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W2058484257 abstract "Annexin V is an abundant eukaryotic protein that binds phospholipid membranes in a Ca2+-dependent manner. In the present studies, site-directed mutagenesis was combined with x-ray crystallography and solution liposome binding assays to probe the functional role of a cluster of interfacial basic residues in annexin V. Four mutants were investigated: R23E, K27E, R61E, and R149E. All four mutants exhibited a significant reduction in adsorption to phospholipid membranes relative to the wild-type protein, and the R23E mutation was the most deleterious. Crystal structures of wild-type and mutant proteins were similar except for local changes in salt bridges involving basic cluster residues. The combined data indicate that Arg23 is a major determinant for interfacial phospholipid binding and participates in an intermolecular salt bridge that is key for trimer formation on the membrane surface. Together, crystallographic and solution data provide evidence that the interfacial basic cluster is a locus where trimerization is synergistically coupled to membrane phospholipid binding. Annexin V is an abundant eukaryotic protein that binds phospholipid membranes in a Ca2+-dependent manner. In the present studies, site-directed mutagenesis was combined with x-ray crystallography and solution liposome binding assays to probe the functional role of a cluster of interfacial basic residues in annexin V. Four mutants were investigated: R23E, K27E, R61E, and R149E. All four mutants exhibited a significant reduction in adsorption to phospholipid membranes relative to the wild-type protein, and the R23E mutation was the most deleterious. Crystal structures of wild-type and mutant proteins were similar except for local changes in salt bridges involving basic cluster residues. The combined data indicate that Arg23 is a major determinant for interfacial phospholipid binding and participates in an intermolecular salt bridge that is key for trimer formation on the membrane surface. Together, crystallographic and solution data provide evidence that the interfacial basic cluster is a locus where trimerization is synergistically coupled to membrane phospholipid binding. fluorescence resonance energy transfer N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) dioleoylphosphatidylserine dioleoylphosphatidylcholine Annexins comprise a large family of homologous eukaryotic proteins with a common core region that promotes Ca2+-dependent binding to phospholipid membrane surfaces (for a review, see Refs. 1Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1032) Google Scholar, 2Seaton, B. A. (ed) (1996) Annexins: Molecular Structure to Cellular Function, R. G. Landes Co., Austin, TXGoogle Scholar, 3Gerke V. Moss S.E. Biochim. Biophys. Acta. 1997; 1357: 129-154Crossref PubMed Scopus (320) Google Scholar, 4Mollenhauer J. Cell Mol. Life Sci. 1997; 53: 506-507Crossref PubMed Scopus (36) Google Scholar, 5Seaton B.A. Dedman J.R. BioMetals. 1998; 11: 399-404Crossref PubMed Scopus (54) Google Scholar). This property underlies many proposed in vivo annexin functions, including membrane trafficking, cell signaling, and roles in inflammatory and coagulation processes. Annexin V is an abundant protein that binds preferentially to acidic phospholipid membranes in the presence of Ca2+. The annexin-phospholipid association is of high affinity but is reversible with the removal of Ca2+. When bound to the membrane surface, annexin V molecules assemble laterally into well organized trimers and higher order arrays of trimers (6Oling F. Bergsma-Schutter W. Brisson A. J. Struct. Biol. 2001; 133: 55-63Crossref PubMed Scopus (93) Google Scholar). This lateral assembly promotes spontaneous two-dimensional crystallization of annexin V on membrane surfaces, a process that is inhibited by extreme membrane surface curvature (7Andree H.A.M. Stuart M.C. Hermans W.T. Reutelingsperger C.P.M. Frederik P.M. Willems G.M. J. Biol. Chem. 1992; 267: 17907-17912Abstract Full Text PDF PubMed Google Scholar, 8Swairjo M.A. Roberts M.F. Campos M.B. Dedman J.R. Seaton B.A. Biochemistry. 1994; 33: 10944-10950Crossref PubMed Scopus (42) Google Scholar). Annexin-coated membrane surfaces undergo changes in their properties, becoming markedly rigid as the crystallization domains increase in size (9Wu F. Gericke A. Flach C.R. Mealy T.R. Seaton B.A. Mendelsohn R. Biophys. J. 1998; 74: 3273-3281Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 10Venien-Bryan C. Lenne P.F. Zakri C. Renault A. Brisson A. Legrand J.F. Berge B. Biophys. J. 1998; 74: 2649-2657Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 11Megli F.M. Selvaggi M. Liemann S. Quagliariello E. Huber R. Biochemistry. 