, in particular, on dielectric and shielding constants; the A-conformation is preferred for low dielectric constant or low shielding. Both the A- and B-conformations are mainly stabilized by electrostatic interactions between favorably juxtaposed atomic charges on base pairs; however, the B-conformation generally has more favorable van der Waals interactions than the A-form. These sequence-dependent conformational preference and environmental effects agree roughly with experimental observations, suggesting that the origin of the conformational polymorphism is attributable to the intrinsic conformational preference of base pairs.     

Role of the buried glutamate in the alpha-helical coiled coil domain of the macrophage scavenger receptor

The macrophage scavenger receptor exhibits a pH-dependent conformational change around the carboxy-terminal half of the alpha-helical coiled coil domain, which has a representative amino acid sequence of a (defgabc)n heptad. We previously demonstrated that a peptide corresponding to this region formed a random coil structure at pH 7 and an alpha-helical coiled coil structure at pH 5 [Suzuki, K., Doi, T., Imanishi, T., Kodama, T., and Tanaka, T. (1997) Biochemistry 36, 15140-15146]. To determine the amino acid responsible for the conformational change, we prepared several peptides in which the acidic amino acids were replaced with neutral amino acids. Analyses of their structures by circular dichroism and sedimentation equilibrium gave the result that the presence of Glu242 at the d position was sufficient to induce the pH-dependent conformational change of the alpha-helical coiled coil domain. Furthermore, we substituted a Glu residue for the Ile residue at the d or a position of a de novo designed peptide (IEKKIEA)4, which forms a highly stable triple-stranded coiled coil. These peptides exhibited a pH-dependent conformational change similar to that of the scavenger receptor. Therefore, we conclude that a buried Glu residue in the hydrophobic core of a triple-stranded coiled coil has the potential to induce the pH-dependent conformational change. This finding makes it possible to elucidate the functions of natural proteins and to create a de novo protein designed to undergo a pH-dependent conformational change.     

Paramagnetic NMR restraints for the characterization of protein structural rearrangements

Mobility is a common feature of biomacromolecules, often fundamental for their function. Thus, in many cases, biomacromolecules cannot be described by a single conformation, but rather by a conformational ensemble. NMR paramagnetic data demonstrated quite informative to monitor this conformational variability, especially when used in conjunction with data from different sources. Due to their long-range nature, paramagnetic data can, for instance, i) clearly demonstrate the occurrence of conformational rearrangements, ii) reveal the presence of minor conformational states, sampled only for a short time, iii) indicate the most representative conformations within the conformational ensemble sampled by the molecule, iv) provide an upper limit to the weight of each conformation.     

A compact intermediate state of calmodulin in the process of target binding

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.     

Evaluation of the cytotoxic activity of polyethers isolated from Laurencia

In this paper, we report on the conformational analysis of several polyether triterpenes with a squalene carbon skeleton which exhibited significant cytotoxic activity using a Monte Carlo conformational search and spectroscopical data. These studies indicate that the conformation of the side chain C-14/C-19 and the arrangement and direction of this chain may be among the fundamental factors related to the activity of this type of metabolites.

We report the conformational analysis of several polyether triterpenes with squalene carbon skeleton which exhibited a significant cytotoxic activity using a Monte Carlo conformational search and spectroscopical data.     

Early steps of the conformational change of influenza virus hemagglutinin to a fusion active state: stability and energetics of the hemagglutinin

A conformational change of the homotrimeric glycoprotein hemagglutinin (HA) of influenza virus mediates fusion between the viral envelope and the endosome membrane. The conformational change of the HA ectodomain is triggered by the acidic pH of the endosome lumen. An essential step of the conformational change is the formation of an extended coiled-coil motif exposing the hydrophobic fusion peptide toward the target membrane. The structures of the neutral-pH, non-fusion active conformation of the HA ectodomain and of a fragment of the ectodomain containing the coiled-coil motif are known. However, it is not known by which mechanism protonation triggers the conformational change of the stable neutral-pH conformation of the ectodomain. Here, recent studies on the stability of the HA ectodomain at neutral pH, the energetics of the conformational change toward the fusion-active state and of the unfolding of the HA ectodomain are summarised. A model for the early steps of the conformational change of the HA ectodomain is presented. The model implicates that protonation leads to a partial dissociation of the distal domains of the HA monomers that is driven by electrostatic repulsion. The opening of the ectodomain enables water to enter the ectodomain. The interaction of water with respective sequences originally shielded from contact with water drives the formation of the coiled-coil structure.     

