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One More Piece in the Fibrillin Puzzle
Structure
Previews One More Piece in the Fibrillin Puzzle Dirk Hubmacher1 and Dieter P. Reinhardt1,2,* 1Department
of Anatomy and Cell Biology, Faculty of Medicine of Biomedical Sciences, Faculty of Dentistry McGill University, Montreal, QC H3A 2B2, Canada *Correspondence: [email protected] DOI 10.1016/j.str.2009.04.002 2Division
Jensen et al. report the crystal structure of a human fibrillin-1 hybrid domain in this issue of Structure. This domain is found exclusively in the fibrillin/latent transforming growth factor-b binding protein superfamily and shares structural features with two other domains in these proteins, the TB/8-Cys and the cbEGF domains. Fibrillins are extended 350-kDa extracellular matrix proteins that constitute the backbone of microfibrils, large molecular machines found in elastic and nonelastic tissues. Microfibrils play important roles in the biogenesis of elastic fibers, in conferring limited elasticity to tissues, and in the regulation of bioavailability of transforming growth factor-b superfamily members. Similar to other matrix glycoproteins, fibrillins display a tandemly arrayed domain organization, with each domain representing an individual folding unit. Fibrillins, together with the latent transforming growth factor-b binding proteins (LTBPs), form a superfamily of proteins characterized by the presence of two unique domains: the transforming growth factor-b binding protein-like (TB or 8-Cys) domains, and the hybrid (hyb) domains (Figure 1). The limitations for high resolution structural studies of the full-length fibrillins are their large molecular mass combined with a relatively low solubility, the extremely high number of cysteine residues (361 in fibrillin-1, equivalent to 12.6%), almost all engaged in disulfide-bridges, and the extensive post-translational modifications, including N-glycosylation (Figure 1). However, it has been demonstrated that individual domains of fibrillin-1 or arrays of a few adjacent domains can be efficiently expressed in bacterial systems and refolded in vitro into correctly folded proteins (Knott et al., 1996). Using this experimental approach, high resolution structures of fibrillin-1 calcium-binding epidermal growth factor-like (cbEGF) domains and TB/8-Cys domains have been solved successfully (Downing et al., 1996; Lee et al., 2004). The hyb1 domain in fibrillins mediates interactions with fibulin-2, 4, and 5,
as well as with LTBP-1 and 4 (El-Hallous et al., 2007; Ono et al., 2009). In addition, it was shown that this domain participates in intermolecular disulfide-bond formation during microfibril assembly (Reinhardt et al., 2000). No functional assignments have been delineated for the hyb2 domain in fibrillins. The initial primary sequence analysis of the TB/8-Cys, hyb, and cbEGF domains revealed similarities of the hyb domains’ N termini with the N terminus of TB/8-Cys domains, and of the hyb domains’ C termini with the C terminus of cbEGF domains (Corson et al., 1993). With the new high resolution (1.8 A˚) crystal structure of a hyb domain from human fibrillin-1 (hyb2) reported by Jensen et al. (2009), it is now clear that these similarities in the primary sequence are fully reflected in the structure, proving that the hyb domain is indeed an evolutionary ‘‘hybrid’’ between TB/8-Cys and cbEGF domains. The overall structure of hyb2 flanked by cbEGF9 and cbEGF10 is similar to the structure of the TB/8-Cys domains in regard to the positions of characteristic side chains. In the central a-helical region of both domains, the ‘‘Cys-Cys-Cys’’ motif of the TB/8-Cys domain and the corresponding ‘‘Cys-Cys’’ motif in hyb2 are found in similar locations. In addition, a conserved cis-proline is placed in a corresponding structural region in both domains. In contrast to the TB/8-Cys structure, the hyb2 domain is missing a second a-helical stretch toward its C-terminal end. Another elementary difference between the two domains was found in the disulfide-bond patterns. Both domains contain eight cysteine residues; however, the hyb2 domain lacks one of the three cysteines in the ‘‘Cys-Cys-Cys’’ motif of the TB/8-Cys domain, but has
