Rat annexin V crystal structure: Ca(2+)-induced conformational changes.

Annexins are a family of calcium- and phospholipid-binding proteins implicated in mediating membrane-related processes such as secretion, signal transduction, and ion channel activity. The crystal structure of rat annexin V was solved to 1.9 angstrom resolution by multiple isomorphous replacement. Unlike previously solved annexin V structures, all four domains bound calcium in this structure. Calcium binding in the third domain induced a large relocation of the calcium-binding loop regions, exposing the single tryptophan residue to the solvent. These alterations in annexin V suggest a role for domain 3 in calcium-triggered interaction with phospholipid membranes.

Annexin V--heparin oligosaccharide complex suggests heparan sulfate--mediated assembly on cell surfaces.

BACKGROUND: Annexin V, an abundant anticoagulant protein, has been proposed to exert its effects by self-assembling into highly ordered arrays on phospholipid membranes to form a protective anti-thrombotic shield at the cell surface. The protein exhibits very high-affinity calcium-dependent interactions with acidic phospholipid membranes, as well as specific binding to glycosaminoglycans (GAGs) such as heparin and heparan sulfate, a major component of cell surface proteoglycans. At present, there is no structural information to elucidate this interaction or the role it may play in annexin V function at the cell surface. RESULTS: We report the 1.9 A crystal structure of annexin V in complex with heparin-derived tetrasaccharides. This structure represents the first of a heparin oligosaccharide binding to a protein where calcium ions are essential for the interaction. Two distinct GAG binding sites are situated on opposite protein surfaces. Basic residues at each site were identified from the structure and site-directed mutants were prepared. The heparin binding properties of these mutants were measured by surface plasmon resonance. The results confirm the roles of these mutated residues in heparin binding, and the kinetic and thermodynamic data define the functionally distinct character of each distal binding surface. CONCLUSION: The annexin V molecule, as it self-assembles into an organized array on the membrane surface, can bind the heparan sulfate components of cell surface proteoglycans. A novel model is presented in which proteoglycan heparan sulfate could assist in the localization of annexin V to the cell surface membrane and/or stabilization of the entire molecular assembly to promote anticoagulation.

Effect of vesicle composition and curvature on the dissociation of phosphatidic acid in small unilamellar vesicles--a 31P-NMR study.

Sonicated small unilamellar vesicles (SUVs) containing phosphatidic acid (PA) give two PA 31P-NMR resonances corresponding to PA molecules in the inner and outer leaflets of the bilayer. This NMR differentiation between the two monolayers is not due to a pH gradient across the membrane but instead reflects differential packing in the inner and outer leaflets imposed by the highly curved SUV surface. The apparent pKa of the outer-leaflet PA increases with decreasing surface curvature and with increasing PA content. The estimated relationship between the apparent pKa of the outer-leaflet PA headgroup and vesicle curvature may provide a qualitative probe for effects related to surface curvature in these model-membrane systems.

Structure refinement of fructose-1,6-bisphosphatase and its fructose 2,6-bisphosphate complex at 2.8 A resolution.

