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A Lewit-Bentley

Publications and source records attributed to A Lewit-Bentley.

At least 19 recordsLinked to original sources
PubMed2001

Annexin A5 D226K structure and dynamics: identification of a molecular switch for the large-scale conformational change of domain III.

The domain III of annexin 5 undergoes a Ca(2+)- and a pH-dependent conformational transition of large amplitude. Modeling of the transition pathway by computer simulations suggested that the interactions between D226 and T229 in the IIID-IIIE loop on the one hand and the H-bond interactions between W187 and T224 on the other hand, are important in this process [Sopkova et al. (2000) Biochemistry 39, 14065-14074]. In agreement with the modeling, we demonstrate in this work that the D226K mutation behaves as a molecular switch of the pH- and Ca(2+)-mediated conformational transition. In contrast, the hydrogen bonds between W187 and T224 seem marginal.

Annexin A5↗
PubMed2000

S100 protein-annexin interactions: a model of the (Anx2-p11)(2) heterotetramer complex.

The (Anx2)(2)(p11)(2) heterotetramer has been implicated in endo- and exocytosis in vivo and in liposome aggregation in vitro. Here we report on the modelling of the heterotetramer complex using docking algorithms. Two types of models are generated-heterotetramer and heterooctamer. On the basis of the agreement between the calculated (X-ray) electron density and the observed projected density from cryo-electron micrographs on the one hand, and calculated energy criteria on the other hand, the heterotetramer models are proposed as the most probable, and one of them is selected as the best model. Analysis of this model at an atomic level suggests that the interaction between the Anx2 core and p11 has an electrostatic character, being stabilised primarily through charged residues.

Algorithms↗
PubMed2000

Structure of membrane-bound annexin A5 trimers: a hybrid cryo-EM - X-ray crystallography study.

Annexins constitute a family of phospholipid- and Ca(2+)-binding proteins involved in a variety of membrane-related processes. The property of several annexins, including annexin A5, to self-organize at the surface of lipid membranes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated the high-resolution structure of annexin A5 trimers in membrane-bound 2D crystals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was discovered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 A resolution, revealing protein densities within each of the four domains characteristic of annexins. A quantitative comparison was performed between this structure and models generated from the structure of the soluble form of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attributed to membrane binding. As a consequence, and contrary to the common view, annexin A5 molecules maintain their bent shape and do not flatten upon membrane binding, which implies either that the four putative Ca(2+) and membrane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural change occurring upon membrane binding. The evidence that 2D arrays of annexin A5 trimers are responsible for its in vitro property of blood coagulation inhibition supports this conclusion.

Animals↗
PubMed2000

Pathway for large-scale conformational change in annexin V.

Crystallographic studies have shown that the binding of calcium to domain III of annexin V is accompanied by a large conformational change involving surface exposure of Trp187. Here we examine this conformational transition using computer simulation. It is found that the burial of Trp187 is accompanied by a large increase in conformational strain, compensated by improved protein-protein interaction energies. A low energy pathway for the conformational change is determined using the conjugate peak refinement method [Fischer, S., and Karplus, M. (1992) Chem. Phys. Lett. 194, 252-261] with solvent effects taken into account using nonuniform charge scaling. The pathway obtained is complex, involving >300 dihedral angle transitions and the complete unwinding of one helix. Acidic residues play a key role in the conformational pathway, via a succession of direct hydrogen bonds with the indole ring of Trp187. This finding is discussed in the light of experimentally determined pH, calcium ion and mutational effects on the conformational transition.

Annexin A5↗
PubMed2000

The annexin A3-membrane interaction is modulated by an N-terminal tryptophan.

The crystal structure of annexin A3 (human annexin III) solved recently revealed a well-ordered folding of its N-terminus with the side chain of tryptophan 5 interacting with residues at the extremity of the central pore. Since the pore of annexins has been suggested as the ion pathway involved in membrane permeabilization by these proteins, we investigated the effect of the N-terminal tryptophan on the channel activity of annexin A3 by a comparative study of the wild-type and the W5A mutant in structural and functional aspects. Calcium influx and patch-clamp recordings revealed that the mutant exhibited an enhanced membrane permeabilization activity as compared to the wild-type protein. Analysis of the phospholipid binding behavior of wild-type and mutant protein was carried out by cosedimentation with lipids and inhibition of PLA(2) activity. Both methods reveal a much stronger binding of the mutant to phospholipids. The structure is very similar for the wild-type and the mutant protein. The exchange of the tryptophan for an alanine results in a disordered N-terminal segment. Urea-induced denaturation of the wild-type and mutant monitored by intrinsic fluorescence indicates a separate unfolding of the N-terminal region which occurs at lower urea concentrations than unfolding of the protein core. We therefore conclude that the N-terminal domain of annexin A3, and especially tryptophan 5, is involved in the modulation of membrane binding and permeabilization by annexin A3.

Alanine↗
PubMed2000

Structural basis of the Ca(2+)-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I.

BACKGROUND: S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions. RESULTS: We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 A resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11-annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100-annexin interaction. CONCLUSIONS: By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.

Acetylation↗
PubMed2000

S100-annexin complexes: some insights from structural studies.

Several annexins have been shown to bind proteins that belong to the S100 calcium-binding protein family. The two best-characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N-terminus and of S100C with annexin I N-terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42-55), we propose a computer generated model for the entire annexin II/p11 complex.

Annexin A2↗
PubMed2000

EF-hand calcium-binding proteins.

The EF-hand motif is the most common calcium-binding motif found in proteins. Several high-resolution structures containing different metal ions bound to EF-hand sites have given new insight into the modulation of their binding affinities. Recently determined structures of members of several newly identified protein families that contain the EF-hand motif in some of their domains, as well as of their complexes with target molecules, are throwing light on the surprising variety of functions that can be served by this simple and ingenious structural motif.

Calcium-Binding Proteins↗
PubMed1999

Conformational flexibility of domain III of annexin V at membrane/water interfaces.

The conformational dynamics of domain III in annexin V bound to negatively charged phospholipid vesicles of 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine and 1-palmitoyl-2-oleoyl-sn-glycerophosphoserine or incorporated into reverse micelles of water/sodium bis(2-ethylhexyl) sulfosuccinate in isooctane, used to mimic the phospholipid/water interface, was studied by steady-state and time-resolved fluorescence of its single tryptophan residue (W187). Upon interaction with sonicated phospholipid vesicles in the presence of calcium, or upon incorporation into reverse micelles without calcium, a progressive 12-14 nm red shift of the fluorescence emission spectrum of W187 is observed. The indole environment becomes therefore more polar than in the unbound protein. Three major lifetime populations describe the fluorescence intensity decays of W187 in both systems. A long-lived excited-state population characterizes the membrane-bound state of the protein. The existence of local conformers with different subnanosecond mobility is suggested by specific association between lifetimes and correlation times both for the protein in buffer and in interaction with the membrane surface. The interaction of the protein with the membrane surface preserves the existence of a rapid unhindered rotational motion, which is coupled with all three lifetimes. The longest lifetime is coupled to restricted motions in subnanosecond and nanosecond time scales. The overall amplitude of rotation of the indole ring is increased in the membrane-bound conformation of the protein. In reverse micelles, the local dynamics reported by W187 is also considerably increased whereas the overall folding of the protein remains unaffected. The same conformational change of domain III can therefore be provoked by different conditions: calcium binding at high concentration, mild acidic pH [Sopkova, J., Vincent, M., Takahashi, M., Lewit-Bentley, A. , and Gallay, J. (1998) Biochemistry 37, 11962-11970] and the interaction of the protein with the membrane surface. The high flexibility of domain III in the membrane-bound protein suggests that this domain may not be crucial for the interaction of the protein with the membrane, in contrast with previous models. Our data are compatible with atomic force microscopy results which suggest that domain III of annexin V does not interact strongly with the membrane surface [Reviakine, I., Bergma-Schutter, W., and Brisson, A. (1998) J. Struct. Biol. 121, 356-361].

Annexin A5↗
PubMed1999

The crystal structure of a complex of p11 with the annexin II N-terminal peptide.

The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11.p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties. We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

Animals↗
PubMed1998

Conformational flexibility of domain III of annexin V studied by fluorescence of tryptophan 187 and circular dichroism: the effect of pH.

The conformation and dynamics of domain III of annexin V was studied by steady-state and time-resolved fluorescence of its single tryptophan residue (Trp187) as a function of pH in the absence of calcium. At neutral pH, the maximum of emission occurs at 326 nm, in agreement with the hydrophobic location of the tryptophan residue seen in the three-dimensional structure. Upon decreasing the pH, a progressive red-shift by about 12 nm of the fluorescence emission spectrum is observed. The effect is complete between pH 6 and 4.5, and most likely involves at least one and maybe two carboxylic group(s). Circular dichroism mesurements give evidence for a preservation of the native folding of the protein in these mild acidic conditions. A fluorescence red-shift of smaller amplitude is also observed at high pH (approximately 11). The aggregation state of the protein is affected by pH: while at neutral pH, the protein is monomeric (rotational correlation time = 14 ns); it forms aggregates larger than a dimer (rotational correlation time > 40 ns) in acidic pH conditions. These results suggest that electrostatic interactions are probably important for the stabilization of the folding of domain III without calcium. The conformational change may be related to the aggregation state of the molecule. Examination of the protein crystal structures with and without calcium ion in domain III shows an interplay of salt bridges implying charged amino acid side chains at the molecule surface of domain III. These observations may provide a further clue to the mechanism of the conformational change of domain III of annexin V induced by high calcium concentrations and interaction at the membrane/water interface.