1998; 37: 10540-10546Crossref PubMed Scopus (30) Google Scholar). This membrane-bound layer of annexin V, which has anticoagulant properties, is believed to play a functional role in processes associated with the protein. In placenta, reduced levels of annexin V occur with anti-phospholipid syndrome, which causes hypercoagulation and recurrent miscarriage. Rand and co-workers (12Rand J.H. Wu X.X. Thromb. Haemost. 1999; 82: 649-655Crossref PubMed Scopus (66) Google Scholar, 13Rand J.H. J. Autoimmun. 2000; 15: 107-111Crossref PubMed Scopus (66) Google Scholar) have proposed a mechanism for anti-phospholipid syndrome in which the disease process strips off the protective layer of annexin V at the maternal-fetal interface, permitting excessive thrombosis to occur. The mechanism of membrane adsorption of annexin V has generated considerable interest from many disciplines, from clinical to structural biology. The protein provides an excellent model for elucidating complex interfacial behavior by peripheral membrane proteins. The molecular structure of the 35-kDa protein has been well characterized in both soluble and membrane-bound forms by x-ray crystallography (14Huber R. Schneider M. Mayr I. Römisch J. Pâques E.P. FEBS Lett. 1990; 275: 15-21Crossref PubMed Scopus (216) Google Scholar, 15Huber R. Berendes R. Burger A. Schneider M. Karshikov A. Luecke H. Römisch J. Pâques E.P. J. Mol. Biol. 1992; 223: 683-704Crossref PubMed Scopus (236) Google Scholar, 16Concha N.O. Head J.F. Kaetzel M.A. Dedman J.R. Seaton B.A. Science. 1993; 261: 1321-1324Crossref PubMed Scopus (167) Google Scholar, 17Sopkova J. Renouard M. Lewit-Bentley A. J. Mol. Biol. 1993; 234: 816-825Crossref PubMed Scopus (114) Google Scholar, 18Bewley M.C. Boustead C.M. Walker J.H. Waller D.A. Huber R. Biochemistry. 1993; 32: 3923-3929Crossref PubMed Scopus (56) Google Scholar) and cryoelectron microscopy (19Olofsson A. Mallouh V. Brisson A. J. Struct. Biol. 1994; 113: 199-205Crossref PubMed Scopus (44) Google Scholar, 20Voges D. Berendes R. Burger A. Demange P. Baumeister W. Huber R. J. Mol. Biol. 1994; 238: 199-213Crossref PubMed Scopus (162) Google Scholar, 21Oling F. Sopkova-de Oliviera Santos J. Govorukhina N. Mazeres-Dubut C. Bergsma-Schutter W. Oostergetel G. Keegstra W. Lambert O. Lewit-Bentley A. Brisson A. J. Mol. Biol. 2000; 304: 561-573Crossref PubMed Scopus (72) Google Scholar), respectively. Monomeric annexin V possesses four internally homologous domains, arranged in pairs. The molecule possesses an extensive membrane binding surface, from which calcium-binding loops protrude. Ca2+ bridges, in which the Ca2+ ion is jointly coordinated by protein and phospholipid oxygen ligands, form at these loops (22Swairjo M.A. Concha N.O. Kaetzel M.A. Dedman J.R. Seaton B.A. Nat. Struct. Biol. 1995; 2: 968-974Crossref PubMed Scopus (283) Google Scholar). Formation of annexin V homotrimers occurs only on membrane surfaces, suggesting that the interfacial surface provides the correct orientation of the bound monomers for oligomerization. The forces that drive this process have not been characterized at the atomic level. The present studies provide evidence that an interfacial cluster of basic residues synergistically couples membrane phospholipid binding and trimerization on the membrane surface. Site-directed mutagenesis was performed using the Clontech transformer kit as described previously (23Campos B., Mo, Y.D. Mealy T.R., Li, C.W. Swairjo M.A. Balch C. Head J.F. Retzinger G. Dedman J.R. Seaton B.A. Biochemistry. 1998; 37: 8004-8010Crossref PubMed Scopus (47) Google Scholar). To produce the R23E, R61E, and R149E mutants, the GGG codon was changed to GAG. For the K27E mutant, the AAA codon was changed to GAA. Each mutation was verified by double-stranded DNA sequence analysis. Proteins were expressed from cultures of Escherichia coli strain JM101 and purified using lipid affinity chromatography as described (23Campos B., Mo, Y.D. Mealy T.R., Li, C.W. Swairjo M.A. Balch C. Head J.F. Retzinger G. Dedman J.R. Seaton B.A. Biochemistry. 1998; 37: 8004-8010Crossref PubMed Scopus (47) Google Scholar). Protein concentration was measured using the Pierce BCA protein assay. The fluorescence resonance energy transfer (FRET)1 assay, which is based upon self-quenching of the NBD chromophore that results from annexin-induced clustering of phospholipids (24Bazzi M.D. Nelsestuen G.L. Biochemistry. 1991; 30: 7961-7969Crossref PubMed Scopus (93) Google Scholar), was performed as described previously (23Campos B., Mo, Y.D. Mealy T.R., Li, C.W. Swairjo M.A. Balch C. Head J.F. Retzinger G. Dedman J.R. Seaton B.A. Biochemistry. 1998; 37: 8004-8010Crossref PubMed Scopus (47) Google Scholar) using large (100-nm) unilamellar vesicles containing 85 mol % phosphatidylcholine and 15 mol % 1–18:1,2-C12-NBD-PS. Measurements were obtained using a 95% confidence level (p = 0.05). The sedimentation assay, which measures the Ca2+ concentration required for half-maximal liposome binding, also was performed as described previously using small unilamellar vesicles of 1:1 mixtures of DOPS/DOPC (Avanti Polar Lipids) (23Campos B., Mo, Y.D. Mealy T.R., Li, C.W. Swairjo M.A. Balch C. Head J.F. Retzinger G. Dedman J.R. Seaton B.A. Biochemistry. 