two cysteine residues in its C terminus as opposed to one. These differently positioned cysteine residues result in a 1-3, 2-5, 4-6, 7-8 disulfide-bond pattern in the hyb domain, versus a 1-3, 2-6, 4-7, 5-8 pattern characteristic for the TB/8-Cys domain. In this context it will be interesting to determine the structure of the hyb1 domain of fibrillin-1, which contains an additional cysteine residue at position 204 and consequently has one unpaired cysteine. This extra cysteine residue in the hyb1 domain is conserved between all fibrillins and between all species. Hyb domains in LTBPs, on the other hand, do not have such an extra cysteine residue. Biochemical data previously suggested that cysteine 204 of fibrillin-1 occurs as a free thiol on the surface of the molecule, making it an ideal candidate for intermolecular crosslink formation (Reinhardt et al., 2000). Molecular modeling of the hyb1 domain of fibrillin-1, based on the experimentally determined hyb2 coordinates presented in Jensen et al. (2009), supports this proposed role for the unpaired cysteine. The affinity for calcium binding to cbEGF domains in fibrillin-1 depends strongly on the nature of the inter-domain interfaces between adjacent domains. These contacts can modulate the affinity for calcium over a wide range (dissociation constants from nanomolar to millimolar). The cbEGF domains directly downstream of the TB/8-Cys domains frequently bind calcium with very high affinity attributed to hydrophobic domain interactions between the TB/8-Cys and the cbEGF domains (Jensen et al., 2005). In Jensen et al. (2009), the authors demonstrate that the cbEGF domains succeeding the hyb1 and hyb2 domains of fibrillin-1 also
Structure 17, May 13, 2009 ª2009 Elsevier Ltd All rights reserved 635
Structure
Previews microfibrils and to correlate the structure with specific functions. To fully understand the structure, macromolecular organization, and, ultimately, the functions of fibrillins, it will be necessary to solve the structure of (1) the proline/glycine-rich domains in fibrillins, (2) the N- and C-terminal unique domains, (3) larger contiguous portions of the proteins, (4) functional protein and selfinteraction sites, and (5) fully assembled fibrillin-1 aggregates giving rise to microfibrils. These are challenges for the future. For now, the fibrillin-1 hyb2 structure is one more piece added to the fibrillin puzzle.
ACKNOWLEDGMENTS We thank Ryan Kirschner for critical reading of the manuscript.
REFERENCES Corson, G.M., Chalberg, S.C., Dietz, H.C., Charbonneau, N.L., and Sakai, L.Y. (1993). Genomics 17, 476–484. Downing, A.K., Knott, V., Werner, J.M., Cardy, C.M., Campbell, I.D., and Handford, P.A. (1996). Cell 85, 597–605. El-Hallous, E., Sasaki, T., Hubmacher, D., Getie, M., Tiedemann, K., Brinckmann, J., Batge, B., Davis, E.C., and Reinhardt, D.P. (2007). J. Biol. Chem. 282, 8935–8946. Jensen, S.A., Corbett, A.R., Knott, V., Redfield, C., and Handford, P.A. (2005). J. Biol. Chem. 280, 14076–14084. Jensen, S.A., Iqbal, S., Lowe, E.D., Redfield, C., and Handford, P.A. (2009). Structure 17, this issue, 759–768.
Figure 1. Domain Organization of the Human Fibrillin/LTBP Superfamily Members The domains discussed in this preview are indicated in color. Jensen et al. (2009) solved the structure of the hyb2 domain (H2, blue) in the context of its flanking cbEGF9 and cbEGF10 domains as indicated. The hyb1 domain (H1, blue) of all fibrillins contain an additional unpaired ninth cysteine residue, while the hyb domains in LTBPs do not contain such an extra cysteine. For simplicity, only the longest splice variant of each LTBP is displayed.