Acrylamide↗
PubMed1998

The isolated H4-H5 cytoplasmic loop of Na,K-ATPase overexpressed in Escherichia coli retains its ability to bind ATP.

The H4-H5 loop of the alpha-subunit of mouse brain Na,K-ATPase was expressed and isolated from Escherichia coli cells. Using fluorescence analogues of ATP, this loop was shown to retain its capability to bind ATP. Isolation of a soluble H4-H5 loop with the native ATP binding site is a crucial step for detailed studies of the molecular mechanism of ATP binding and utilisation.

Adenosine Triphosphate↗
PubMed1998

Interactions of benzodiazepine derivatives with annexins.

Human annexins III and V, members of the annexin family of calcium- and membrane-binding proteins, were complexed within the crystals with BDA452, a new 1,4-benzodiazepine derivative by soaking and co-crystallization methods. The crystal structures of the complexes were analyzed by x-ray crystallography and refined to 2.3- and 3.0-A resolution. BDA452 binds to a cleft which is located close to the N-terminus opposite to the membrane binding side of the proteins. Biophysical studies of the interactions of various benzodiazepine derivatives with annexins were performed to analyze the binding of benzodiazepines to annexins and their effects on the annexin-induced calcium influx into phosphatidylserine/phosphatidylethanolamine liposomes. Different effects were observed with a variety of benzodiazepines and different annexins depending on both the ligand and the protein. Almost opposite effects on annexin function are elicited by BDA250 and diazepam, its 7-chloro-derivative. We conclude that benzodiazepines modulate the calcium influx activity of annexins allosterically by stabilizing or destabilizing the conducting state of peripherally bound annexins in agreement with suggestions by Kaneko (Kaneko, N., Ago, H., Matsuda, R., Inagaki, E., and Miyano, M. (1997) J. Mol. Biol., in press).

Amino Acid Sequence↗
PubMed1998

The isolated H4-H5 cytoplasmic loop of Na,K-ATPase overexpressed in Escherichia coli retains its ability to bind ATP.

The H4-H5 loop of the alpha-subunit of mouse brain Na,K-ATPase was expressed and isolated from E.coli cells. Using fluorescence analogues of ATP, this loop was shown to retain its capability to bind ATP. Isolation of a soluble H4-H5 loop with the native ATP binding site is a critical step for detailed studies of the molecular mechanism of ATP binding and utilisation.

Adenosine Triphosphate↗
PubMed1997

Can enzymatic activity, or otherwise, be inferred from structural studies of annexin III?

Annexin III, a putative inositol (1,2)-phosphohydrolase, was co-crystallized with inositol 2-phosphate, the inhibitor of the reaction, and its structure was solved to 1.95 A resolution. No enzyme active site was observed in the structure. Assays for enzymatic activity were also negative. Search for annexin III-inositol phosphate interactions using the BIAcoreTM system revealed an affinity for inositol cyclic (1,2)-phosphate, suggesting annexin III may sequester the molecule in the cell. The BIAcoreTM system used with different phospholipids showed that annexin III displays specificity for phosphatidylethanolamine, but not for phosphatidylinositols. Interestingly, a molecule of ethanolamine was found bound to the protein in the crystal structure. Coupled with the fact that this is a particularly abundant phospholipid in granules specific to neutrophils, cells where annexin III is highly expressed, our finding could be pointing to a physiological role of annexin III.

Annexin A3↗
PubMed1996

The high-resolution crystal structure of human annexin III shows subtle differences with annexin V.

The structure of recombinant human annexin III was solved to 1.8 A resolution. Though homologous to annexin I and V, the annexin III structure shows significant differences. The tryptophan in the calcium loop of the third domain is exposed to the solvent, as in the structure of annexin V crystallized in high calcium concentrations, although the annexin III crystals were prepared at low calcium concentrations. The position of domain III relative to the other domains is different from both annexin V and I, suggesting further flexibility of the molecule. The entire N-terminus of the protein is well-defined in the present structure. The side chain of tryptophan 5 interacts with the hinge region of the hydrophillic channel, which could have an effect on the potential mobility of this region, as well as on its possible calcium channel behavior.

Annexin A1↗
PubMed1994

Calcium-induced changes in annexin V behaviour in solution as seen by proton NMR spectroscopy.

The behaviour of human annexin V in the presence of calcium was studied by NMR. We observe the formation of well defined dimers, as well as a change in the local dynamics of one His side chain. We assign the observed changes to either His98 or His267 residues and conclude that they could be related either to the hinge-bending motion reported from crystal structures, or to a local side chain rearrangement within the calcium-binding loops concerned. Dimerization was also confirmed by a small-angle neutron-scattering experiment. Under the experimental conditions used, we do not observe the conformational change involving Trp187 seen in previous studies, which occurs at higher relative calcium concentrations.

Amino Acid Sequence↗