1998; 37: 8004-8010Crossref PubMed Scopus (47) Google Scholar). Briefly, mixtures of annexin V and phospholipid vesicles were incubated together under a range of CaCl2 concentrations, where CaCl2 is added in 5–10 μm increments to buffer containing 0.1 mm EGTA, and then centrifuged. The Centricon-filtered pellet and its filtrate were collected at each CaCl2concentration and subjected to SDS-PAGE. The half-maximal Ca2+ concentration is found where the relative band intensities of the filtrate and retentate (reflecting free and bound protein populations, respectively) are approximately equal as detected by eye and by densitometry. The half-maximal Ca2+concentration is measured experimentally as falling within a concentration range, where, at the lower concentration, the filtrate band is greater than that of the retentate, and, at the higher concentration, the reverse is true. The same buffer solutions are used for all the protein samples, providing a qualitative but highly reproducible hierarchy for the set of annexin V wild-type and mutant proteins. It should be noted that the Ca2+-dependent association of wild-type annexin V with these vesicles is sufficiently tight that no protein can be detected in filtrate samples prepared as described, even with no CaCl2 added to the EGTA-containing buffer. For the DOPS/DOPC vesicle binding studies, centrifugation and filtration were used again to separate free annexin V from that portion bound to the vesicles using the protocol of Feng et al.(25Feng J. Wehbi H. Roberts M.F. J. Biol. Chem. 2002; 277: 19867-19875Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). A 10 mm stock of unilamellar vesicles was prepared by multiple passages of aqueous 1:4 DOPS/DOPC solution through 100-nm pore diameter polycarbonate membranes (annexin V binds only to the DOPS component). Samples for the binding assays were prepared with 0.4 μm annexin V in 5 mm KCl, 2.5 mmHEPES-buffer, pH 7.5, 1 mm CaCl2. The bulk concentrations of DOPS were 0, 1, 2, 3, 4, 6, or 8 μm for all annexin V samples. After a 45-min incubation at room temperature, the samples were applied to 2-ml Centricon YM100 (100-kDa molecular mass cut-off) filters, which were centrifuged at 990 ×g for 90 min or until all of the solution had passed through the filter. The filtrate, which contained the unbound protein, was collected and lyophilized overnight. SDS-PAGE (12.5% polyacrylamide) was used to quantitate the free annexin V. Samples were prepared by adding 35 μl of sample buffer that did not contain any buffering agent. Gels were stained with Coomassie Blue. After destaining, the gels were dried, scanned, and imaged using the public domain software, ImageJ (by Wayne Rasband; available on the World Wide Web at rsb.info.nih.gov/ij) to monitor annexin V band intensities. Comparison of band intensities with an annexin V sample incubated without vesicles was used to measure the fraction of free protein (P f/P t, whereP t is the total amount of protein). The bound fraction, P b, was evaluated as (1 −P f/P t). The wild-type data fit a standard “noncooperative” Langmuir adsorption isotherm (26Cho W. Bittova L. Stahelin R.V. Anal. Biochem. 2001; 296: 153-161Crossref PubMed Scopus (114) Google Scholar), which yielded the dissociation constant, K d, and the number of lipid binding sites per annexin molecule. For the mutants, significant sigmoidicity was observed in their binding curves, so the apparent dissociation constants,K d(app), defined as the lipid concentration giving rise to half-maximal membrane binding of the protein, were estimated graphically derived from the binding curves. In addition, the binding isotherms were fit to the equationP b/P t = 1/(1 + ((K d)app/[DOPS]) h ) to yieldK d(app) and the Hill coefficient,h. For all three liposome binding assays, measurements were obtained in triplicate. Three of the four recombinant rat annexin V mutants, R23E, K27E, and R149E, crystallized under the same conditions as the wild-type protein (i.e. vapor diffusion against a reservoir of 34–40% saturated ammonium sulfate, 50 mm HEPES buffer, pH 8.2, 2 mm dithiothreitol, and 10 mmCaCl2). The final protein and CaCl2concentrations after equilibration were 14 mg/ml and 20 mm, respectively. The R61E mutant failed to crystallize. X-ray data were collected at room temperature on an R-Axis IIc imaging plate detector mounted on a Rigaku RU-300 rotating anode generator. Data were indexed, analyzed, and scaled using DENZO and SCALEPACK (27Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The mutant proteins produced R3 crystals isomorphous with wild-type rat annexin V (16Concha N.O. Head J.F. Kaetzel M.A. Dedman J.R. Seaton B.A. Science. 1993; 261: 1321-1324Crossref PubMed Scopus (167) Google Scholar), with one molecule per asymmetric unit. Partial merohedral twinning was detected (28Yeates T.O. Methods Enzymol. 1997; 276: 344-358Crossref PubMed Scopus (358) Google Scholar) in the R23E mutant data. A twin fraction of 0.213 was obtained using the twinning operator h, -h-k, -l, and the data were detwinned using CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Initial rigid body refinement using the four domains as separate bodies produced R-free values of ∼0.3. Manual model building and refinement were carried out iteratively using O (30Jones T.A. Kjeldgaard M. O: The Manual. Uppsala, Uppsala, Sweden1992Google Scholar) and CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), respectively, using simulated annealing protocols, difference electron density maps, and simulated annealing omit maps (31Brünger A.T. Krukowski A. Erickson J. Acta Crystallogr. Sect. A. 1990; 46: 585-593Crossref PubMed Scopus (600) Google Scholar). Model quality was assessed throughout refinement using PROCHECK (32Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 282-291Crossref Google Scholar) and by monitoring R-free (33Brünger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3872) Google Scholar). Data collection and refinement statistics for the mutant structures are presented in Table I.Table ICrystallographic statistics for annexin V mutant structuresParameterValueR23EK27ER149EData collection Unit cell dimensions, a and c(Å)157.76, 36.76157.60, 37.06157.54, 36.38 Maximum resolution (Å)2.702.002.00 No. of reflections measured84602192422384 Completeness (%), overall (highest resolution shell)93.8 (89.8)96.7 (92.0)99.7 (99.5) R merge, 1-aRmerge = Σ ‖I i − 〈I〉 ‖ /ΣI i × 100, where I i is the intensity of an individual reflection and 〈I〉 is the mean intensity of that reflection.overall (highest resolution shell)12.6 (23.4)6.4 (23.6)7.7 (29.2)Refinement Resolution range (Å)50–3.050–2.150–2.1 Rcryst1-bRcryst = Σ ∥F p ‖ − ‖ F calc ∥ / Σ ‖F p‖ × 100, where ‖F calc‖ is the calculated structure factor.0.16310.18700.1927 R free1-cRfree is as defined by Brünger (33).0.24120.22340.2395 No. of protein atoms (mean B in Å2)2502 (27.2)2502 (34.7)2502 (40.1) No. of atoms total (mean B in Å2)2514 (27.3)2717 (35.7)2662 (40.1) No. of ions (Ca2+/SO42−)2/25/35/3 No. of water molecules0195140 r.m.s.d.1-dr.m.s.d., root mean square deviation. angles (Å2)0.0096490.0055310.005859 r.m.s.d.1-dr.m.s.d., root mean square deviation. bonds (degrees)1.528001.101841.11217Ramachandran plot regions, No. of residues (%) Most favored84.895.294.1 Additional allowed15.24.25.5 Generously allowed0.00.70.3 Disallowed0.00.00.01-a Rmerge = Σ ‖I i − 〈I〉 ‖ /ΣI i × 100, where I i is the intensity of an individual reflection and 〈I〉 is the mean intensity of that reflection.1-b Rcryst = Σ ∥F p ‖ − ‖ F calc ∥ / Σ ‖F p‖ × 100, where ‖F calc‖ is the calculated structure factor.1-c Rfree is as defined by Brünger (33Brünger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3872) Google Scholar).1-d r.m.s.d., root mean square deviation. Open table in a new tab The four mutants and wild-type annexin V were assayed in the presence of calcium and 1:1 DOPS/DOPC liposomes. In FRET measurements, which reflect the extent of annexin-induced clustering of acidic phospholipids following membrane adsorption, the mutants produced less quenching than wild-type annexin V. The order of quenching in the FRET experiments was as follows: wild type > R149E > K27E > R61E > R23E (Fig. 1). A similar hierarchy was apparent from the liposome sedimentation assay measuring the half-maximal Ca2+ concentrations required for membrane binding (Fig. 2), except that R61E was more similar to the K27E than to the R23E phenotype. In this assay, higher half-maximal Ca2+ concentrations reflect lower intrinsic affinity for membranes (i.e. higher levels of Ca2+ offset weaker membrane binding affinity due to the synergism between Ca2+ and membrane binding in this system). Taken together, these assays demonstrate that the mutants exhibit lower membrane affinity than wild-type protein, with R23E showing the weakest interaction.Figure 2Liposome sedimentation assay comparing calcium dependence of the membrane association of annexin V wild-type and mutants. The arrows indicate the midpoint of the range in which the half-maximal calcium concentration for membrane binding falls. The stated CaCl2 concentrations refer to the amount added to a buffer containing 0.1 mm EGTA.View Large Image Figure ViewerDownload (PPT) Quantitative analysis of the binding of annexin V proteins to DOPS/DOPC vesicles showed the wild-type protein bound most tightly, with aK d of 20 nm. The wild-type data fit well to a Langmuir isotherm and yielded a value of 4.2 ± 0.3 forn, the number of lipid molecules bound per annexin V molecule. In contrast, the binding isotherms for the mutants were distinctly sigmoidal (Fig. 3), consistent with their weaker binding and greater dependence upon clustering of acidic lipids for adsorption. Hill coefficients were similar (h = 3–4) in all four mutants. The apparentK d values were in the range of 1–5 μmand showed a trend: R149E < K27E < R61E = R23E (TableII). The apparent K dvalues measured at this fixed protein concentration are all higher than the apparent K d extracted for the wild-type protein (<1 μm; data not shown).Table IIComparative binding of annexin V mutants to liposomes containing DOPSMutantApparentK dHill coefficientμmR149E1.56 ± 0.073.75 ± 0.46K27E3.32 ± 0.173.20 ± 0.43R61E4.76 ± 0.233.35 ± 0.35R23E4.73 ± 0.323.90 ± 0.75 Open table in a new tab In crystallization trials, K27E and R149E mutants behaved nearly as well as wild-type annexin V, forming crystals that grew quickly and diffracted well (2.0-Å resolution versus 1.8 Å for wild type), whereas the R23E and R61E mutants performed very poorly during crystallization trials. The R61E mutant failed to crystallize, and the R23E mutant produced over several months only a single, merohedrally twinned crystal. This crystal produced no observable diffraction beyond 2.7-Å resolution, and data were only usable to 3.0 Å. The tertiary structures of the R23E, K27E, and R149E mutants are essentially identical to that of the wild-type protein, with root mean square deviations in C-α atoms of 0.494, 0.356, and 0.344 Å2, respectively. Two to five Ca2+ ions are observed in the structures. The Ca2+ ion bound in the IAB loop is present in all of the structures. Another shared feature is a Ca2+ ion found in the IIIAB loop (residues 182–188) and a sulfate ion coordinating that Ca2+. Structural differences between the wild-type and mutant structures are localized to the immediate vicinity of the basic cluster (Fig. 4). 1) In R149E, the Arg61forms a strong salt bridge with the mutated Glu149′ side-chain (residues from the adjacent subunit are designated with a prime). To form this new bridge, the Arg61 side chain moves 0.5 Å away from Arg23. 2) In K27E, the mutated Glu27 interacts only with solvent. The substitution removes the intermolecular Lys27–Glu190′ salt bridge. 3) In R23E, more significant alterations are observed: (i) Arg61 forms a new intramolecular salt bridge with the mutated Glu23 side chain; (ii) neither Arg23nor Arg61 bind the sulfate ion, which remains associated with the protein only via the Gly28 main chain; and (iii) the Arg23-Glu190′ interaction is lost, and Glu190′ forms an intermolecular salt bridge only with Lys27. Surface clusters of basic amino acids occur infrequently in proteins, where they usually play specialized roles. Proteins that utilize basic clusters for their association with phospholipid membranes include some forms of secretory phospholipase A2(34Snitko Y. Koduri R.S. Han S.K. Othman R. Baker S.F. Molini B.J. Wilton D.C. Gelb M.H. Cho W. Biochemistry. 1997; 36: 14325-14333Crossref PubMed Scopus (110) Google Scholar, 35Lee B.I. Dua R. Cho W. Biochemistry. 1999; 38: 7811-7818Crossref PubMed Scopus (10) Google Scholar), pp60 src (36Sigal C.T. Zhou W. Buser C.A. McLaughlin S. Resh M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12253-12257Crossref PubMed Scopus (218) Google Scholar), calcineurin B (37Martin B.A. Oxhorn B.C. Rossow C.R. Perrino B.A. J. Biochem. (Tokyo). 2001; 129: 843-849Crossref PubMed Scopus (10) Google Scholar), and type IIβ phosphatidylinositol phosphate kinase (38Burden L.M. Rao V.D. Murray D. Ghirlando R. Doughman S.D. Anderson R.A. Hurley J.H. Biochemistry. 1999; 38: 15141-15149Crossref PubMed Scopus (31) Google Scholar). In those studies, the roles of membrane interfacial cationic residues were investigated using site-directed mutagenesis and membrane binding and enzyme activity assays. The present study takes this approach a step further by additionally evaluating the structural consequences of each mutation at the atomic level with x-ray crystallography to make detailed structure-function correlations. Membrane adsorption properties for annexin V mutants were determined through liposome binding assays. The use of x-ray crystallography in the present work provides not only high resolution structural details but also a relative measure of annexin V trimer formation capabilities for the mutants. Since annexin V trimer formation is reversible and occurs only on membrane surfaces, trimerization is difficult to measure directly in solution, and no reliable assay has been developed. However, the ability to form stable trimers is requisite to form R3 crystals. Studies combining high resolution cryoelectron microscopy with x-ray crystallography have demonstrated that for annexin V, the trimers obtained from two-dimensional crystals on lipid monolayers or three-dimensional crystals from solution have the same structure (21Oling F. Sopkova-de Oliviera Santos J. Govorukhina N. Mazeres-Dubut C. Bergsma-Schutter W. Oostergetel G. Keegstra W. Lambert O. Lewit-Bentley