bind calcium with very high affinity with dissociation constants in the low nanomolar range, which is again explained by the extensive domain interactions at least observed between hyb2 and cbEGF10. Under physiological calcium concentrations (1.5 mM), the region around hyb2 adopts a near linear shape. Together with previously published structural data available for the TB/8-Cys domains and
several cbEGF domains in their natural domain context, the authors predict the region between cbEGF3 and cbEGF43 (i.e., the majority of the fibrillin-1 molecule) to form an extended structure, as suggested by low resolution methods that include electron microscopy after rotary shadowing (Sakai et al., 1991). This information will be very useful to validate existing models for fibrillin organization in
636 Structure 17, May 13, 2009 ª2009 Elsevier Ltd All rights reserved
Knott, V., Downing, A.K., Cardy, C.M., and Handford, P. (1996). J. Mol. Biol. 255, 22–27. Lee, S.S., Knott, V., Jovanovic, J., Harlos, K., Grimes, J.M., Choulier, L., Mardon, H.J., Stuart, D.I., and Handford, P.A. (2004). Structure 12, 717–729. Ono, R.N., Sengle, G., Charbonneau, N.L., Carlberg, V., Bachinger, H.P., Sasaki, T., LeeArteaga, S., Zilberberg, L., Rifkin, D.B., Ramirez, F., et al. (2009). J. Biol. Chem., in press. Published online April 6, 2009. 10.1074/jbcM809348200. Reinhardt, D.P., Gambee, J.E., Ono, R.N., Ba¨chinger, H.P., and Sakai, L.Y. (2000). J. Biol. Chem. 275, 2205–2210. Sakai, L.Y., Keene, D.R., Glanville, R.W., and Ba¨chinger, H.P. (1991). J. Biol. Chem. 266, 14763–14770.
Previews One More Piece in the Fibrillin Puzzle Dirk Hubmacher1 and Dieter P. Reinhardt1,2,* 1Department
of Anatomy and Cell Biology, Faculty of Medicine of Biomedical Sciences, Faculty of Dentistry McGill University, Montreal, QC H3A 2B2, Canada *Correspondence: [email protected] DOI 10.1016/j.str.2009.04.002 2Division
Jensen et al. report the crystal structure of a human fibrillin-1 hybrid domain in this issue of Structure. This domain is found exclusively in the fibrillin/latent transforming growth factor-b binding protein superfamily and shares structural features with two other domains in these proteins, the TB/8-Cys and the cbEGF domains. Fibrillins are extended 350-kDa extracellular matrix proteins that constitute the backbone of microfibrils, large molecular machines found in elastic and nonelastic tissues. Microfibrils play important roles in the biogenesis of elastic fibers, in conferring limited elasticity to tissues, and in the regulation of bioavailability of transforming growth factor-b superfamily members. Similar to other matrix glycoproteins, fibrillins display a tandemly arrayed domain organization, with each domain representing an individual folding unit. Fibrillins, together with the latent transforming growth factor-b binding proteins (LTBPs), form a superfamily of proteins characterized by the presence of two unique domains: the transforming growth factor-b binding protein-like (TB or 8-Cys) domains, and the hybrid (hyb) domains (Figure 1). The limitations for high resolution structural studies of the full-length fibrillins are their large molecular mass combined with a relatively low solubility, the extremely high number of cysteine residues (361 in fibrillin-1, equivalent to 12.6%), almost all engaged in disulfide-bridges, and the extensive post-translational modifications, including N-glycosylation (Figure 1). However, it has been demonstrated that individual domains of fibrillin-1 or arrays of a few adjacent domains can be efficiently expressed in bacterial systems and refolded in vitro into correctly folded proteins (Knott et al., 1996). Using this experimental approach, high resolution structures of fibrillin-1 calcium-binding epidermal growth factor-like (cbEGF) domains and TB/8-Cys domains have been solved successfully (Downing et al., 1996; Lee et al., 2004). The hyb1 domain in fibrillins mediates interactions with fibulin-2, 4, and 5,
as well as with LTBP-1 and 4 (El-Hallous et al., 2007; Ono et al., 2009). In addition, it was shown that this domain participates in intermolecular disulfide-bond formation during microfibril assembly (Reinhardt et al., 2000). No functional assignments have been delineated for the hyb2 domain in fibrillins. The initial primary sequence analysis of the TB/8-Cys, hyb, and cbEGF domains revealed similarities of the hyb domains’ N termini with the N terminus of TB/8-Cys domains, and of the hyb domains’ C termini with the C terminus of cbEGF domains (Corson et al., 1993). With the new high resolution (1.8 A˚) crystal structure of a hyb domain from human fibrillin-1 (hyb2) reported by Jensen et al. (2009), it is now clear that these similarities in the primary sequence are fully reflected in the structure, proving that the hyb domain is indeed an evolutionary ‘‘hybrid’’ between TB/8-Cys and cbEGF domains. The overall structure of hyb2 flanked by cbEGF9 and cbEGF10 is similar to the structure of the TB/8-Cys domains in regard to the positions of characteristic side chains. In the central a-helical region of both domains, the ‘‘Cys-Cys-Cys’’ motif of the TB/8-Cys domain and the corresponding ‘‘Cys-Cys’’ motif in hyb2 are found in similar locations. In addition, a conserved cis-proline is placed in a corresponding structural region in both domains. In contrast to the TB/8-Cys structure, the hyb2 domain is missing a second a-helical stretch toward its C-terminal end. Another elementary difference between the two domains was found in the disulfide-bond patterns. Both domains contain eight cysteine residues; however, the hyb2 domain lacks one of the three cysteines in the ‘‘Cys-Cys-Cys’’ motif of the TB/8-Cys domain, but has
two cysteine residues in its C terminus as opposed to one. These differently positioned cysteine residues result in a 1-3, 2-5, 4-6, 7-8 disulfide-bond pattern in the hyb domain, versus a 1-3, 2-6, 4-7, 5-8 pattern characteristic for the TB/8-Cys domain. In this context it will be interesting to determine the structure of the hyb1 domain of fibrillin-1, which contains an additional cysteine residue at position 204 and consequently has one unpaired cysteine. This extra cysteine residue in the hyb1 domain is conserved between all fibrillins and between all species. Hyb domains in LTBPs, on the other hand, do not have such an extra cysteine residue. Biochemical data previously suggested that cysteine 204 of fibrillin-1 occurs as a free thiol on the surface of the molecule, making it an ideal candidate for intermolecular crosslink formation (Reinhardt et al., 2000). Molecular modeling of the hyb1 domain of fibrillin-1, based on the experimentally determined hyb2 coordinates presented in Jensen et al. (2009), supports this proposed role for the unpaired cysteine. The affinity for calcium binding to cbEGF domains in fibrillin-1 depends strongly on the nature of the inter-domain interfaces between adjacent domains. These contacts can modulate the affinity for calcium over a wide range (dissociation constants from nanomolar to millimolar). The cbEGF domains directly downstream of the TB/8-Cys domains frequently bind calcium with very high affinity attributed to hydrophobic domain interactions between the TB/8-Cys and the cbEGF domains (Jensen et al., 2005). In Jensen et al. (2009), the authors demonstrate that the cbEGF domains succeeding the hyb1 and hyb2 domains of fibrillin-1 also
Structure 17, May 13, 2009 ª2009 Elsevier Ltd All rights reserved 635
Structure
Previews microfibrils and to correlate the structure with specific functions. To fully understand the structure, macromolecular organization, and, ultimately, the functions of fibrillins, it will be necessary to solve the structure of (1) the proline/glycine-rich domains in fibrillins, (2) the N- and C-terminal unique domains, (3) larger contiguous portions of the proteins, (4) functional protein and selfinteraction sites, and (5) fully assembled fibrillin-1 aggregates giving rise to microfibrils. These are challenges for the future. For now, the fibrillin-1 hyb2 structure is one more piece added to the fibrillin puzzle.
ACKNOWLEDGMENTS We thank Ryan Kirschner for critical reading of the manuscript.
REFERENCES Corson, G.M., Chalberg, S.C., Dietz, H.C., Charbonneau, N.L., and Sakai, L.Y. (1993). Genomics 17, 476–484. Downing, A.K., Knott, V., Werner, J.M., Cardy, C.M., Campbell, I.D., and Handford, P.A. (1996). Cell 85, 597–605. El-Hallous, E., Sasaki, T., Hubmacher, D., Getie, M., Tiedemann, K., Brinckmann, J., Batge, B., Davis, E.C., and Reinhardt, D.P. (2007). J. Biol. