(Received for publication, October 6, 1994; and in revised form, January 20, 1995)
From the
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
H-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.
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(
)-like domains, 7
domains that have homology to transforming growth factor
1 binding
protein (8-cysteine motif), 2 hybrid domains with features of both the
transforming growth factor
1 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
-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
H-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.
Figure 2:
One-dimensional NMR spectra showing the
chemical shift displacement of the resonance for the 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 () of the H
* resonance of Tyr-2149
plotted against [Ca
]
.
, wild-type domain;
, mutant
domain.
Figure 4: Electron micrographs of normal (a) and mutant (b) fibrillin-containing microfibrils after rotary shadowing. Bars = 200 nm.
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.
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.
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,
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,
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.