A. Brisson A. J. Mol. Biol. 2000; 304: 561-573Crossref PubMed Scopus (72) Google Scholar). Three-dimensional crystallization thus can provide a useful comparative measure of the propensity of wild-type and mutant annexin V proteins to form trimers and higher order aggregates. In wild-type annexin V, the basic cluster is situated between trimer subunits at the largely hydrophilic subunit interface (Figs. 4 a and 5). The cationic side chains of the cluster reside in a cleft slightly above the plane of the Ca2+-binding loops that comprise the membrane surface. The cationic functional groups of Lys27, Arg23, Arg61, and Arg149′ form a closely spaced row, each separated by 4–5 Å (Fig. 4 a). Arg23, Lys27, and Arg61, all from domain I, each participate in one or more electrostatic interactions, mostly strong salt bridges with lengths of <3 Å (TableIII). An intermolecular salt bridge is made between Arg61 and Asp66, whereas Glu190′ from the adjacent subunit makes intermolecular salt bridges with Arg23 and Lys27 side chains. Arg149′ and Glu190′ are from domains II and III, respectively. Arg149′ does not form any salt bridges, although it makes up part of the cluster.Table IIIElectrostatic interactions at the annexin V basic clusterWild typeK27ER149ER23EArg23SO4 O1(NH1)3.28SO4 O2(NH1)3.15Mutated to GluSO4 O2(NH2)2.85SO4 O1(NH2)2.59SO4 O1(NH2)2.98E190′ OE1(NH2)3.07E190′ OE1(NH2)3.13E190′ OE1(NH2)3.46E190′ OE1(NE)2.93E190′ OE1(NE)3.03E190′ OE2(NE)3.15Arg61SO4 O1(NH2)3.59SO4 O2(NH2)3.98SO4 O2(NH2)3.50SO4 O1(NH1)3.92SO4 O2(NH2)4.04D66 OD1(NH1)3.86D66 OD1(NH2)3.34D66 OD2(NH2)3.43D66 OD1(NE)3.87D66 OD2(NH1)3.98D66 OD2(NE)2.82D66 OD1(NE)2.96D66 OD1(NH1)2.86E149′ OE2(NH1)2.66E23 OE1(NH2)3.77E23 OE2(NH1)2.36Lys27E190′ OE2(NZ)2.97Mutated to GluE190′ OE1(NZ)2.48E190′ OE2(NZ)3.76E190′ OE1(NZ)3.56E23 OE1(NZ)2.65Arg149′Mutated to GluGly28SO4 O3(N)2.31SO4 O3(N)2.81SO4 O4(N)2.92SO4 O4(N)2.69 Open table in a new tab Bound sulfate ions in several annexin V crystal structures have usefully located potential phospholipid binding sites. In structures of wild-type annexin V crystallized from ammonium sulfate, a strongly bound sulfate ion forms one of seven ligands in the primary coordination shell of the Ca2+ ion in the IIIAB loop (15Huber R. Berendes R. Burger A. Schneider M. Karshikov A. Luecke H. Römisch J. Pâques E.P. J. Mol. Biol. 1992; 223: 683-704Crossref PubMed Scopus (236) Google Scholar, 17Sopkova J. Renouard M. Lewit-Bentley A. J. Mol. Biol. 1993; 234: 816-825Crossref PubMed Scopus (114) Google Scholar). In the crystal structures of two ternary complexes formed between annexin V, Ca2+, and phospholipid head group analogs, this sulfate ion site is occupied by a phosphoryl group (21Oling F. Sopkova-de Oliviera Santos J. Govorukhina N. Mazeres-Dubut C. Bergsma-Schutter W. Oostergetel G. Keegstra W. Lambert O. Lewit-Bentley A. Brisson A. J. Mol. Biol. 2000; 304: 561-573Crossref PubMed Scopus (72) Google Scholar). Another significant binding site for sulfate ion is observed at the basic cluster in wild-type annexin V (Fig. 4 a). This sulfate ion forms several interactions with the protein: salt bridges with Arg23 and Arg61 side chains and a hydrogen bond to the amide nitrogen of Gly28 in the IAB Ca2+-binding loop (residues 26–32). Despite its close proximity (6 Å) to the IAB Ca2+ ion, there is no direct coordination between the metal and sulfate ions as occurs in the IIIAB loop. Instead, Gly28 coordinates the metal ion via its carbonyl oxygen while forming a bond to the sulfate ion through its amide nitrogen. This bridging arrangement is an example of the synergy between Ca2+ and phospholipids in the annexin system. The membrane binding behavior of annexin V is complex, with many interrelated variables, including calcium concentration, lipid composition, and membrane curvature. We used three different liposome binding assays to probe the effect of mutation on annexin-membrane interactions. In our studies, all four mutants exhibit significantly reduced adsorption to acidic membranes, compared with wild type, confirming the importance of the basic cluster in membrane binding. Since these are charge reversal mutations, some loss of affinity in binding to acidic membranes would be expected simply from reducing the overall net positive charge of the cluster from +4 to +2. However, the varied impairment of the different phenotypes demonstrates that the mutated residues are not functionally interchangeable. In all three binding assays, R149E is the most similar to wild type, followed by K27E, and the R23E mutant is the most deleterious, whereas the properties of R61E range between those of K27E and R23E, depending upon the assay. In terms of calcium dependence, R61E behaves more like K27E; in the FRET assay, it falls in between K27E and R23E, and its K d(app) for DOPS binding is equivalent to that of R23E. The structural basis of the R61E properties could not be determined since R61E failed to crystallize. For the structurally characterized mutants, a direct correlation can be made between membrane binding affinity (R149E > K27E > R23E) and the number of bonds formed between the sulfate ion and Arg23 (R149E, 2; K27E, 1; R23E, 0). The number of Arg61–sulfate bonds appears to be less critical but has sufficient influence to reduce membrane binding affinity of R149E relative to wild-type annexin V. In wild-type protein, the sulfate ion is held by two sets of interactions: (i) a hydrogen bond with Gly28 and (ii) salt bridges to the Arg23 and Arg61 guanidinium groups, each providing two bonds to the sulfate. In the mutants, the sulfate–Gly28 hydrogen bond is unaffected, but interactions with the basic cluster vary by phenotype (Table III, Fig. 4). For R149E, the least deleterious mutant phenotype, the Arg23–sulfate bond is unperturbed. However, the Arg61 guanidinium group shifts 0.5 Å (centroid-centroid distance) away from the sulfate ion, removing one bond to the sulfate and lengthening the bond distance for the other. In K27E, the Arg23 guanidinium group reorients its position to form a salt bridge with the mutated Glu27. This shift removes one Arg23–sulfate bond and lengthens both Arg61–sulfate bonds. For R23E, contact is lost completely between the sulfate ion and the basic cluster; the mutation has removed the Arg23 side chain and shifted the Arg61guanidinium group >2 Å away from the sulfate. Apart from distinctions in their membrane adsorption properties, the mutants also exhibit varying abilities to form quarternary structure through self-association. In this property, Arg23 again appears to play a key role, this time through formation of an intermolecular salt bridge with Glu190′. The mutational analysis suggests that the Arg23-Glu190′ ion pair is a key feature in promoting oligomerization and crystallization. In wild-type annexin V, the importance of having an intermolecular bridge at this site is supported by its apparent functional redundancy; both Arg23and Lys27 form salt-bridges to Glu190′. The arginine guanidinium group makes more interactions with Glu190′ than does the lysine ε-amino group, giving the Arg23-Glu190′ bridge greater stabilizing power. The combined results show that if Lys27 is removed but Arg23 is retained, as in the K27E mutant, crystallization is not much different from wild type. However, in the reverse case, when Arg23 is removed, leaving only Lys27, as in the R23E mutant, crystallization is severely impaired. The net number of intermolecular salt bridges, surprisingly, is less important to crystallization. The R149E mutant forms one more salt bridge (Arg61-Glu149′) than is found in wild-type, yet R149E crystals are more similar to those of K27E, diffracting to resolution than wild-type. The effects of the R61E mutation on trimer formation can only be speculative in the absence of crystallographic data. In the wild-type protein, Arg61 makes no intermolecular bonds and would seem to have little influence on protein-protein interactions. However, it is possible that in R61E, the mutated Glu61 competes with Glu190′ for the Arg23 guanidinium group, thereby interfering with Arg23-Glu190′ salt bridge formation and inhibiting crystallization. The basic cluster is not invariant in annexins but appears to be restricted to the subset that have been shown to form trimers on membrane surfaces: annexins IV (39Kaetzel M.A., Mo, Y.D. Mealy T.R. Campos B. Bergsma-Schutter W. Brisson A. Dedman J.R. Seaton B.A. Biochemistry. 2001; 40: 4192-4199Crossref PubMed Scopus (73) Google Scholar), V (21Oling F. Sopkova-de Oliviera Santos J. Govorukhina N. Mazeres-Dubut C. Bergsma-Schutter W. Oostergetel G. Keegstra W. Lambert O. Lewit-Bentley A. Brisson A. J. Mol. Biol. 2000; 304: 561-573Crossref PubMed Scopus (72) Google Scholar), and VI (40Driessen H.P. Newman R.H. Freemont P.S. Crumpton M.J. FEBS Lett. 1992; 306: 75-79Crossref PubMed Scopus (25) Google Scholar). This distribution suggests that the basic cluster plays a specialized role in these particular annexins (i.e. stabilizing trimer subunit interactions). The crystal structure of annexin IV (Thr6→ Asp mutant; PDB code 1I4A) crystallized from ammonium sulfate presents the four conserved basic residues in a subtly different spatial arrangement than in annexin V. However, the cluster appears to be functionally similar in the two proteins. In annexin IV (Fig. 4 e), the Arg23, Lys27, Arg61, and Arg149′ side chains form a circular cationic patch that binds a sulfate ion, forming numerous strong salt bridges with Arg23, Lys27, and Arg149′. The Arg61 side chain does not interact with the sulfate ion but may contribute to the net cationic character of the cluster, as does Arg149′ in annexin V. Glu190′, the important salt bridge partner of Arg23 and Lys27 in annexin V, is not conserved in annexin IV and is replaced by valine. Instead of the Arg23-Glu190′ salt bridge, the sulfate ion cross-links the two subunits by forming a complex network of strong salt bridges with Arg23, Lys27, and Arg149′. In serving as a subunit cross-linker, the sulfate ion moves away from the calcium binding loop to take a position spatially analogous to the carboxylate group of Glu190′ in annexin V. This alternate but effective arrangement in annexin IV provides another example of how phospholipid binding and trimerization are coupled at the basic cluster site. In annexin VI, the spatial arrangement of basic cluster residues more closely resembles that of annexin V than IV. Annexin VI is a unique, eight-repeat annexin in which each half of the molecule (designated VIA or VIB) corresponds to the core region of a tetrad repeat annexin. In annexin VI, the two tetrad lobes A and B are rotated about 90° from each other, connected by a linker region. However, the two lobes can adopt different relative orientations (41Benz J. Berger A. Hofmann A. Demange P. Gottig P. Liemann S. Huber R. Voges D. J. Mol. Biol. 1996; 260: 638-643Crossref PubMed Scopus (91) Google Scholar, 42Avila-Sakar A.J. Kretsinger R.H. Creutz C.E. J. Struct. Biol. 2000; 130: 54-62Crossref PubMed Scopus (35) Google Scholar). In the x-ray crystal structure of annexin VI (PDB code 1AVC) (43Avila-Sakar A.J. Creutz C.E. Kretsinger R.H. Biochim. Biophys. Acta. 1998; 1387: 103-116Crossref PubMed Scopus (49) Google Scholar), the basic cluster occurs only in the VIB lobe, where Arg373, Lys377, and Arg411 correspond to Arg23, Lys27, and Arg61 in annexins IV and V. The existence of a functionally equivalent residue to Arg149′ in annexins IV and V is difficult to predict, since the tertiary and quaternary structure of annexin VI is more complex. Furthermore, in this structure, the crystallization medium did not contain sulfate ions (43Avila-Sakar A.J. Creutz C.E. Kretsinger R.H. Biochim. Biophys. Acta. 1998; 1387: 103-116Crossref PubMed Scopus (49) Google Scholar). Nonetheless, despite the structural differences, most features of the basic cluster are retained in VI, including the intramolecular salt bridge between Arg411 and Arg416 (equivalent to Arg61-Arg66 annexins IV and V) and an intermolecular salt bridge between Arg373 and Glu27′ (equivalent to Arg23-Glu190′ in annexin V). Whereas the precise details may differ among these trimer-forming annexins, their oligomerization mechanism on membrane surfaces may involve the same key elements. The central influence of Arg23 or its equivalent in mediating and facilitating protein-protein contacts is a common feature in annexins IV, V, and VI. Whereas data are not available yet for annexin VI, annexins IV and V both couple phospholipid binding and subunit stabilization through mechanisms involving the basic cluster; in annexin V, cross-linking of subunits is promoted via the intermolecular Arg23-Glu190′ salt bridge and in annexin IV, cross-linking is promoted through a sulfate ion that bridges basic cluster side chains in adjacent subunits." @default.
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W2058484257 title "Interfacial Basic Cluster in Annexin V Couples Phospholipid Binding and Trimer Formation on Membrane Surfaces" @default.
W2058484257 cites W1485863323 @default.
W2058484257 cites W1539796472 @default.
W2058484257 cites W1552375346 @default.
W2058484257 cites W1586792701 @default.
W2058484257 cites W1970057409 @default.
W2058484257 cites W1970821321 @default.
W2058484257 cites W1973124178 @default.
W2058484257 cites W1986051643 @default.
W2058484257 cites W1986191025 @default.
W2058484257 cites W1990704484 @default.
W2058484257 cites W1992400924 @default.
W2058484257 cites W1994155247 @default.
W2058484257 cites W1995017064 @default.
W2058484257 cites W1996607909 @default.
W2058484257 cites W1998712463 @default.
W2058484257 cites W2002953560 @default.
W2058484257 cites W2004152842 @default.
W2058484257 cites W2020994777 @default.
W2058484257 cites W2021420028 @default.
W2058484257 cites W2028736165 @default.
W2058484257 cites W2033735740 @default.
W2058484257 cites W2034957051 @default.
W2058484257 cites W2038368604 @default.
W2058484257 cites W2039388732 @default.
W2058484257 cites W2040832198 @default.
W2058484257 cites W2041181454 @default.
W2058484257 cites W2049193784 @default.
W2058484257 cites W2054721967 @default.
W2058484257 cites W2059626243 @default.
W2058484257 cites W2060448324 @default.
W2058484257 cites W2069751978 @default.
W2058484257 cites W2072637321 @default.
W2058484257 cites W2079295410 @default.
W2058484257 cites W2083248905 @default.
W2058484257 cites W2095572871 @default.
W2058484257 cites W2113005146 @default.
W2058484257 cites W2131082171 @default.
W2058484257 cites W2148340685 @default.
W2058484257 cites W2156303945 @default.
W2058484257 cites W2169459924 @default.
W2058484257 cites W4251847509 @default.
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