The N1317H Substitution Associated with Leber Congenital Amaurosis Results in Impaired Interdomain Packing in Human CRB1 Epidermal Growth Factor-like (EGF) Domains
2007; Elsevier BV; Volume: 282; Issue: 39 Linguagem: Inglês
10.1074/jbc.m704015200
ISSN1083-351X
AutoresJason A. Davis, Penny A. Handford, Christina Redfield,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoThe calcium-binding epidermal growth factor-like (cbEGF) domain is a widely occurring module in proteins of diverse function. Amino acid substitutions that disrupt its structure or calcium affinity have been associated with various disorders. The extracellular portion of CRB1, the human homologue of Drosophila Crumbs, exhibits a modular domain organization that includes EGF and cbEGF domains. The N1317H substitution in the 19th cbEGF domain of CRB1 is associated with the serious visual disorder Leber congenital amaurosis. We have investigated the structure and Ca2+ binding of recombinant wild-type and N1317H CRB1 fragments (EGF18-cbEGF19) using NMR and find that Ca2+ binding is altered, resulting in disruption of long range interactions between adjacent EGF domains in CRB1. From these observations, we propose that this substitution affects the structural integrity of CRB1 in the inter-photoreceptor matrix of the retina, where it is expressed. Furthermore, we identify disease-causing substitutions in other cbEGF-containing proteins that are likely to result in similar disruption of interdomain packing, supporting the hypothesis that the tandem cbEGF domain linkages are critical for the structure and function of proteins containing cbEGF domains. The calcium-binding epidermal growth factor-like (cbEGF) domain is a widely occurring module in proteins of diverse function. Amino acid substitutions that disrupt its structure or calcium affinity have been associated with various disorders. The extracellular portion of CRB1, the human homologue of Drosophila Crumbs, exhibits a modular domain organization that includes EGF and cbEGF domains. The N1317H substitution in the 19th cbEGF domain of CRB1 is associated with the serious visual disorder Leber congenital amaurosis. We have investigated the structure and Ca2+ binding of recombinant wild-type and N1317H CRB1 fragments (EGF18-cbEGF19) using NMR and find that Ca2+ binding is altered, resulting in disruption of long range interactions between adjacent EGF domains in CRB1. From these observations, we propose that this substitution affects the structural integrity of CRB1 in the inter-photoreceptor matrix of the retina, where it is expressed. Furthermore, we identify disease-causing substitutions in other cbEGF-containing proteins that are likely to result in similar disruption of interdomain packing, supporting the hypothesis that the tandem cbEGF domain linkages are critical for the structure and function of proteins containing cbEGF domains. CRB1 is the human homologue of Drosophila Crumbs, a well conserved gene with homologues across multiple phyla (1Richard M. Roepman R. Aartsen W.M. van Rossum A.G. den Hollander A.I. Knust E. Wijnholds J. Cremers F.P. Hum. Mol. Genet. 2006; 15: R235-R243Crossref PubMed Scopus (108) Google Scholar). In Drosophila, the Crumbs protein is expressed in all ectodermally derived epithelial cells, which includes photoreceptors, and its disruption results in a discontinuous cuticle and extensive cell death (2Jürgens G. Wieschaus E. Nüsslein-Volhard C. Kluding H. Roux's Arch. Dev. Biol. 1984; 193: 283-295Crossref PubMed Scopus (615) Google Scholar, 3Tepass U. Theres C. Knust E. Cell. 1990; 61: 787-799Abstract Full Text PDF PubMed Scopus (555) Google Scholar). In higher organisms, the orthologues CRB2 and CRB3 have a similarly broad expression profile (4Makarova O. Roh M.H. Liu C.J. Laurinec S. Margolis B. Gene (Amst.). 2003; 302: 21-29Crossref PubMed Scopus (164) Google Scholar, 5van den Hurk J.A.J.M. Rashbass P. Roepman R. Davis J.A. Voesenek K.E.J. Arends M.L. Zonneveld M.N. van Roekel M.H.G. Cameron K. Rohrschneider K. Heckenlively J.R. Koenekoop R.K. Hoyng C.B. Cremers F.P. den Hollander A.I. Mol. Vis. 2005; 11: 263-273PubMed Google Scholar), but CRB1 is restricted to brain and retina (6den Hollander A.I. Johnson K. de Kok Y.J. Klebes A. Brunner H.G. Knust E. Cremers F.P. Hum. Mol. Genet. 2001; 10: 2767-2773Crossref PubMed Scopus (65) Google Scholar). The expression of CRB1 in retina has been localized to the subapical region adjacent to the outer limiting membrane, a region analogous to the zonula adherens in Drosophila where a similar localization of the protein is observed (7Izaddoost S. Nam S.C. Bhat M.A. Bellen H.J. Choi K.W. Nature. 2002; 416: 178-183Crossref PubMed Scopus (225) Google Scholar, 8Pellikka M. Tanentzapf G. Pinto M. Smith C. McGlade C.J. Ready D.F. Tepass U. Nature. 2002; 416: 143-149Crossref PubMed Scopus (352) Google Scholar, 9Van De Pavert S.A. Kantardzhieva A. Malysheva A. Meuleman J. Versteeg I. Levelt C. Klooster J. Geiger S. Seeliger M.W. Rashbass P. Le Bivic A. Wijnholds J. J. 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Denton M.J. Stone E.M. Ophthalmic Genet. 2001; 22: 163-169Crossref PubMed Scopus (56) Google Scholar) preserved para-arteriolar retinal pigment epithelium, paravenous pigmented chorioretinal atrophy (13McKay G.J. Clarke S. Davis J.A. Simpson D.A. Silvestri G. Investig. Ophthalmol. Vis. Sci. 2005; 46: 322-328Crossref PubMed Scopus (75) Google Scholar), and Leber congenital amaurosis (LCA) 3The abbreviations used are: LCALeber congenital amaurosisEGFepidermal growth factor-like domaincbEGFcalcium-binding epidermal growth factor-like domainHPLChigh pressure liquid chromatographyDQFCOSYdouble-quantum filtered correlation spectroscopyNOEnuclear Overhauser enhancementNOESYNOE spectroscopy. (14den Hollander A.I. Heckenlively J.R. van den Born L.I. de Kok Y.J. van der Velde-Visser S.D. Kellner U. Jurklies B. van Schooneveld M.J. Blankenagel A. Rohrschneider K. Wissinger B. Cruysberg J.R. Deutman A.F. Brunner H.G. Apfelstedt-Sylla E. Hoyng C.B. Cremers F.P. Am. J. Hum. Genet. 2001; 69: 198-203Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 15Lotery A.J. Jacobson S.G. Fishman G.A. Weleber R.G. Fulton A.B. Namperumalsamy P. Heon E. Levin A.V. Grover S. Rosenow J.R. Kopp K.K. Sheffield V.C. Stone E.M. Arch. Ophthalmol. 2001; 119: 415-420Crossref PubMed Scopus (191) Google Scholar). Of these disorders, LCA is the most severe form of inherited retinal dystrophy and is characterized by severe visual impairment from birth or very early life and a decreased or absent electroretinogram response (16Cremers F.P. van den Hurk J.A. den Hollander A.I. Hum. Mol. Genet. 2002; 11: 1169-1176Crossref PubMed Scopus (176) Google Scholar). LCA is generally inherited in an autosomal recessive manner (16Cremers F.P. van den Hurk J.A. den Hollander A.I. Hum. Mol. Genet. 2002; 11: 1169-1176Crossref PubMed Scopus (176) Google Scholar). Mutations in CRB1 represent a significant cause (10-13%) of LCA among the identified LCA loci; missense, nonsense, and splice site mutations together with insertions and deletions have been identified (17Hanein S. Perrault I. Gerber S. Tanguy G. Barbet F. Ducroq D. Calvas P. Dollfus H. Hamel C. Lopponen T. Munier F. Santos L. Shalev S. Zafeiriou D. Dufier J.L. Munnich A. Rozet J.M. Kaplan J. Hum. Mutat. 2004; 23: 306-317Crossref PubMed Scopus (289) Google Scholar, 18den Hollander A.I. Davis J. van der Velde-Visser S.D. Zonneveld M.N. Pierrottet C.O. Koenekoop R.K. Kellner U. van den Born L.I. Heckenlively J.R. Hoyng C.B. Handford P.A. Roepman R. Cremers F.P. Hum. Mutat. 2004; 24: 355-369Crossref PubMed Scopus (148) Google Scholar). The majority of disease-associated missense mutations in CRB1 have been localized to the extracellular region of the protein, and substitutions associated with the various disorders are approximately evenly distributed along its length, with no unambiguous genotype-phenotype correlation (Fig. 1) (17Hanein S. Perrault I. Gerber S. Tanguy G. Barbet F. Ducroq D. Calvas P. Dollfus H. Hamel C. Lopponen T. Munier F. Santos L. Shalev S. Zafeiriou D. Dufier J.L. Munnich A. Rozet J.M. Kaplan J. Hum. Mutat. 2004; 23: 306-317Crossref PubMed Scopus (289) Google Scholar, 18den Hollander A.I. Davis J. van der Velde-Visser S.D. Zonneveld M.N. Pierrottet C.O. Koenekoop R.K. Kellner U. van den Born L.I. Heckenlively J.R. Hoyng C.B. Handford P.A. Roepman R. Cremers F.P. Hum. Mutat. 2004; 24: 355-369Crossref PubMed Scopus (148) Google Scholar). Leber congenital amaurosis epidermal growth factor-like domain calcium-binding epidermal growth factor-like domain high pressure liquid chromatography double-quantum filtered correlation spectroscopy nuclear Overhauser enhancement NOE spectroscopy. CRB1 is a transmembrane protein whose large extracellular portion exhibits a modular domain organization consisting of epidermal growth factor-like, calcium-binding epidermal growth factor-like, and laminin A globular domain-like modules (Fig. 1) (6den Hollander A.I. Johnson K. de Kok Y.J. Klebes A. Brunner H.G. Knust E. Cremers F.P. Hum. Mol. Genet. 2001; 10: 2767-2773Crossref PubMed Scopus (65) Google Scholar). Both Drosophila and human variants possess these domain types, although the extracellular portion of the Drosophila homologue is larger than the corresponding human protein (3Tepass U. Theres C. Knust E. Cell. 1990; 61: 787-799Abstract Full Text PDF PubMed Scopus (555) Google Scholar, 6den Hollander A.I. Johnson K. de Kok Y.J. Klebes A. Brunner H.G. Knust E. Cremers F.P. Hum. Mol. Genet. 2001; 10: 2767-2773Crossref PubMed Scopus (65) Google Scholar). Deletion studies in Drosophila have shown the extracellular region to be essential to limit light-dependent photoreceptor degeneration and have shown the highly conserved cytoplasmic tail to participate in a scaffolding complex necessary for the morphogenesis and maintenance of epithelial polarity (19Johnson K. Grawe F. Grzeschik N. Knust E. Curr. Biol. 2002; 12: 1675-1680Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Proteins homologous to those identified in the cytoplasmic Drosophila scaffold complex have been identified in mammals and shown to interact in a similar way with the cytoplasmic tail of CRB1 (9Van De Pavert S.A. Kantardzhieva A. Malysheva A. Meuleman J. Versteeg I. Levelt C. Klooster J. Geiger S. Seeliger M.W. Rashbass P. Le Bivic A. Wijnholds J. J. Cell Sci. 2004; 117: 4169-4177Crossref PubMed Scopus (202) Google Scholar, 20Kantardzhieva A. Gosens I. Alexeeva S. Punte I.M. Versteeg I. Krieger E. Neefjes-Mol C.A. den Hollander A.I. Letteboer S.J. Klooster J. Cremers F.P. Roepman R. Wijnholds J. Investig. Ophthalmol. Vis. Sci. 2005; 46: 2192-2201Crossref PubMed Scopus (57) Google Scholar, 21Kantardzhieva A. Alexeeva S. Versteeg I. Wijnholds J. FEBS. J. 2006; 273: 1152-1165Crossref PubMed Scopus (30) Google Scholar). No interacting partner has been identified for the extracellular region of mammalian CRB1 and, therefore, its precise function remains unknown. However, a missense substitution that occurs in this region of CRB1 has recently been associated, in a mouse model, with a down-regulation of a mitotic checkpoint protein (PTTG1) (22van de Pavert S.A. Meuleman J. Malysheva A. Aartsen W.M. Versteeg I. Tonagel F. Kamphuis W. McCabe C.J. Seeliger M.W. Wijnholds J. J. 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Disruption of this domain type through genetic mutations has been implicated in many disease states including Marfan syndrome (31Dietz H.C. Cutting G.R. Pyeritz R.E. Maslen C.L. Sakai L.Y. Corson G.M. Puffenberger E.G. Hamosh A. Nanthakumar E.J. Curristin S.M. Stetten G. Meyers D.A. Francomano C.A. Nature. 1991; 352: 337-339Crossref PubMed Scopus (1631) Google Scholar), familial hypercholesterolemia (32Hobbs H.H. Brown M.S. Goldstein J.L. Hum. Mutat. 1992; 1: 445-466Crossref PubMed Scopus (940) Google Scholar), hemophilia B (33Giannelli F. Green P.M. Sommer S.S. Poon M.C. Ludwig M. Schwaab R. Reitsma P.H. Goossens M. Yoshioka A. Brownlee G.G. Nucleic Acids Res. 1996; 24: 103-118Crossref PubMed Scopus (44) Google Scholar), and the ocular disorders Malattia Leventinese and age-related macular degeneration (34Stone E.M. Braun T.A. Russell S.R. Kuehn M.H. Lotery A.J. Moore P.A. Eastman C.G. Casavant T.L. Sheffield V.C. N. Engl. J. Med. 2004; 351: 346-353Crossref PubMed Scopus (280) Google Scholar, 35Schultz D.W. Weleber R.G. Lawrence G. Barral S. Majewski J. Acott T.S. Klein M.L. Ophthalmic. Genet. 2005; 26: 101-105Crossref PubMed Scopus (36) Google Scholar). The missense mutations in cbEGF domains of CRB1 can be divided into two broad categories, cysteine and non-cysteine sequence changes (Fig. 1) (15Lotery A.J. Jacobson S.G. Fishman G.A. Weleber R.G. Fulton A.B. Namperumalsamy P. Heon E. Levin A.V. Grover S. Rosenow J.R. Kopp K.K. Sheffield V.C. Stone E.M. Arch. Ophthalmol. 2001; 119: 415-420Crossref PubMed Scopus (191) Google Scholar, 17Hanein S. Perrault I. Gerber S. Tanguy G. Barbet F. Ducroq D. Calvas P. Dollfus H. Hamel C. Lopponen T. Munier F. Santos L. Shalev S. Zafeiriou D. Dufier J.L. Munnich A. Rozet J.M. Kaplan J. Hum. Mutat. 2004; 23: 306-317Crossref PubMed Scopus (289) Google Scholar, 18den Hollander A.I. Davis J. van der Velde-Visser S.D. Zonneveld M.N. Pierrottet C.O. Koenekoop R.K. Kellner U. van den Born L.I. Heckenlively J.R. Hoyng C.B. Handford P.A. Roepman R. Cremers F.P. Hum. Mutat. 2004; 24: 355-369Crossref PubMed Scopus (148) Google Scholar). Substitutions that involve the replacement of a cysteine are likely to lead to misfolding, changes in calcium binding, and an increase in proteolytic susceptibility (36Vollbrandt T. Tiedemann K. El-Hallous E. Lin G. Brinckmann J. John H. Batge B. Notbohm H. Reinhardt D.P. J. Biol. Chem. 2004; 279: 32924-32931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 37Suk J.Y. Jensen S. McGettrick A. Willis A.C. Whiteman P. Redfield C. Handford P.A. J. Biol. Chem. 2004; 279: 51258-51265Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The second category includes conservative and non-conservative substitutions (with respect to charge and size) distributed throughout the cbEGF module. Interestingly, in contrast to cbEGF domains from other proteins such as fibrillin-1, no mutations have been identified in CRB1 that directly affect a calcium-binding residue. Structural studies on cbEGF-containing fragments from fibrillin-1, low density lipoprotein receptor, and Notch have identified a calcium-dependent interdomain interface that is important in maintaining a rigid, rod-like arrangement of tandem domains (27Hambleton S. Valeyev N.V. Muranyi A. Knott V. Werner J.M. McMichael A.J. Handford P.A. Downing A.K. Structure (Camb.). 2004; 12: 2173-2183Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 38Saha S. Boyd J. Werner J.M. Knott V. Handford P.A. Campbell I.D. Downing A.K. Structure (Camb.). 2001; 9: 451-456Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 39Smallridge R.S. Whiteman P. Werner J.M. Campbell I.D. Handford P.A. Downing A.K. J. Biol. Chem. 2003; 278: 12199-12206Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This interface may also facilitate protein-protein interactions and protect against proteolytic cleavage (39Smallridge R.S. Whiteman P. Werner J.M. Campbell I.D. Handford P.A. Downing A.K. J. Biol. Chem. 2003; 278: 12199-12206Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 40Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 41Reinhardt D.P. Ono R.N. Sakai L.Y. J. Biol. Chem. 1997; 272: 1231-1236Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In the absence of bound Ca2+, this interface does not exist, and the rigidity of the molecule is lost (42Jensen S.A. Corbett A.R. Knott V. Redfield C. Handford P.A. J. Biol. Chem. 2005; Google Scholar). The interdomain interface involves the conserved aromatic residue located between cysteines 5 and 6 of the N-terminal domain (Phe-1289 in EGF18 of CRB1) and two residues located in the turn in the major β-hairpin between cysteines 3 and 4 of the C-terminal domain (Leu-1316 and Asn-1317 in cbEGF19 of CRB1) (see Fig. 2a). The proper formation of this interdomain interface has been implicated in high affinity calcium binding (42Jensen S.A. Corbett A.R. Knott V. Redfield C. Handford P.A. J. Biol. Chem. 2005; Google Scholar, 43Smallridge R.S. Whiteman P. Doering K. Handford P.A. Downing A.K. J. Mol. Biol. 1999; 286: 661-668Crossref PubMed Scopus (45) Google Scholar), and it has been suggested that alteration or disruption of this interface may have some involvement in disease (38Saha S. Boyd J. Werner J.M. Knott V. Handford P.A. Campbell I.D. Downing A.K. Structure (Camb.). 2001; 9: 451-456Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 40Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). In CRB1, missense mutations that might affect this putative interdomain interface are observed (Fig. 1). Here, we have undertaken an investigation to assess the effect of a disease-causing missense mutation in CRB1 on calcium binding and the interface between tandemly linked domains. The calcium binding properties of the 19th EGF module (cbEGF19) were studied in the minimum structure approximating native conditions (the tandemly linked domain pair EGF18-cbEGF19) both for the wild-type fragment and for a fragment into which the LCA-associated N1317H missense mutation (15Lotery A.J. Jacobson S.G. Fishman G.A. Weleber R.G. Fulton A.B. Namperumalsamy P. Heon E. Levin A.V. Grover S. Rosenow J.R. Kopp K.K. Sheffield V.C. Stone E.M. Arch. Ophthalmol. 2001; 119: 415-420Crossref PubMed Scopus (191) Google Scholar) has been introduced. The data reveal a site of moderate affinity for Ca2+ in the wild-type fragment and reveal that this affinity is significantly reduced in the mutant construct. The effect of this particular substitution can be further exacerbated by changes in the protonation state of His-1317, providing further evidence of the nature and importance of the native interdomain interface. We consider these results in the context of the calcium environment in the region of the retina to which CRB1 has been localized and propose a model for the molecular basis of the effects of this substitution. Production of Wild-type and N1317H Variant Recombinant Fragments EGF18-cbEGF19—A cDNA fragment (nucleotides 3898-4143, numbering according to GenBank™ accession AY043325 (6den Hollander A.I. Johnson K. de Kok Y.J. Klebes A. Brunner H.G. Knust E. Cremers F.P. Hum. Mol. Genet. 2001; 10: 2767-2773Crossref PubMed Scopus (65) Google Scholar)), encoding CRB1 domains EGF18 and cbEGF19 (residues 1255-1336), was amplified from a plasmid derived from a human fetal brain cDNA library using Pfu polymerase (Stratagene) and was cloned into bacterial expression vector pQE30 (Qiagen). Recombinant protein was expressed as a His6 affinity-tagged fusion in Escherichia coli NM554 and purified using Ni2+ affinity chromatography. The protein fragment was then reduced with dithiothreitol, purified by reverse-phase HPLC, and refolded in vitro, and the His6 affinity tag was removed by cleavage with Factor Xa as has been described for production of similar cbEGF-containing protein fragments (44Knott V. Downing A.K. Cardy C.M. Handford P. J. Mol. Biol. 1996; 255: 22-27Crossref PubMed Scopus (100) Google Scholar). The purity and identity of the final oxidized product were confirmed by reducing and non-reducing SDS-PAGE analysis and electrospray ionization mass spectrometry. The missense mutant N1317H was created from the corresponding wild-type plasmid described above. Protein expression, refolding, and purification were carried out as described above for the wild-type protein. Limited Proteolysis of the Wild-type EGF18-cbEGF19 Domain Pair—Proteolysis with elastase (1:5, w/w) was performed as described previously (45Whiteman P. Smallridge R.S. Knott V. Cordle J.J. Downing A.K. Handford P.A. J. Biol. Chem. 2001; 276: 17156-17162Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) in 50 mm Tris-HCl buffer at pH 8.0 with 150 mm NaCl. Reaction mixtures contained 10 mm CaCl2 or 10 mm EGTA, and the mixtures were incubated at room temperature. Aliquots were removed at time intervals up to 4 h; the reaction was stopped by the addition of reducing protein-loading dye with boiling for 5 min, and samples were kept on ice until SDS-PAGE analysis and staining with Coomassie Blue. The identification of cleavage sites was performed by N-terminal sequencing of aliquots taken after 2.5 h of incubation with elastase and purified under non-reducing conditions by reverse phase HPLC. NMR Spectroscopy—Samples for NMR spectroscopy contained 300 μm protein in a 600-μl volume of matrix solution (99.9% D2O containing 5 mm Tris-DCl and 150 mm NaCl at pH 7.0 or 7.5). All NMR experiments were performed at 35 °C on a home-built 500-MHz spectrometer in the Department of Biochemistry NMR facility. Calcium titrations were performed by adding small aliquots (1-5 μl) of CaCl2 solutions in D2O. The pKa of His-1317 in the EGF18-cbEGF19 mutant fragment in the absence of Ca2+ was determined by measuring the chemical shifts of the Hδ2 and Hϵ1 peaks arising from His-1317 in the aromatic region of one-dimensional spectra as a function of pH. One-dimensional NMR spectra were collected with a spectral width of 5494.51 Hz, 4096 complex points, and 512 scans. Data were zero-filled to 8192 points, resulting in a digital resolution of 0.67 Hz/point. Two-dimensional DQFCOSY were collected with a spectral width of 5494.51 Hz in F2 and F1, with 1024 complex points in F2, with 512 complex t1 increments, and with 48 scans per increment. Two-dimensional NOESY were collected with 1024 complex points in F2, with 256 complex t1 increments, 96 scans per increment, and a mixing time of 150 ms. All spectra were processed using Felix 2.3 (Accelrys, Inc.). DQFCOSY and NOESY spectra were zero-filled to 2048 points in each dimension, yielding a digital resolution of 2.68 Hz/point. Assignment of peaks in one-dimensional and two-dimensional spectra was performed by analysis of the DQFCOSY and NOESY spectra in conjunction with site-directed mutagenesis (F1289Y) and comparison of wild-type and N1317H-mutant spectra. Determination of Calcium Dissociation Constants—Changes in chemical shifts of intra- and interdomain markers for Ca2+ binding were measured in one-dimensional and two-dimensional spectra and fitted to the equation Δ/Δo = [Ca2+]free/([Ca2+]free + Kd), where Δ is the observed chemical shift change at each value of [Ca2+]free and Δo is the maximum observed chemical shift change (44Knott V. Downing A.K. Cardy C.M. Handford P. J. Mol. Biol. 1996; 255: 22-27Crossref PubMed Scopus (100) Google Scholar). For the wild-type EGF18-cbEGF19 fragment, changes in the chemical shift of the Hϵ resonance of the aromatic packing residue Phe-1289 from EGF18 were monitored in two-dimensional DQFCOSY spectra. The uncertainty in the calculated Kd value for the single Ca2+-binding site in wild-type cbEGF19, arising from errors in chemical shifts, was determined using standard error propagation formulae and Monte Carlo simulations. For the mutant N1317H fragment, changes in the chemical shifts of the Hϵ1 and Hδ2 peaks arising from the substituted His-1317 residue were monitored by one-dimensional spectra in addition to changes in the Phe-1289 Hϵ peak from two-dimensional DQFCOSY spectra. The Kd value was determined by averaging the values obtained for each of these three markers; the uncertainty in the Kd is defined by the standard deviation from the mean. Expression and Characterization of EGF18-cbEGF19 Wild-type and N1317H Missense Mutant Fragments—The EGF18-cbEGF19 wild-type and mutant constructs of CRB1 were expressed and purified using established methods (44Knott V. Downing A.K. Cardy C.M. Handford P. J. Mol. Biol. 1996; 255: 22-27Crossref PubMed Scopus (100) Google Scholar). The addition of Ca2+ was found to be essential for the in vitro refolding of the fragments to form the disulfide bond-stabilized fold. After removal of the His6 affinity tag by cleavage with Factor Xa, a single peak was observed after HPLC purification. Masses determined experimentally using electrospray ionization mass spectrometry (9132.00 ± 0.07 Da for the EGF18-cbEGF19 wild-type and 9154.46 ± 0.08 Da for the N1317H mutant) were in good agreement with the predicted masses for the oxidized form of each protein (9132.12 and 9155.20 Da, respectively). NMR spectroscopy confirmed the presence of a stably folded single conformation for both wild-type and mutant proteins; peaks in the one-dimensional spectrum were well dispersed and at significantly different positions from those expected for an unstructured protein. In two-dimensional NOESY spectra, downfield shifted Hα-Hα cross-peaks confirmed the presence of antiparallel β-sheet, a structural feature common to all EGF domains (37Suk J.Y. Jensen S. McGettrick A. Willis A.C. Whiteman P. Redfield C. Handford P.A. J. Biol. Chem. 2004; 279: 51258-51265Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). cbEGF domains in fibrillin-1 demonstrate a protection against proteolytic cleavage in the presence of saturating Ca2+ concentrations (37Suk J.Y. Jensen S. McGettrick A. Willis A.C. Whiteman P. Redfield C. Handford P.A. J. Biol. Chem. 2004; 279: 51258-51265Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 41Reinhardt D.P. Ono R.N. Sakai L.Y. J. Biol. Chem. 1997; 272: 1231-1236Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 45Whiteman P. Smallridge R.S. Knott V. Cordle J.J. Downing A.K. Handford P.A. J. Biol. Chem. 2001; 276: 17156-17162Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The effect of Ca2+ binding in the wild-type EGF18-cbEGF19 fragment was assessed by limited proteolysis with elastase; a time course of proteolysis shows cleavage both in the absence and in the presence of Ca2+, but significant protection is observed in the presence of Ca2+ (Fig. 2c). Edman degradation of digested protein reveals four sites of elastase cleavage (Fig. 2a). The majority of these occur in the non-calcium-binding domain EGF18, with a single site observed in cbEGF19. It is interesting to note that differences in the degree of proteolysis between the Ca2+-free (10 mm EGTA) and Ca2+-saturated (10 mm Ca2+) samples were observed for the sites in both EGF18 and cbEGF19 (Fig. 2a). This indicates that Ca2+ binding in cbEGF19 leads to a more stable structure throughout the molecule. NMR Reveals Formation of an Interdomain Interface Associated with Moderate Affinity Calcium Binding in Wild-type EGF18-cbEGF19—NMR spectroscopy has been used very successfully in previous studies of cbEGF domains of fibrillin-1 to probe changes in structure associated with calcium binding at the level of individual residues. It has also been used to determine calcium affinity by following changes in chemical shift of consensus aromatic residues (37Suk J.Y. Jensen S. McGettrick A. Willis A.C. Whiteman P. Redfield C. Handford P.A. J. Biol. Chem. 2004; 279: 51258-51265Abstract Full
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