CHAPTER 15

Insights into Fibrillin-1 Structure and Function from Domain Studies

Pat Whiteman and Penny A. Handford

Structure of Fibrillin-1 Domains

Introduction

Structural information is required to understand the assembly of fibrillin-1 into 10-12 nm microfibrils and to gain insight into the consequences of Marfan syndrome (MFS)-causing mutations. Since fibrillin-1 is a modular protein (Fig. 1), a dissection approach has been used to generate structural information for individual or small numbers of domains, where an

analysis of the complete protein is unlikely to be feasible due to its physicochemical properties (size, disulphide-rich, post-translational modifications, rapid macromolecular association). From these data, one can begin to produce a homology model of fibrillin-1.

The two predominant domain types 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-cysteine) domain. The properties of individual cbEGF domains (Fig. 2) have been well studied previously due to their widespread distribution amongst extracellular and transmembrane proteins. A common feature of these domains is their involvement in protein-protein interactions. They contain a calcium-binding consensus sequence which is defined as D/N-X-D/ N-E/Q-Xm-D*/N*-Xn-Y/F where m and n can be a variable number of residues and * denotes a potentially β-hydroxylated residue.1 These residues, located in the N-terminal region of the cbEGF domain, donate side-chain oxygen ligands to Ca2+ or stabilise the Ca2+-binding pocket by hydrogen bonding or hydrophobic interactions.2 In contrast to the cbEGF domain, the TB domain has a much more restricted distribution and is found only in members of the fibrillin/ LTBP (latent transforming growth factor β binding protein) family of proteins. Since these two domain types comprise the majority of fibrillin-1 structure and most of MFS-causing missense mutations affect one or other domain type, studies have focussed on identifying their contribution to native fibrillin-1 structure.

A prokaryotic (E. coli) expression system has been employed to produce the large quantities of protein (~10 mg) required for high resolution structural studies.3 Use of a 6xHis-tag sequence at the N-terminus of each domain construct has facilitated purification by Ni2+ affinity chromatography. The intracellular environment of E. coli is reducing, and since both the cbEGF and TB domains require oxidation to form their native structures which are stabilised by disulphide bridges, peptides purified from this prokaryotic source have been reduced and subsequently oxidised using an in vitro refolding procedure. In addition, because these fragments are expressed in E.coli they lack the post translational modifications associated with these domains in vivo (N- and O-linked glycosylation, β-hydroxylation).

High resolution structures of fibrillin-1 domain fragments have been solved using either X-ray crystallography or nuclear magnetic resonance (NMR), the latter identifying the solu-

Marfan Syndrome: A Primer for Clinicians and Scientists, edited by Peter N. Robinson and Maurice Godfrey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishers.

Figure 2. The secondary structure and consensus sequence of the cbEGF domain. Conserved cysteine residues are in yellow and the calcium-binding consensus residues in red. Ligands predicted to bind directly to calcium are indicated. Hydrophobic residues involved in stabilising inter-domain interactions are in blue and other conserved residues in green.The missense mutations discussed in the text and mapped onto Figure 5 are indicated. Those associated with neonatal MFS (nMFS) are underlined. A double mutation is indicated by asterisks.

calcium-stabilised tetragonal pyramidal conformation (Fig. 3c). The “RGD” integrin binding site localises, as predicted from the structure of TB6, to the tip of a β-sheet 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.

From these data, homology modelling has been used to generate a structural model of a large region of fibrillin-1 (cbEGF 11-TB5). The model 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 TB4.13 Information such as this will allow more precise models of fibrillin organisation within microfibrils to be constructed.

Figure 3. Structures of fibrillin-1 domain fragments: a) cbEGF32-33, b) TB6, c) cbEGF22-TB4- cbEGF23. The cbEGF domains are coloured green, TB domains blue and Ca2+ ions red. The position of the RGD motif in TB4 is indicated. Figures were produced using Bobscript26 and rendered with Raster3D.27

Calcium Binding Properties of Fibrillin-1

Different methods have been used to measure the calcium-binding properties of cbEGF domain fragments from fibrillin-1. These include NMR, intrinsic protein fluorescence and equilibrium dialysis. NMR allows assignment of a calcium-binding site to a specific domain and has been used to analyse the effects of different domain linkages on calcium-binding affinity. It is known that single cbEGF domains expressed in isolation from fibrillin-1 and other proteins display low affinity binding in the mM range. However in fibrillin-1, and 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.14 The bound calcium, together with the hydrophobic packing interaction, performs a key structural role in

restricting interdomain flexibility4,15 and therefore also protects the modules against proteolytic cleavage.16,17 Dynamics studies show that the most stable region of a cbEGF pair is in the

vicinity of the interdomain calcium-binding site.15 Analysis of different cbEGF domain pairs has however identified a range of affinities associated with the C-terminal domain from 350M (cbEGF32-33) to < 30 M (cbEGF12-13), suggesting that primary sequence variation, in addition to the pairwise domain interaction, must also influence affinity. Similar variation appears to exist within heterologous cbEGF domain pairs since TB6-cbEGF32 was observed to have an affinity of 1.6 mM,18 while preliminary data indicate that the affinity of TB4-cbEGF23 is at least two orders of magnitude higher. These data suggest that under physiological condi-

tions of I=0.15 and [Ca2+]free ~1.5 mM, some of the fibrillin-1 cbEGF domains will not be fully saturated and may impart flexibility/extensibility to the native protein. These properties

may be important for protein-protein interactions involved in higher order assembly or for biomechanical function within the specific tissues that contain fibrillin microfibrils. Further examination of other homologous and heterologous cbEGF domain pairs will be required to reveal the extent of variation in affinity.

Structural Consequences of FBN1 Mutations

The availability of structural knowledge of the cbEGF and TB domain types has provided the basis on which to study the consequences of disease-causing FBN1 mutations and understand their role in the pathogenesis of MFS and related disorders. Missense mutations represent the majority of identified FBN1 mutations and most of these affect one of the 43 cbEGF modules of fibrillin-1. Consequently most studies have focussed on understanding their effects on cbEGF structure and function. A significant number of these mutations lead to a substitution of one of the six highly conserved cysteine residues hence disrupting one of the three disulphide bonds of the affected domain. These missense mutations are therefore predicted to cause domain misfolding, those that result in the introduction of extra cysteines are likely to have similar effects. The second most common type of missense mutation affects residues of the calcium-binding consensus sequence and thereby reduces the calcium-binding affinity of the affected domain.

Mutations which lead to substitution of glycine or proline residues adjacent to one of the six conserved cysteine residues have been reported. Glycine residues have greater conformational freedom than other amino acids while the structure of proline renders it the least flexible, consequently substitution of these residues would also be predicted to have effects on domain structure. These and other conformational mutations may compromise the fold of the cbEGF domain without disrupting the native arrangement of disulphide bonds and could therefore affect interor intra-domain interactions.

Other mutations causing amino acid substitutions with no obvious structural significance have been reported. These may be involved in intermolecular protein-protein interactions, either within the microfibril or with other components in the matrix.

Calcium Binding As a Marker of Structural Integrity—NMR Studies

NMR methodology is particularly suited to the study of the effects of disease-causing mutations in single and multidomain constructs. Following the determination of calcium-binding affinities for wild-type cbEGF domains, the effects of missense mutations that alter predicted ligands can be investigated. This provides information on the contribution of the ligand to calcium binding and identifies any effect that its substitution has on the global fold of the domain. Reduction of calcium binding caused by substitution of a calcium ligand would be predicted to destabilise the interdomain linkage and produce a less extended, more flexible structure within a region of fibrillin-1. This may result in increased proteolytic susceptibility and/or distort potential protein binding interfaces.

The heterologous domain pair TB6-cbEGF32 has been found by NMR analysis to have a calcium-binding site of low affinity (see above). This suggests that the linkage between TB6 and cbEGF32 is flexible and that pairwise interactions between TB6 and cbEGF32 are weak or absent. The structural effects of a calcium-binding substitution N2144S which is located on a

β-strand in the N-terminal region of cbEGF32 have been determined. The substitution did not affect the structure of the mutant domain, or the adjacent N-terminal TB6 or C-terminal cbEGF33 when analysed in domain pairs. However, the affinity for calcium was decreased nine-fold in a TB6-cbEGF32 pair while calcium binding in the C-terminal cbEGF33 in a cbEGF 32-33 pair was unaffected.18 The structural effect of the N2144S substitution thus appears to be localised.

Limited Proteolysis Studies of Domain Fragments

The observation that FBN1 missense mutations often increase the susceptibility of fibrillin-1 peptides to proteolysis in the presence of Ca2+ has provided a useful tool with which to probe structural effects of both folding and calcium-binding substitutions. Analyses of the proteolytic degradation products obtained from digests of recombinant fibrillin-1 fragments have suggested that enzyme-specific cryptic cleavage sites are exposed when the calcium-binding properties are altered by the presence of a missense mutation. This is also observed in wild-type cbEGF domain fragments treated with a chelator such as EDTA/EGTA prior to protease digestion, and is presumably due to an increase in conformational flexibility. Identification of how far reaching the loss of calcium-dependent protection is, by mapping the sites of cleavage onto a three-dimensional model of the domain fragments, can be used to assess the short versus long range consequences of different mutations. The specific effect of a missense mutation on protease susceptibility of a cbEGF domain can 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. Although a low resolution method, proteolysis can be used to probe structural effects of mutations in relatively large fragments which are not readily amenable to NMR analysis. Further, since it requires only small amounts of material it can be more easily applied to a number of different mutations.

Folding Substitutions

The structural consequences of a G1127S substitution in cbEGF13, which is associated with familial ascending aortic aneurysm, have been investigated by a combination of both NMR and limited proteolysis. NMR analysis of a peptide containing the G1127S substitution demonstrated that it disrupted the folding of cbEGF13. This is most likely due to the exchange of glycine for a less flexible residue at the start of the major β-hairpin.19 Its localised consequences were demonstrated by lack of interference of the folding of the adjacent cbEGF12 or cbEGF14 in cbEGF12-13 and cbEGF13-14 domain pairs. In the mutant cbEGF12-13 pair, cbEGF13 retained the ability to bind calcium suggesting that the domain preserves a ‘native-like’ fold. This was supported by a more detailed NMR study of the mutant and wild-type cbEGF12-13 pair which showed only minor structural differences underscoring the subtlety of the substitution.20

The short-range effect was confirmed by protease digestion assays of cbEGF12-13 and also a cbEGF12-14 triple construct. In the mutant pair both cbEGF12 and 13 retained similar calcium-binding properties and thus tertiary structure to the wild type domain pair. In addition, all identified cleavage sites (determined by N-terminal sequence analysis of purified digestion products) showed calcium-dependent protection from proteolysis. These results were consistent with the NMR data for this domain pair described above and suggested that the mutant domain has suffered some disruption but is not severely misfolded. Additional cleavage sites identified in cbEGF12-14 G1127S triple construct indicated further subtle changes within the mutant domain but not the flanking domains. Calcium-dependent protection from proteolysis of all cleavage sites including those in mutant cbEGF13 was observed. The SDS-PAGE analysis (Fig. 4a) of the tryptic digests of the mutant cbEGF12-14 triple construct illustrates the protection against digestion conferred by Ca2+. This correlation between the NMR analyses and protease digestion assays demonstrates the usefulness of proteolysis for probing the structural effects of mutations.

Figure 4. Limited proteolysis as a tool for determining structural consequences of FBN1 missense mutations. A comparison of wild-type (wt) and mutant (m) tryptic digests in the presence of 10 mM Ca2+ of the cbEGF12-14 triple domain fragment containing (a) a G1127S or (b) a N1131Y substitution in cbEGF13 (indicated with an asterisk). Cleavage sites identified by N-terminal sequence analysis are shown by arrows above a schematic representation of the fragment.

Calcium Binding Substitutions

The N2144S calcium-binding substitution in cbEGF32 described earlier did not alter the proteolytic susceptibility of a TB6-cbEGF32 pair. This is in contrast to the increased proteolytic susceptibility observed for a cbEGF32-33 pair containing an analogous N2183S substitution in cbEGF33.17 This finding is consistent with the key role for interdomain calcium binding in stabilising tandem cbEGF domain linkages. The mechanism by which N2144S causes disease thus appears different from that of other calcium-binding substitutions which occur in the context of a cbEGF pair. Thus seemingly similar calcium-binding substitutions may cause variable intramolecular effects, dependent upon domain context.

In contrast to the localised effect of the G1127S substitution, significant structural changes can result from disruption of individual calcium-binding sites within fibrillin-1. Protease analysis of a recombinantly expressed cbEGF10-22 fragment containing a calcium-binding substitution E1073K in cbEGF12 demonstrated that the cleavage sites within the mutant domain showed enhanced susceptibility to proteolysis compared with the wild type. Furthermore, an additional cleavage site was revealed N-terminal to cbEGF11 in the mutant fragment indicative of a longer range structural effect of this mutation.21

A comparison of the SDS-PAGE analysis of tryptic digestion of an N1131Y calcium-binding substitution with the G1127S folding substitution, both in cbEGF13 of the cbEGF12-14 triple fragment, is shown in Figure 4. The more disruptive nature of N1131Y is evident and its longer range consequences are demonstrated by a lack of calcium protection in cbEGF12 as well as in cbEGF13 (manuscript in preparation).

Figure 6. Fibroblast expression of a ~100 kDa wild-type (wt) recombinant fragment (NterPro-cbEGF11-22) from fibrillin-1 and comparison with a fragment containing a C1117Y, C1129Y or G1127S substitution in cbEGF13. MSU-1.1 (MSU) is the untransfected cell-line. The fragment was detected in a) conditioned media or b) cell lysates using an anti-Pro antibody specific for fibrillin-1 following Western blot analysis.

of studies reported in the literature have determined the effects of cysteine substitutions and have usually detected normal synthesis of fibrillin-1, but a delay in its secretion leading to a severe reduction of matrix deposition. However, in some cell lines containing cysteine substitutions a normal secretion profile has been characterised.22-24 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. Consequently it is difficult to identify if the mutant fibrillin-1 is retained in the cell or is delayed and eventually secreted into the extracellular space. To better understand the fate of mutant fibrillin-1 a recombinant system has been developed using a fibroblast host cell. This has been used to study the defects in intracellular trafficking and secretion associated with disease-causing mutations.25

In the recombinant system, fibrillin-1 fragments containing two cysteine substitutions associated with classic MFS, C1117Y and C1129Y in cbEGF13, were retained inside the cell. 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 (Fig. 6a, b). This, together with the pulse-chase studies of patient fibroblasts containing G1127S, which showed normal synthesis and secretion of fibrillin-1, suggests that this substitution has an extracellular dominant negative effect. A greater disruption to cbEGF13 presumably results from the presence of an unpaired cysteine than from the localised structural effects of G1127S (see above) and consequently variable effects on cellular trafficking and secretion result. The recombinant system can thus be used to study functional effects of the structural changes introduced by missense mutations and to provide additional information on the pathogenic mechanisms leading to MFS.

Conclusions

An interdisciplinary approach has been used to gain insight into the structure of fibrillin-1 and the consequences of MFS-causing missense mutations. These studies have highlighted in particular the variable properties of cbEGF domains and the importance of domain context and type of amino acid substitution in determining the structural effects of mutations. Different pathogenic mechanisms appear to be associated with MFS-causing mutations and the future challenge will be to understand to what degree the complex phenotypes exhibited by patients are due to expression of a mutant protein or to other modulating factors.

Acknowledgements

We thank Stephen Lee and Kristy Downing for assistance with figures. We thank Jemima Cordle and Ji Young Suk for critical reading of this manuscript. This work was supported by the Medical Research Council, British Heart Foundation and the Wellcome Trust.

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