©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Calcium Binding Properties and Molecular Organization of Epidermal Growth Factor-like Domains in Human Fibrillin-1 (*)

(Received for publication, October 6, 1994; and in revised form, January 20, 1995)

Penny Handford (1)(§)(¶) A. Kristina Downing (2) Zihe Rao(§) (3) Duncan R. Hewett (4) Bryan C. Sykes (4) Cay M. Kielty (5)(**)

From the  (1)Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, the (2)Department of Biochemistry, University of Oxford, South Parks Road, Oxford, the (3)Laboratory of Molecular Biophysics, Rex Richards Building, South Parks Road, Oxford, the (4)Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, and the (5)School of Biological Sciences, University of Manchester, Manchester, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human fibrillin-1 is a 350-kDa glycoprotein found in 10-nm connective tissue microfibrils. Mutations in the gene encoding this protein cause the Marfan syndrome, a disease characterized by cardiovascular, ocular, and skeletal abnormalities. Fibrillin-1 has a modular structure that includes 47 epidermal growth factor-like (EGF-like) domains, 43 of which contain a consensus sequence associated with calcium binding. A mutation causing an Asn-2144 Ser amino acid change in one of the potential calcium binding residues has been described in a patient with the Marfan syndrome. We have chemically synthesized a wild-type EGF-like domain (residues 2126-2165 of human fibrillin-1) and a mutant EGF-like domain containing the Asn-2144 Ser amino acid change and measured calcium binding to each using ^1H-NMR spectroscopy. The wild-type domain binds calcium with a similar affinity to isolated EGF-like domains from coagulation factors IX and X; however, the mutant domain exhibits >5-fold reduction in affinity. Rotary shadowing of fibrillin-containing microfibrils, isolated from dermal fibroblast cultures obtained from the Marfan patient, shows that the mutation does not prevent assembly of fibrillin into microfibrils but does alter the appearance of the interbead region. We have modeled a region of fibrillin-1 (residues 2126-2331) encompassing five calcium binding EGF-like domains, using data derived from the recently determined crystal structure of a calcium binding EGF-like domain from human factor IX. Our model suggests that these fibrillin-1 EGF-like domains adopt a helical arrangement stabilized by calcium and that defective calcium binding to a single EGF-like domain results in distortion of the helix. We propose a mechanism for the interaction of contiguous arrays of calcium binding EGF-like domains within the microfibril.


INTRODUCTION

Fibrillin-1 is a major structural component of connective tissue microfibrils that have an average diameter of 10 nm (Sakai et al., 1986). When visualized by rotary shadowing electron microscopy, these microfibrils have a distinctive ``beads on a string'' appearance with an average beaded periodicity of 50-55 nm (Kielty et al., 1993). Immunohistochemical data show that fibrillin monomers are assembled in a repetitive manner along the length of the microfibril, although the exact organization and molecular interactions remain undetermined (Sakai et al., 1991). The importance of fibrillin-1 in the maintenance of connective tissue architecture is emphasized by the linkage of the gene for fibrillin-1 to the Marfan syndrome, an autosomal dominant disease of connective tissue that occurs at a frequency of at least 1 in 10,000 in the population (Lee et al., 1991; Dietz et al., 1991; Maslen et al., 1991).

The presumed complete cDNA sequence (8.6 kilobases) has been recently determined (Pereira et al., 1993; Corson et al., 1993) and shows fibrillin-1 to be a modular protein comprising 47 EGF(^1)-like domains, 7 domains that have homology to transforming growth factor beta1 binding protein (8-cysteine motif), 2 hybrid domains with features of both the transforming growth factor beta1 binding protein motif and EGF-like domain motif, a proline-rich region, and 2 unique regions located at the predicted N and C terminus, respectively (Fig. 1). 43 of the 47 EGF-like domains contain the consensus sequence Asp-Asp/Asn-Glu/Gln-Asp*/Asn*-Tyr-Phe (where * indicates a beta-hydroxylated residue), which was shown to be required for effective calcium binding to an equivalent domain from human coagulation factor IX (Rees et al., 1988; Handford et al., 1991a). Mutations altering calcium binding consensus residues in the N-terminal factor IX EGF-like domain cause hemophilia B, demonstrating the physiological importance of this calcium binding domain (Handford et al., 1991a; Mayhew et al., 1992). We previously proposed that fibrillin EGF-like domains with a similar consensus sequence bound calcium and that disruption of calcium binding could be one cause of the Marfan syndrome (Handford et al., 1991b). Recently, mutations changing potential calcium binding consensus residues in EGF-like domains have been identified in patients with the Marfan syndrome (Dietz et al., 1993; Hewett et al., 1993; Kainulainen et al., 1994), and abnormal microfibrils have been isolated from fibroblast cultures established from one such patient (Kielty and Shuttleworth, 1993). Although recent data have indeed confirmed that fibrillin is a calcium binding protein (Corson et al., 1993; Maslen et al., 1993), the calcium binding properties of individual wild-type and mutant fibrillin EGF-like domains have not been investigated in detail.


Figure 1: Domain organization of human fibrillin-1 based on the cDNA sequence (Lee et al., 1991; Maslen et al., 1991; Corson et al., 1993; Pereira et al., 1993). Calcium binding EGF-like domains are defined as those containing the Asp-Asp/Asn-Gln/Glu-Asp*/Asn*-Tyr/Phe consensus sequence (Rees et al., 1988; Handford et al., 1991a) in addition to 6 conserved cysteine residues (Campbell and Bork, 1993).



In this study, we have chemically synthesized a wild-type calcium binding EGF-like domain from fibrillin-1 (residues 2126-2165, numbering according to Pereira et al., 1993) and a mutant domain containing an Asn-2144 Ser amino acid change identified in a patient with Marfan syndrome (Hewett et al., 1993). We have used ^1H-NMR techniques to demonstrate that the calcium binding properties of the fibrillin EGF-like domain are similar to those of the calcium binding EGF-like domains from coagulation factors IX and X (Persson et al., 1989; Handford et al., 1991a). We show that removal of one of the proposed ligands for calcium leads to a reduced affinity for calcium with a small conformational distortion of the calcium binding site. In addition, we have examined fibrillin-containing microfibrils from dermal fibroblasts derived from the patient and show that, although assembly of fibrillin into microfibrils is not prevented, the microfibrils have an altered interbead appearance. Finally, we have used molecular modeling to demonstrate that contiguous EGF-like domains in fibrillin-1 could form helical structures. We suggest a mechanism by which removal of a calcium ligand in a single EGF-like domain could result in altered fibrillin monomer interactions and microfibril structure, thus causing the Marfan syndrome, and we propose a model for the interaction of calcium binding EGF-like domains within the microfibril.


MATERIALS AND METHODS

Numbering of the Human Fibrillin-1 Amino Acid Sequence

For our studies on the calcium binding EGF-like domains of fibrillin, we use the numbering system according to Pereira et al.(1993). Where previously published amino acid changes in fibrillin, identified in Marfan patients, are referred to in the text, we have converted the numbering of the amino acid sequence to that of Pereira et al.(1993).

Chemical Synthesis of Peptides

Two peptides (residues 2126-2165 of human fibrillin) were synthesized using conventional Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied Biosystems 430A synthesizer. One peptide corresponded to the wild-type sequence, and the second contained an Asn-2144 Ser amino acid change. Peptides were deprotected and cleaved from the resin as described by Mayhew et al.(1992), except the cleavage time was extended to 3 h.

Purification and Refolding of Peptides

EGF-like domains contain 6 cysteine residues, which form three characteristic disulfide bonds in the native structure. Each synthetic peptide was fully reduced and purified by reverse-phase HPLC away from failure sequences, prior to refolding to form the native structure (Mayhew et al., 1992). Each peptide was refolded for 4 h at 25 °C in a solution containing 3 mM cysteine, 0.3 mM cystine, 0.1 M Tris-HCl, pH 8.3, 1 mM EDTA (Jaenicke and Rudolph, 1989). Products were analyzed by reverse-phase HPLC using a previously described buffer system (Handford et al., 1990).

Analysis of Calcium Binding to Peptides by^1H-NMR

Calcium titrations of the mutant and wild-type peptides were carried out as previously described (Handford et al., 1991a). Two sets of titrations were performed at pH 7.4 and constant ionic strength (I = 0.15 M) for each peptide. The first set was designed to give an approximate measure of calcium binding affinity, and thus protein concentration was not kept constant throughout the titration. In the second set of titrations, protein was added to the stock CaCl(2) solutions to maintain a constant protein concentration in each NMR sample. The concentrations of the wild-type and mutant protein samples in these latter titrations were 254 and 374 µM, respectively. To ensure that the calcium binding site was saturated, it was necessary to exceed the ionic strength (I = 0.15) for the final 2 points of each titration. Standard regression analysis was used to fit curves to the data using the equation Delta = Delta(0)[Ca]/(K(d) + [Ca]). Due to the relatively high concentrations of protein used in these experiments, the experimental calcium concentration was corrected using the equation [Ca] = [Ca] - Delta/Delta(0) [protein], where Delta/Delta(0) is the fractional change in the chemical shift of the 2,6 proton (H*) resonance for Tyr-2149. Spectra were acquired on an AM600 mHz Bruker spectrometer at 30 °C.

Cell Culture

Normal and Marfan dermal fibroblasts were maintained in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum (FCS), glutamine (2 mM final), penicillin (50 units/ml), and streptomycin (50µg/ml).

Immunofluorescence Staining of Microfibrils

Confluent dermal fibroblasts were fixed in cold acetone for 10 min and incubated for 1 h with a 1:50 dilution of rabbit anti-bovine fibrillin polyclonal antiserum (Kielty et al., 1993) in phosphate-buffered saline (PBS), 10% FCS. The samples were washed 3 times in PBS, 10% FCS before the addition of 1:100 dilution in PBS, 10% FCS of fluorescein isothiocyanate-anti-rabbit conjugate (Sigma). Cells were incubated with this secondary antibody for 45 min. The samples were washed 3 times in PBS, 10% FCS, covered with Citifluor (Citifluor, UKC Chemical Laboratory, Canterbury, UK), and viewed under the microscope.

Ultrastructural Analysis of Microfibrils

Microfibrils were solubilized from post-confluent cell cultures and chromatographed as previously described (Kielty and Shuttleworth, 1993). Void volume fractions were analyzed for their microfibril content by rotary shadowing (Kielty et al., 1993). In addition, normal and mutant microfibril preparations were incubated with the following: (i) 10 mM CaCl(2), (ii) 50 mM CaCl(2), (iii) 100 mM CaCl(2) (each 5 min, 20 °C), (iv) 50 mM CaCl(2) followed by 50 mM EDTA, (v) 100 mM CaCl(2) followed by 100 mM EDTA (each 5 min; 20 °C for CaCl(2), then 2 min at 20 °C for EDTA) prior to rotary shadowing.

Modeling of EGF-like Domains of Fibrillin-1

Structural models of the wild-type and mutant EGF-like domains used in this study were constructed by homology (Ring and Cohen, 1993) based on the coordinates of the human factor IX calcium binding EGF-like domain (^2)using the program Insight 2.3 (Biosym, Inc.). Similarly, a model of five consecutive calcium binding EGF-like domains (residues 2126-2331) from human fibrillin was constructed, based on the orientation of the two EGF-like domains in the asymmetric unit of the factor IX EGF-like domain crystals.


RESULTS

Synthesis and Purification of Mutant and Wild-type EGF-like Domains

A wild-type peptide (residues 2126-2165 of human fibrillin) and a mutant peptide containing the amino acid substitution Asn-2144 Ser were chemically synthesized (see ``Materials and Methods''). Each peptide was reduced and purified prior to in vitro refolding. On refolding each peptide, one major protein species and two minor species were observed following HPLC analysis of the refolding mix (data not shown). The major species, in each case, was purified to homogeneity and used for calcium binding experiments. The similar HPLC profiles obtained on refolding of the mutant and wild-type peptides demonstrated that the introduced mutation did not affect the efficiency of in vitro refolding.

Calcium Binding to Wild-type and Mutant EGF-like Domains

Chemical shift displacement of the H* resonance of Tyr-2149, one of the consensus calcium binding residues (Rees et al., 1988), was monitored as a function of calcium concentration for the wild-type and mutant EGF-like domains. These protons were assigned by homology based on the chemical shifts for the calcium-dependent Tyr-69 resonance of the human factor IX calcium binding EGF-like domain (Handford et al., 1990, Baron et al., 1992) and the calcium-dependent Tyr-68 resonance of the equivalent domain from factor X (Persson et al., 1989, Selander et al., 1990). A comparison of the aromatic region of ^1H-NMR spectra recorded in the presence and absence of calcium for both the wild-type domain and the mutant domain is shown (Fig. 2). In the absence of calcium, there are only minor differences in the chemical shifts of resonances derived from the mutant and wild-type peptides, demonstrating that the amino acid change does not affect the fold of the domain. The wild-type spectrum, relative to the mutant spectrum, shows a large chemical shift displacement of the H* resonance for Tyr-2149 upon saturation of the binding site with Ca. The change in chemical shift (Delta) of the H* resonance for Tyr-2149 was plotted as a function of free calcium concentration (Fig. 3) to calculate K(d) values for calcium. K(d) values of 4.3 mM (wild-type) and 22.1 mM (mutant) were obtained measured at pH 7.4 and at constant ionic strength (I = 0.15). Interestingly, the degree of H* chemical shift displacement observed with the mutant peptide was reduced on saturation of the calcium binding site. This has previously been observed in the analysis of factor IX mutant EGF-like domains (Handford et al., 1991; Mayhew et al., 1992) and suggests that there is a distortion of the geometry of the calcium binding site.


Figure 2: One-dimensional NMR spectra showing the chemical shift displacement of the resonance for the Delta ring protons (H*) of Tyr-2149 upon addition of saturating amounts of calcium to wild-type and mutant EGF-like domains. The resonance for these protons shifts dramatically upon calcium binding to the wild-type EGF-like domain. The amino acid change Asn-2144 Ser disrupts calcium binding to the EGF-like domain, hence the Tyr-2149 H* chemical shift displacement observed for the mutant domain is relatively minor.




Figure 3: The change in chemical shift (Delta) of the H* resonance of Tyr-2149 plotted against [Ca]. bullet, wild-type domain; circle, mutant domain.



Ultrastructural Analysis of Fibrillin-containing Microfibrils

Preliminary examination of fibrillin-containing microfibrils by immunofluorescence demonstrated a qualitative difference in staining between primary dermal fibroblast cell lines derived from the Marfan patient and a normal control (data not shown). To examine the microfibrils in more detail, an ultrastructural analysis was undertaken. Intact microfibrils isolated from post-confluent fibroblast cell layers were previously shown to be morphologically indistinguishable from those isolated from tissues (Kielty and Shuttleworth, 1993) with a well defined regular beaded periodicity with interbead striations visible. Intact fibrillin-containing microfibrils, extracted from post-confluent cell layers, were isolated by size-exclusion chromatography (see ``Materials and Methods''). Examination of the void volume fraction by rotary shadowing electron microscopy revealed that intact microfibrils isolated from fibroblasts derived from the Marfan cell line were present (Fig. 4), although low in number. In addition, a number of what appeared to be collapsed assemblies were visible (data not shown). The intact microfibrils had what appeared to be a normal beaded periodicity, as previously described for other microfibril preparations, but had a diffuse interbead region (Fig. 4). The diffuse appearance was observed even after treatment of mutant microfibrils with high concentrations of Ca (10, 50, and 100 mM) (see ``Materials and Methods'' and data not shown). A similar morphology has previously been observed in normal microfibrils treated with EDTA (Kielty and Shuttleworth, 1993). The abnormal appearance of mutant microfibrils indicated that the mutant form of fibrillin was secreted by the patient cell line.


Figure 4: Electron micrographs of normal (a) and mutant (b) fibrillin-containing microfibrils after rotary shadowing. Bars = 200 nm.



Modeling of Calcium Binding EGF-like Domains (Residues 2126-2331) from Human Fibrillin-1

The recently solved crystal structure of the calcium binding EGF-like domain from human factor IX revealed two EGF-like domains in the asymmetric unit.^2 Each domain had a major calcium binding site where the calcium was coordinated by 7 oxygen ligands, which formed a classic pentagonal bipyramidal calcium binding cluster. One of these ligands was provided by the neighboring EGF-like domain in the crystal. In addition, the packing of EGF-like domains in the crystal generated a helical arrangement, which was proposed as a general structural model for consecutive calcium binding EGF-like repeats in proteins with multiple copies of this domain, such as fibrillin. Furthermore, it was suggested that interactions of adjacent helices of EGF-like domains, in either a parallel or anti-parallel orientation, might facilitate the polymerization of fibrillin monomers to form microfibrils. To test the plausibility of this hypothesis, a structural model of five consecutive calcium binding EGF-like domains (calcium binding EGF-like domains 32-36) (see Fig. 1, residues 2126-2331) was constructed by homology (Fig. 5a), based on the factor IX EGF-like domain coordinates. The amino acid sequence of this region of fibrillin is compatible with the proposed helical arrangement of EGF-like domains, despite the fact that the number of residues between the cysteine residues involved in disulfide bond formation is not always conserved (Fig. 5b). By analogy to the crystal structure, each calcium bound to a fibrillin EGF-like domain should be coordinated by a side chain ligand located between the second and third conserved cysteine residues in each EGF-like domain (see boxedregion, Fig. 5b), donated from the adjacent N-terminal EGF-like domain. The variation in loop length between these cysteine residues precludes the identification of this ligand, although in all but one case a suitable side chain oxygen donor is present in this region (Fig. 5b). Where there is an absence of a suitable side chain donor, we cannot rule out that either a main chain carbonyl group or H(2)O could complete the coordination sphere of calcium. We consider it unlikely that the coordination is completed by a ligand from different fibrillin monomer since the calcium ions are relatively buried in the helical structure (Fig. 5a). It should be noted that a ligand could be provided by a different type of domain, e.g. an 8-cysteine repeat if this domain is connected to the N terminus of an EGF-like domain (Fig. 1). In native factor IX, the N-terminal -carboxyglutamate (Gla) domain is thought to provide a ligand, completing the coordination of calcium bound to the adjacent EGF-like domain (Valcarce et al., 1993).^2


Figure 5: a, helical model of five consecutive calcium binding EGF-like domains from human fibrillin (residues 2126-2331) constructed using the program Insight 2.3 (Biosym, Inc.). Calcium ions occupying putative calcium binding sites are shown in green. Charged and hydrophobic side chains (including aromatic rings) are shown in cyan and red, respectively. Each EGF-like domain was aligned by eye and modeled by homology based on the crystal structure of the calcium binding EGF-like domain of factor IX. b, sequence alignments of fibrillin calcium binding EGF-like domains 32-36 (residues 2126-2331) and the human factor IX calcium binding EGF-like domain (residues 46-82). The region between the second and third cysteine residues of each EGF-like domain predicted to contain a calcium binding ligand is boxed; in the factor IX EGF-like domain crystal structure, this ligand is Asn-58.




DISCUSSION

We have demonstrated that a single EGF-like domain from fibrillin, with a consensus sequence associated with calcium binding, has similar ligand binding properties to equivalent EGF-like domains found in the coagulation proteins factor IX and factor X. The affinity of the isolated fibrillin domain is a moderate 4 mM at physiological ionic strength and pH 7.4; this probably reflects the absence of one of the seven protein ligands required to complete the pentagonal bipyramidal coordination of calcium recently demonstrated in the crystal structure of the calcium binding EGF-like domain from human factor IX.^2 Interestingly, the arrangement of calcium binding ligands within fibrillin EGF-like domains is different from that of the coagulation factors IX and X, with the positions of a carboxyamide side chain and a carboxylate side chain reversed (e.g. Asn-2144 in fibrillin-1, Asp-64 in human factor IX, Glu-2130 in fibrillin-1, Gln-50 in human factor IX). This has only a minor effect on the affinity of the isolated domain for calcium, a result that is compatible with the crystal structure since these ligands lie in a common plane adjacent to one other. These experiments confirm that an EGF-like domain with the consensus Asp-Asp/Asn-Gln/Glu-Asp*/Asn*-Tyr/Phe is a general extracellular calcium binding structure and not just a feature of vitamin K-dependent coagulation proteins, which have previously been studied.

An Asn-2144 Ser amino acid change at a potential calcium binding residue within a fibrillin EGF-like domain has recently been identified in a patient with the Marfan syndrome (Hewett et al., 1993). This amino acid change causes a large reduction in the affinity of the isolated domain for calcium, consistent with the removal of a calcium ligand. From the analysis of one-dimensional NMR data, it is apparent that this amino acid substitution does not disrupt the fold of the domain (see Fig. 2and ``Results''). However, the reduced upfield shift of the Tyr resonance seen in the presence of saturating Ca suggests that there is a small local change in the conformation of the mutant domain. Hence, we propose that the phenotype displayed by this Marfan patient is caused by a failure of the EGF-like domain to adopt a wild-type conformation, even in the presence of bound Ca. This is consistent with our microfibril analyses, which show that the mutant microfibrils retain a diffuse interbead region after treatment with 100 mM Ca (see ``Results'').

The diffuse morphology has previously been observed in microfibrils isolated from a Marfan cell line (Kielty and Shuttleworth, 1993), which has an Asn-2144 Ser amino acid change within the same domain, also at a predicted calcium binding residue (Kainulainen et al., 1994). These data, taken together, localize this EGF-like domain to the interbead region of the microfibril.

We have modeled the region of fibrillin-1 containing the Asn-2144 Ser and Asp-2127 Glu amino acid changes (see ``Results'' and Fig. 5a) to try and explain the morphological changes observed in the mutant microfibrils and to derive information about the role of calcium binding EGF-like domains in the organization of fibrillin monomers within a microfibril. Since Ca ions are relatively buried within the structure, we suggest that calcium binding to EGF-like domains imparts a structural stability to each fibrillin monomer rather than cross-linking fibrillin monomers. This is consistent with the appearance of the interbead regions of mutant microfibrils examined in this study. In the normal microfibril, helical arrangements of EGF-like domains within each fibrillin monomer, predicted by recent crystallographic data,^2 would be stabilized by calcium. Loss of a calcium ligand and any change in the conformation of the domain produced as a result would distort the helical structure of a mutant fibrillin monomer (increasing the flexibility of the polypeptide chain) and result in a disordered interbeaded region. Since this may increase the susceptibility of the monomer to proteolysis, it could provide an explanation for the collapsed assemblies present in our microfibril preparations (see ``Results'').

These data are consistent with the observation that the morphological changes that occur in normal microfibrils on treatment with EDTA are reversible (Kielty and Shuttleworth, 1993). The formation of helical arrays of calcium binding EGF-like domains would be dependent on the presence of calcium. Removal of calcium by EDTA treatment of microfibril preparations would destabilize helices, resulting in the frayed or diffuse appearance of the interbead region. Subsequent addition of calcium would promote helical formation, leading once more to a structured interbead region.

Our structural model suggests that the association of calcium binding EGF-like domains is not the driving force for the assembly of microfibrils since the scattered distribution of charged and hydrophobic side chains preclude an obvious mechanism for association of fibrillin monomers through either hydrophobic or electrostatic interactions (Fig. 5a). Previous immunohistochemical data suggested that multiple fibrillin monomers might be arranged in a parallel head to tail alignment along the microfibril (Sakai et al., 1991). However, although a parallel alignment of fibrillin monomers would be possible if helical arrays of EGF-like domains form in vivo, the arrangement of helices observed in the crystal packing of the factor IX calcium binding EGF-like domain suggests an alternative anti-parallel model for the lateral association of fibrillin EGF-like domains in the interbead regions of the microfibril. This model retains the head to tail alignment of monomers and facilitates closer packing of calcium-stabilized EGF-like domain helices than would be achieved by a parallel alignment. The simplest arrangement of EGF-like domains would comprise two anti-parallel pairs of fibrillin monomers, which would form a 4-helix bundle (Fig. 6). The diameter across this bundle, based on dimensions of helix bundles within crystals of the factor IX calcium binding EGF-like domain, would be 8 nm,^2 without making an allowance for carbohydrate modifications. Since variable microfibril diameters have been reported (10-15 nm), a larger number of anti-parallel pairs of fibrillin monomers could comprise the core of the microfibril. Covalent cross-links and/or hydrophobic interactions between adjacent fibrillin monomers would be required to stabilize the structure. Recently, transglutaminase-derived cross-links localized to the interbead region of microfibrils have been reported (Glanville and Quian, 1994). We suggest that a staggered anti-parallel alignment of fibrillin monomers would maintain the close packing of helices formed by multiple calcium binding EGF-like domains (Fig. 6) while allowing N termini projecting from each 4-helix bundle to interact, thus providing a potential mechanism for polymerizing and stabilizing the microfibril. If this is correct, we would predict that fibrillin N termini are located in the beaded regions of the microfibril. We are currently conducting further experiments to test this model.


Figure 6: A model for the interaction of multiple calcium binding EGF-like domains within the interbead region of the microfibril. A, anti-parallel pairing of two fibrillin monomers; regions encompassing multiple calcium binding EGF-like repeats are shaded. The N and C terminus of each monomer is labeled. B, the association of two anti-parallel pairs of monomers to form a 4-helix bundle in the interbead region. C, a cross-section through the 4-helix bundle showing helices labeled A-D. Helices A and B are anti-parallel relative to C and D.




FOOTNOTES

*
This paper is a contribution from the Oxford Center for Molecular Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Members of the Oxford Center for Molecular Sciences.

Supported by a Royal Society University Research Fellowship. To whom correspondence should be addressed. Tel.: 865-275583, Fax: 865-275556.

**
An MRC Senior Research Fellow.

(^1)
The abbreviations used are: EGF, epidermal growth factor; HPLC, high pressure liquid chromatography; FCS, fetal calf serum; PBS, phosphate-buffered saline.

(^2)
Z. Rao, P. A. Handford, M. Mayhew, V. Knott, G. G. Brownlee, and D. Stuart, submitted for publication.


ACKNOWLEDGEMENTS

We thank Prof. I. D. Campbell, Dr. R. J. O. Davies, and C. Cardy for helpful discussions. We thank Dr. M. Pitkeathly for peptide synthesis and Tony Willis for Edman degradations.


REFERENCES

  1. Baron, M., Norman, D. G., Harvey, T. S., Handford, P. A., Mayhew, M., Tse, A. G. D., Brownlee, G. G., and Campbell, I. D. (1992) Protein Sci. 1, 81-90 [Abstract/Free Full Text]
  2. Campbell, I. D., and Bork, P. (1993) Curr. Opin. Struct. Biol. 3, 385-392
  3. Corson, G. M., Chalberg, S. C., Dietz, H. C., Charbonneau, N. L., and Sakai, L. S. (1993) Genomics 17, 476-484 [CrossRef][Medline] [Order article via Infotrieve]
  4. Dietz, 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., and Francomano, C. A. (1991) Nature 352, 337-339 [CrossRef][Medline] [Order article via Infotrieve]
  5. Dietz, H. C., McIntosh, I., Sakai, L. Y., Corson, G. M., Chalberg, S. C., Pyeritz, R. E., and Francomano, C. (1993) Genomics 17, 468-475 [CrossRef][Medline] [Order article via Infotrieve]
  6. Glanville, R. W., and Quian, R. Q. (1994) 3rd International Symposium on the Marfan Syndrome (Abstr. 16) Berlin
  7. Handford, P. A., Baron, M., Mayhew, M., Willis, A., Beesley, T., Brownlee, G. G., and Campbell, I. D. (1990) EMBO J. 9, 475-480 [Abstract]
  8. Handford, P. A., Mayhew, M., Baron, M., Winship, P. R., Campbell, I. D., and Brownlee, G. G. (1991a) Nature 351, 164-167 [CrossRef][Medline] [Order article via Infotrieve]
  9. Handford, P. A., Mayhew, M., and Brownlee, G. G. (1991b) Nature 353, 395 [Medline] [Order article via Infotrieve]
  10. Hewett, D. R., Lynch, J. R., Smith, R., and Sykes, B. C. (1993) Hum. Mol. Genet. 2, 475-477 [Medline] [Order article via Infotrieve]
  11. Jaenicke, R., and Rudolph, R. (1989) in Protein Structure: A Practical Approach (Creighton, T. E., ed) pp. 208-209, IRL Press, Oxford
  12. Kainulainen, K., Karttunen, L., Puhakka, L., Sakai, L., and Peltonen, L. (1994) Nat. Genet. 6, 64-69 [Medline] [Order article via Infotrieve]
  13. Kielty, C. M., and Shuttleworth, C. A. (1993) FEBS Lett. 336, 323-326 [CrossRef][Medline] [Order article via Infotrieve]
  14. Kielty, C. M., Berry, L., Whittaker, S. P., and Shuttleworth, C. A. (1993) Matrix 13, 103-112 [Medline] [Order article via Infotrieve]
  15. Lee, B., Godfrey, M., Vitale, E., Hori, H., Mattei, M.-G, Sarfarazi, M., Tsipouras, P., Ramirez, F., and Hollister, D. W. (1991) Nature 352, 330-334 [CrossRef][Medline] [Order article via Infotrieve]
  16. Maslen, C. L., Corson, G. M., Maddox, B. K., Glanville, R. W., and Sakai, L. Y. (1991) Nature 352, 334-337 [CrossRef][Medline] [Order article via Infotrieve]
  17. Maslen, C. L., Qian, R-G., McClure, D. W., and Glanville, R. W. (1993) 9th Annual Conference of National Marfan Foundation (Abstr. 4) Portland, OR
  18. Mayhew, M., Handford, P. A., Baron, M., Tse, A., Campbell, I. D., and Brownlee, G. G. (1992) Protein Eng. 5, 489-494 [Abstract]
  19. Pereira, L., D'Alessio, M., Ramirez, F., Lynch, J. R., Sykes, B., Pangilinan, T., and Bonadio, J. (1993) Hum. Mol. Genet. 2, 961-968 [Abstract]
  20. Persson, E., Selander, M., Linse, S., Drakenberg, T., Ohlin, A., and Stenflo, J. (1989) J. Biol. Chem. 264, 16897-16904 [Abstract/Free Full Text]
  21. Rees, D. J. G., Jones, I. M., Handford, P. A., Walter, S. J., Esnouf, M. P., Smith, K. J., and Brownlee, G. G. (1988) EMBO J. 7, 2053-2061 [Abstract]
  22. Ring, C. S., and Cohen, F. E. (1993) FASEB J. 7, 783-790 [Abstract/Free Full Text]
  23. Sakai, L. Y., Keene, D. R., and Envall, E. (1986) J. Cell Biol. 103, 2499-2509 [Abstract]
  24. Sakai, L. S., Keene, D. R., Glanville, R. W., and Bachinger, H. P. (1991) J. Biol. Chem. 266, 14763-14770 [Abstract/Free Full Text]
  25. Selander, M., Persson, E., Stenflo, J., and Drakenberg, T. (1990) Biochemistry 29, 8111-8118 [Medline] [Order article via Infotrieve]
  26. Valcarce, C., Selander-Sunnerhagen, M., Tamlitz, A-M., Drakenberg, T., Bjork, I., and Stenflo, J. (1993) J. Biol. Chem. 268, 26673-26678 [Abstract/Free Full Text]

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