The structures of the native fructose-1,6-bisphosphatase (Fru-1,6-Pase), from pig kidney cortex, and its fructose 2,6-bisphosphate (Fru-2,6-P2) complexes have been refined to 2.8 A resolution to R-factors of 0.194 and 0.188, respectively. The root-mean-square deviations from the standard geometry are 0.021 A and 0.016 A for the bond length, and 4.4 degrees and 3.8 degrees for the bond angle. Four sites for Fru-2,6-P2 binding per tetramer have been identified by difference Fourier techniques. The Fru-2,6-P2 site has the shape of an oval cave about 10 A deep, and with other dimensions about 18 A by 12 A. The two Fru-2,6-P2 binding caves of the dimer in the crystallographically asymmetric unit sit next to one another and open in opposite directions. These two binding sites mutually exchange their Arg243 side-chains, indicating the potential for communication between the two sites. The beta, D-fructose 2,6-bisphosphate has been built into the density and refined well. The oxygen atoms of the 6-phosphate group of Fru-2,6-P2 interact with Arg243 from the adjacent monomer and the residues of Lys274, Asn212, Tyr264, Tyr215 and Tyr244 in the same monomer. The sugar ring primarily contacts with the backbone atoms from Gly246 to Met248, as well as the side-chain atoms, Asp121, Glu280 and Lys274. The 2-phosphate group interacts with the side-chain atoms of Ser124 and Lys274. A negatively charged pocket near the 2-phosphate group includes Asp118, Asp121 and Glu280, as well as Glu97 and Glu98. The 2-phosphate group showed a disordered binding perhaps because of the disturbance from the negatively charged pocket. In addition, Asn125 and Lys269 are located within a 5 A radius of Fru-2,6-P2. We argue that Fru-2,6-P2 binds to the active site of the enzyme on the basis of the following observations: (1) the structure similarity between Fru-2,6-P2 and the substrate; (2) sequence conservation of the residues directly interacting with Fru-2,6-P2 or located at the negatively charged pocket; (3) a divalent metal site next to the 2-phosphate group of Fru-2,6-P2; and (4) identification of some active site residues in our structure, e.g. tyrosine and Lys274, consistent with the results of the ultraviolet spectra and the chemical modification. The structures are described in detail including interactions of interchain surfaces, and the chemically modifiable residues are discussed on the basis of the refined structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Annexin V forms calcium-dependent trimeric units on phospholipid vesicles.

The quaternary structure of annexin V, a calcium-dependent phospholipid binding protein, was investigated by chemical cross-linking. Calcium was found to induce the formation of trimers, hexamers, and higher aggregates only when anionic phospholipids were present. Oligomerization occurred under the same conditions annexin-vesicle binding. A model is proposed in which cell stimulation leads to calcium-induced organization of arrays of annexin V lining the inner membrane surface, thus altering properties such as permeability and fluidity.

Interaction of heparin with annexin V.

The energetics and kinetics of the interaction of heparin with the Ca2+ and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca2+ dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a Kd of approximately 21 nM for the interaction of annexin V with end-chain immobilized heparin and a Kd of approximately 49 nM for the interaction with end-chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The Kd of a heparin octasaccharide binding to annexin V was approximately 1 microM with a binding stoichiometry of 1:1.

Mutation of highly conserved arginine residues disrupts the structure and function of annexin V.

BACKGROUND: Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Annexin V contains four such domains. Each of these domains has a highly conserved arginine (R). METHODS: To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. RESULTS: Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation. R43E and R115E exhibited much greater susceptibility to proteolytic digestion than the wild type. While Ca(2+)-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 microM Ca2+), R43E and R115E exhibited a 6- and 2-fold decrease in phospholipid affinity, respectively. Consistent with the different phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. CONCLUSIONS: Our data suggest that mutation of these highly conserved arginine residues in each of the four canonical domains of annexin have differential effects on the phospholipid binding, tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation, R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of annexin V by establishing hydrogen bonds and ionic bridges.

X-ray diffraction analysis on single crystals of recombinant Escherichia coli ornithine transcarbamoylase.

Single crystals of recombinant Escherichia coli ornithine transcarbamoylase suitable for x-ray analysis have been grown from polyethylene glycol and 2-methyl-2,4-pentanediol. The space group has been determined as P3(1) or P3(2), with one protein trimer of three identical 36.8-kDa subunits in the asymmetric unit. The unit cell dimensions are a = b = 105.1 A and c = 87.8 A. The crystals diffract well to 3-A resolution and are quite resistant to radiation damage. Single crystals have also been grown of a genetically engineered site-specific mutant for which the replacement of an arginine (Arg-57) to a glycine has been shown to not only drastically affect the enzyme activity but also its kinetic mechanism (Kuo, L. C., Miller, A. W., Lee, S., and Kozuma, C. (1988) Biochemistry 27, 8823-8832). The crystals of the Arg-57----Gly mutant protein are isomorphous to those of the wild type. Crystal soaking experiments using both wild-type and Arg-57----Gly crystals in the presence of various ligands have provided evidence of specific conformational changes upon substrate binding which supports our previous kinetic and spectroscopic observations.

Chemical rescue by guanidine derivatives of an arginine-substituted site-directed mutant of Escherichia coli ornithine transcarbamylase.

Escherichia coli ornithine transcarbamylase (OTCase) catalyzes the production of L-citrulline and phosphate from carbamyl phosphate and L-ornithine in L-arginine biosynthesis. We show that exogenous guanidines can restore activity to (chemically rescue) a catalytically-impaired site-directed mutant OTCase, R57G, in which glycine replaces an an active site arginine. The best rescue agent is guanidine hydrochloride, which enhances the rate of the mutant 2000-fold. The turnover number for the guanidine-rescued R57G mutant is 10% that of wild-type. The addition of guanidine to the R57G mutant has little effect on KMCP values, and the rescue effect is therefore attributed principally to an increase in kcat. Other compounds were screened as potential rescue agents, but rate enhancement is highly selective for guanidines. Not all guanidines show large increases in kcat. For a comparative series that includes guanidine and alkylguanidines, substituent size is inversely related to kcat. Brønsted analysis of guanidines with varying pKa values indicates that a partial positive charge is implicated in rescue, consistent with the proposed role of arginine 57 in catalysis. In UV difference and 31P-NMR spectra, carbamyl phosphate-induced effects associated with wild-type OTCase are observed in the R57G mutant only in the presence of guanidine. The kinetic mechanism of the mutant is random in the presence or absence of guanidine, in contrast to the sequential ordered mechanism of the wild-type enzyme. Thus, chemical rescue of R57G by guanidine hydrochloride restores many but not all wild-type properties to the mutant enzyme.

Annexins.

The annexins are a family of proteins that bind anionic phospholipid surfaces in a Ca(2+)-dependent manner (general reviews include Raynal & Pollard 1994, Swairjo & Seaton 1994, Seaton 1996, Mollenhauer, 1997). Due to this functional property, individual annexins have been discovered independently by numerous laboratories with diverse experimental goals. Ca2+ characteristically causes the annexins to shift from a soluble to membrane associated state. This shift is believed to be the mechanism that underlies annexin cellular function.

Binding of proteolytically processed phospholipase D from Streptomyces chromofuscus to phosphatidylcholine membranes facilitates vesicle aggregation and fusion.

Ca(2+)-dependent phospholipase D is secreted from Streptomyces chromofuscus as an intact enzyme of 57 kDa (PLD(57)). Under certain growth conditions, PLD is proteolytically cleaved and activated to form PLD(42/20) (named for the apparent size of the peptides). The PLD(42) catalytic core and 20 kDa C-terminal domain remain tightly associated through noncovalent interactions. In the presence of Ba(2+) (to enhance protein binding to zwitterionic vesicles without hydrolysis of substrate), PLD(42/20), but not PLD(57), induces POPC vesicle leakiness as measured by entrapped CF leakage. PLD(42/20) also induces vesicle fusion (as measured by light scattering, fluorescence quenching, and cryo-TEM) under these conditions (1 mM POPC, 5 mM Ba(2+)); neither PLD(42) nor PLD(20) alone can act as a fusogen. For intact PLD(57) to cause CF leakiness, the soluble activator diC(4)PA must be present. However, even with diC(4)PA, PLD(57) does not induce significant vesicle fusion. In the absence of metal ions, all PLD forms bind to PC vesicles doped with 10 mol % PA. Again, only PLD(42/20) is fusogenic and causes aggregation and fusion on a rapid time scale. Taken together, these data suggest that activated PLD(42/20) inserts more readily into the lipid bilayer than other PLD forms and creates structures that allow bilayers to fuse. Cleavage of the PLD(57) by a secreted protease to generate PLD(42/20) occurs in the late stages of S. chromofuscus cell cultures. Production of this more active and fusogenic enzyme may play a role in nutrient scavenging in stationary phase cultures.

Inactivation of chymotrypsin by 5-benzyl-6-chloro-2-pyrone: 13C NMR and X-ray diffraction analyses of the inactivator-enzyme complex.

The inactivation of chymotrypsin by 5-benzyl-6-chloro-2-pyrone has been studied. Chloride analysis of the inactivated enzyme suggests that chlorine is no longer present in the complex. 13C NMR spectroscopy of chymotrypsin inactivated with 5-benzyl-6-chloro-2-pyrone-2,6-13 C2 shows the presence of two new resonances from the protein-bound inactivator. The chemical shift values of these resonances are consistent with an intact pyrone ring on the enzyme as well as the replacement of the C-6 chlorine by a different heteroatom. X-ray diffraction analysis at 1.5-A resolution of the inactivator-enzyme complex demonstrates that the gamma-oxygen of the active site serine residue (serine 195) is covalently attached to C-6 of the inactivator and that the pyrone ring is intact. The 5-benzyl group of the inactivator is bound to the enzyme in the hydrophobic specificity pocket. The conformational changes that occur in the protein as a result of complexation with the inactivator are discussed.

Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle X-ray scattering.

We have used solution small-angle X-ray scattering to characterize bovine brain calmodulin in the presence and absence of calcium. In the presence of calcium, calmodulin exists in solution as an elongated molecule with a radius of gyration of 21.5 A and a maximum vector length of approximately 62 A. These values are consistent with the dimensions recently determined for the crystal form of rat testis calmodulin. In the absence of calcium, the calmodulin molecule is shorter, the radius of gyration decreases to 20.6 A, and the maximum vector length decreases to approximately 58 A. This change in dimensions is consistent with an overall contraction of the protein through movement of the two lobes closer to each other upon removal of calcium from calmodulin.

Purification, crystallization, and preliminary X-ray diffraction analysis of rat kidney annexin V, a calcium-dependent phospholipid-binding protein.

We have purified annexin V, a monomeric 35-kDa protein, from rat kidney using calcium-dependent phospholipid chromatography. The identity of annexin V was confirmed by immunoblot analysis using monospecific anti-annexin V antibody. Large single crystals of annexin V in the presence of calcium have been grown from ammonium sulfate under a variety of conditions, with an optimum pH range of 7.5-8.0. The crystals diffract to at least 2.2 A Bragg spacing and are stable to x-rays. Preliminary crystallographic analysis reveals the space group to be R3, with hexagonal cell dimensions of a = b = 156.8 A and c = 36.9 A, and there is one molecule/asymmetric unit.

Melittin binding causes a large calcium-dependent conformational change in calmodulin.

The interaction between calmodulin and its target protein is a key step in many calcium-regulated cellular functions. Melittin binds tightly to calmodulin in the presence of calcium and is a competitive inhibitor of calmodulin function. Using melittin as a model for the target peptide of calmodulin, we have found a large Ca2+-dependent conformational change of calmodulin in solution induced by peptide binding. Mg2+ does not substitute for Ca2+ in producing the conformation change. Small-angle x-ray scattering has shown that calmodulin exists as a dumbbell in solution, similar to that observed in the crystalline state. Our present measurements reveal that the overall structure of the Ca2+-calmodulin-melittin complex is not a dumbbell but a globular shape. Upon binding melittin, the radius of gyration decreases from 20.9 to 18.0 A and the largest dimension decreases from 60 to 47.5 A. In the absence of calcium, however, melittin has little effect on the solution structure of calmodulin.

Molecular structure of fructose-1,6-bisphosphatase at 2.8-A resolution.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) from the cortex of pig kidney and its complexes with either fructose 2,6-bisphosphate (Fru-2,6-P2) or adenosine monophosphate (AMP) have been crystallized in the space group P3(2)21. The three-dimensional structure of the native enzyme has been solved at 3.0-A resolution by the multiple isomorphous replacement method and refined at 2.8-A resolution to a crystallographic R factor of 0.194. A total of 316 of 335 residues, omitting disordered regions 1-5 and 54-67, have been built into the monomer, which has average dimensions of about 30 A by 50 A by 35 A. Four monomeric units aggregate into a molecular tetramer with D2 symmetry, which approximates a disk about 35 A thick. Each monomer consists of about 33% alpha-helix, 23% beta-strand, and 6% beta-turn. Four sites for Fru-2,6-P2 and two major sites for AMP binding per tetramer have been identified by difference Fourier techniques. The binding site for Fru-2,6-P2 is shared by two neighboring monomers and consists of side-chain atoms of Asn-212, Tyr-244, Tyr-264, and Lys-274; backbone atoms of Gly-246 through Met-248; and only Arg-243 from the adjacent subunit. In addition, Asn-125, Tyr-215, and Lys-269 are located within a distance of about 5 A of Fru-2,6-P2. A negatively charged pocket near this binding site includes Asp-118, Asp-121, Glu-280, Glu-97, and Glu-98. The AMP binding site is located near Val-17, Gln-20, Gly-21, Ala-24 through Met-30, Lys-112, Tyr-113, Arg-140, and Met-177.

Studies of calmodulin structure: laser raman spectroscopy of biomolecules.

The structure of bovine brain calmodulin was probed by using laser Raman spectroscopy to elucidate cation-induced conformational changes in the protein. Local changes, most likely reflecting metal binding but not rearrangement of the peptide backbone, were observed in the presence of calcium or magnesium. A conformational change involving the peptide backbone and secondary structure content of calmodulin was observed only in the presence of calcium. The calcium-induced conformational change in the peptide backbone involves increased alpha helix and beta sheet. This was the only major calcium-specific change observed in the Raman spectrum, which suggests that the flexibility of the backbone conformation may play a critical role in the physiological activity of calmodulin.

Stability of annexin V in ternary complexes with Ca2+ and anionic phospholipids: IR studies of monolayer and bulk phases.

Annexin V (AxV) is a member of a family of proteins that exhibit functionally relevant Ca2+-dependent binding to anionic phospholipid membranes. Protein structure and stability as a function of Ca2+ and phospholipids was studied by bulk phase infrared (IR) spectroscopy and by IR reflection-absorption spectroscopy (IRRAS) of monolayers in situ at the air/water (A/W) interface. Bulk phase experiments revealed that AxV undergoes an irreversible thermal denaturation at approximately 45-50 degreesC, as shown by the appearance of amide I bands at 1617 and 1682 cm-1. However, some native secondary structure is retained, even at 60 degreesC, consistent with a partially unfolded "molten globule" state. Formation of the Ca2+/phospholipid/protein ternary complex significantly protects the protein from thermal denaturation as compared to AxV alone, Ca2+/AxV, or lipid/AxV mixtures. Stabilization of AxV secondary structure by a DMPA monolayer in the presence of Ca2+ was also observed by IRRAS. Spectra of an adsorbed AxV film in the presence or absence of Ca2+ showed a 10 cm-1 shift in the amide I mode, corresponding to loss of ordered structure at the A/W interface. In both the bulk phase and IRRAS experiments, protection against H-->D exchange in AxV was enhanced only in the ternary complex. The combined data suggest that the secondary structure of AxV is strongly affected by the Ca2+/membrane component of the ternary complex whereas lipid conformational order is unchanged by protein.

Preliminary X-ray crystallographic studies of pig kidney fructose-1,6-bisphosphatase.

Preliminary x-ray data have been obtained from large single crystals of pig kidney fructose-1,6-bisphosphatase, grown from polyethylene glycol. The crystals have the symmetry of space group P3(1)21 or its enantiomorph P3(2)21, contain two subunits of the 146,000-dalton tetramer/asymmetric unit, and diffract to 2.9-A resolution on still photographs. The unit cell dimensions are a = b = 132.5 A and c = 68.0 A. Small single crystals have been grown in the presence of the inhibitor fructose 2,6-bisphosphate, with and without the allosteric effector AMP added. Crystals grown in the presence of both ligands are isomorphous with native crystals and generate diffraction patterns that show significant intensity changes.