The Uncoupling of the Effects of Formins on the Local and Global Dynamics of Actin Filaments

In this study, experiments were carried out in the conventional and saturation-transfer electron paramagnetic resonance (EPR) time domains to explore the effect of mDia1-FH2 formin fragments on the dynamic and conformational properties of actin filaments. Conventional EPR measurements showed that addition of formin to actin filaments produced local conformational changes in the vicinity of Cys-374 by increasing the flexibility of the protein matrix in the environment of the label. The results indicated that it was the binding of formin to the barbed end that resulted in these conformational changes. The conventional EPR results obtained with actin labeled on the Lys-61 site showed that the binding of formins could only slightly affect the structure of the subdomain 2 of actin, reflecting the heterogeneity of the formin-induced conformational changes. Saturation transfer EPR measurements revealed that the binding of formins decreased the torsional flexibility of the actin filaments in the microsecond time range. We concluded that changes in the local and the global conformational fluctuations of the actin filaments are associated with the binding of formins to actin. The results on the two EPR time domains showed that the effects of formins on the substantially different types of motions were uncoupled.     

Theoretical conformation analysis of tetrasaccharide-repeated links of the Shigella flexneri O-antigenic polysaccharide

Theoretical conformational analysis of four tetrasaccharide repeating units of the Shigella flexneri serogroup Y polysaccharide has been carried out. Interdependency of conformational states of neighbouring disaccharide units in the oligosaccharides has been investigated and conformational distribution of tetrasaccharides in solution calculated. Taking into account the entropy of oligosaccharide chains is shown to lead to significant correction of the results.     

Conformational proofreading: the impact of conformational changes on the specificity of molecular recognition

To perform recognition, molecules must locate and specifically bind their targets within a noisy biochemical environment with many look-alikes. Molecular recognition processes, especially the induced-fit mechanism, are known to involve conformational changes. This raises a basic question: Does molecular recognition gain any advantage by such conformational changes? By introducing a simple statistical-mechanics approach, we study the effect of conformation and flexibility on the quality of recognition processes. Our model relates specificity to the conformation of the participant molecules and thus suggests a possible answer: Optimal specificity is achieved when the ligand is slightly off target; that is, a conformational mismatch between the ligand and its main target improves the selectivity of the process. This indicates that deformations upon binding serve as a conformational proofreading mechanism, which may be selected for via evolution.     

Spatial organization of the N-terminal beta-endorphin tridecapeptide

Using conformational analysis spatial structure and conformational properties of the N-terminal tridecapeptide--endorphine molecules were investigated. Calculations were based on the fragmental analysis using non-valent, electrostatic, torsional interactions and hydrogen bonds. It was shown that tridecapeptide could exist in several low-energetical conformational states. Enkephaline fragments structure depends on the most perspective structure of free metioninenkephaline. The results can be used for conformational analysis of endorphine molecules, for structure--function relations study.     

Diversity of conformational states and changes within the EF-hand protein superfamily

The EF-hand motif, which assumes a helix-loop-helix structure normally responsible for Ca2+ binding, is found in a large number of functionally diverse Ca2+ binding proteins collectively known as the EF-hand protein superfamily. In many superfamily members, Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. In calmodulin and troponin C, this is described as a change from the closed conformational state in the absence of Ca2+ to the open conformational state in its presence. It is now clear from structures of other EF-hand proteins that this "closed-to-open" conformational transition is not the sole model for EF-hand protein structural response to Ca2+. More complex modes of conformational change are observed in EF-hand proteins that interact with a covalently attached acyl group (e.g., recoverin) and in those that dimerize (e.g., S100B, calpain). In fact, EF-hand proteins display a multitude of unique conformational states, together constituting a conformational continuum. Using a quantitative 3D approach termed vector geometry mapping (VGM), we discuss this tertiary structural diversity of EF-hand proteins and its correlation with target recognition.     

Three-dimensional structure of the lipotropic active fragment of sea whale somatotropin

Using semiempirical conformational analysis method, the spatial structure and conformational properties of the sea-whale somatotropin lipotropic action were investigated. The calculations were based on the fragmental analysis using non-valent, electrostatic interactions, torsional barriers and hydrogen bonds. The computer data suggest that this molecule can exist only in several low energy conformational states. The experimental results can be used for the analysis of structural-and-functional relationships.     

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O‐glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O‐glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn‐antigen (GalNAc‐O‐Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding‐competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution‐state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre‐selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.     

Recent advances in the structural biology of the 26S proteasome

There is growing appreciation for the fundamental role of structural dynamics in the function of macromolecules. In particular, the 26S proteasome, responsible for selective protein degradation in an ATP dependent manner, exhibits dynamic conformational changes that enable substrate processing. Recent cryo-electron microscopy (cryo-EM) work has revealed the conformational dynamics of the 26S proteasome and established the function of the different conformational states. Technological advances such as direct electron detectors and image processing algorithms allowed resolving the structure of the proteasome at atomic resolution. Here we will review those studies and discuss their contribution to our understanding of proteasome function.     

Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin

The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.     

Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states

The beta2 adrenoreceptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified beta2AR. Here we show that upon catecholamine binding, beta2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions.