In order to understand the effects of fibrillin mutations on the structure and function of the tissues affected by MFS, it will ultimately be necessary to understand the effects of mutations on protein structure. It has not been possible to determine the structure of the entire fibrillin‐1 protein or even of larger fragments thereof. However, studies on recombinant polypeptides comprising up to several cbEGF or 8‐Cys modules have provided significant insight.
The two predominant structural modules in fibrillin‐1 are the calcium‐binding epidermal growth factor‐like domain (cbEGF) and the transforming growth factor β binding protein‐like (TB or 8‐Cys) domain. High resolution structures of the fibrillin‐1 domain fragments cbEGF32–33, TB6, and cbEGF12–13 have been solved previously using nuclear magnetic resonance (NMR) which identifies the solution structure of the molecule.17,35,206
More recently, x ray crystallography has been used to solve the structure of the triple domain fragment, cbEGF22‐TB4‐cbEGF2336
and demonstrated a calcium‐stabilised tetragonal pyramidal conformation. An RGD integrin binding site localises to the tip of a β‐sheet within TB4 and is thus accessible to cell‐surface integrins. Comparative sequence alignments of the linker regions from cbEGF‐TB domains within fibrillin‐1 suggest that the relative orientation of cbEGF22‐TB4 is likely to be preserved at homologous sites within fibrillin‐1. In contrast, the variation in amino acid number and composition of TB‐cbEGF linker sequences suggests that these pairs will adopt different orientations with respect to one another within fibrillin‐1, and may contribute to the biomechanical properties of microfibrils. This is supported by the variability of Kd
values of TB‐cbEGF pairs from fibrillin‐1 (see below). A model of a large region of fibrillin‐1 (cbEGF11‐TB5) has been generated from this combined structural information, which suggests that although the protein is in an extended conformation, it is not simply linear. A significant bend is introduced by the packing of cbEGF22 against TB4. Based on this structure, a staggered model for assembly of fibrillin‐1 into the microfibril has been suggested as an alternative to the proposed organisation based on electron microscopic studies.36
Single cbEGF domains expressed from fibrillin‐1 and other proteins usually display low affinity binding in the mM range. However, in fibrillin‐1, and in many other proteins, the cbEGF domains are often arranged as repeating tandem arrays. On covalent linkage of an N‐terminal cbEGF, the affinity of the C‐terminal cbEGF increases.207
The bound calcium, together with the hydrophobic packing interaction, performs a key structural role in restricting interdomain flexibility35,208
and therefore protects the modules against proteolytic cleavage.209,210
Dynamics studies show that the most stable region of a cbEGF pair is in the vicinity of the interdomain calcium‐binding site.208
Analysis of different cbEGF domain pairs has, however, identified a range of affinities from 350 μM to 300 nM, suggesting that primary sequence variation, in addition to the pairwise domain interaction, must also influence affinity.211,212
A study of heterologous TB‐cbEGF domain pairs37
has shown that most of these domain pairs bind Ca2+
considerably more tightly than previously observed, with Kd
values as low as 9 nM. These data suggest that under physiological conditions, many fibrillin‐1 cbEGF domains will be fully saturated and may impart rigidity to the native protein. However, the TB6‐cbEGF32 domain pair, with a Kd
of 1.6 mM,213
appears the most likely of the TB‐cbEGF domain pairs to be flexible and may contribute to the extensibility and elasticity of the microfibrils.
Insights into the role of a number of disease‐causing FBN1 mutations in the pathogenesis of MFS have been gained from NMR, calcium chelation, and limited proteolysis studies of recombinantly expressed fragments of fibrillin‐1. Reduction of calcium binding caused by substitution of a calcium ligand or destabilisation of the interdomain interface would be predicted to produce a less extended, more flexible structure within a region of fibrillin‐1. This may result in increased proteolytic susceptibility due to exposure of enzyme‐specific cryptic cleavage sites. The effect of a missense mutation on protease susceptibility of a cbEGF domain can, however, be influenced by a number of factors such as the particular residue mutated and the position of the mutant domain within the fibrillin‐1 peptide.
The structural effects of the pathogenic mutations C1977Y and C1977R which disrupt the 1–3 disulphide bond of cbEGF30 and are therefore predicted to cause misfolding, have been studied in a cbEGF29–31 triple construct using the combined methods of NMR, chelation, and limited proteolysis.212
The substitutions caused loss of Ca2+
binding to cbEGF30, consistent with intradomain misfolding and disrupted cbEGF29–30 domain‐domain packing. Surprisingly, the calcium binding properties of cbEGF29 and cbEGF31 were unaffected, suggesting these cysteine substitutions have relatively localised effects confined to the N‐terminal end of the mutant domain (fig 3A). However, a disruption of the 5–6 disulphide bond by a C750G substitution which affects the C‐5 residue of cbEGF7 in an EGF4‐TB3 fragment caused increased proteolytic susceptibility of cbEGF8.214
This is presumably due to disruption of domain packing between cbEGF7 and 8 and hence reduction of the calcium binding affinity of cbEGF8. Cysteine substitutions are therefore likely to have different structural effects, which depend on the particular disulphide bond affected, and hence result in a variety of pathogenic mechanisms.
Figure 3Schematic illustration of the variable effects of missense mutations on calcium binding in multi‐domain fragments from fibrillin‐1. Calcium binding properties were assessed by NMR, limited proteolysis, and (for A) calcium (more ...)
These studies, together with earlier reports, emphasise the structural heterogeneity that can be introduced into fibrillin‐1 by different FBN1
mutations. In the case of the folding substitution, G1127S, in cbEGF13, it was shown215,216
that the mutant domain retained the ability to bind calcium (fig 3B). Studies210,213
of the effects of the calcium binding substitution, N2144S, in domain pairs demonstrated that, while the structure of the mutant domain was unaffected, its ability to bind Ca2+
was reduced (fig 3C). Calcium binding substitutions which occur in the context of a cbEGF domain pair can, however, result in more significant structural changes.210,217
For example, the protein engineered N2183S substitution in cbEGF33 (fig 3D) resulted in an increased proteolytic susceptibility of the cbEGF32–33 domain pair and the lack of calcium dependent protection indicated the absence of Ca2+
binding to the mutant domain.210
Thus, as with cysteine substitutions, calcium‐binding substitutions may cause variable intramolecular effects dependent upon domain context.
It is evident that the structural effects of different FBN1
missense mutations are complex. In the neonatal region of fibrillin‐1, for example, missense mutations which affect structurally analogous calcium ligands in different cbEGF domains or cause substitution of different ligands coordinating the same Ca2+
(D1113G and N1131Y) produce varying phenotypes. Three missense mutations, K1043R, I1048T, and V1128I, which have no clear structural effect (although the I1048T substitution does introduce a glycosylation consensus sequence) are found to cluster on one face of a model constructed for the cbEGF11–15 region of fibrillin‐1. An unstructured, extended loop, present in cbEGF12 between cysteines 5 and 6, may also localise to this face of the model and be involved in intra‐ or intermolecular contacts.206
Analysis of the model shows that substitutions that may affect the calcium‐binding properties of cbEGF12 give rise to severe phenotypes. An increase in the intrinsic flexibility of this region resulting from defective calcium binding could distort a potential binding interface, which may be important for the microfibril assembly process and/or interactions with other microfibril components.
A correlation between the in vitro structural effects of amino acid substitutions with their cellular behaviour and consequences for intracellular trafficking and secretion is important for understanding the pathogenesis of MFS. Fibrillin‐1 biosynthesis, processing, and matrix deposition have been studied by pulse‐chase analyses of patient fibroblast cell cultures.44,45,218
The interpretation of such pulse‐chase studies, however, is complicated by the presence of normal fibrillin‐1 produced from the wild type allele, which cannot be distinguished from the mutant product. In order to study the fate of mutant fibrillin‐1, a recombinant system has been developed using a fibroblast host cell.219
In this system, fibrillin‐1 fragments containing two cysteine substitutions associated with classic MFS, C1117Y and C1129Y in cbEGF13, were retained intracellularly in the endoplasmic reticulum when expressed as a shortened form (100 kDa) of fibrillin‐1. This suggests that the delay in secretion observed in the patient cells is due to selective retention of mutant protein in the cell. In contrast, the G1127S folding substitution in the same domain was secreted into conditioned medium. This, together with the pulse‐chase studies of patient fibroblasts containing G1127S, which showed normal synthesis and secretion of fibrillin‐1 but reduced deposition in the extracellular matrix, suggests that this substitution has an extracellular dominant negative effect during or after incorporation of fibrillin‐1 into the microfibril. A greater disruption to cbEGF13 presumably results from the presence of an unpaired cysteine than from the localised structural effects of G1127S. Mutant proteins retained as a consequence of misfolding may result in functional haploinsufficiency or, alternatively, have an intracellular dominant negative effect. These functional studies of the structural changes introduced by missense mutations provide further insights into the pathogenic mechanisms leading to MFS.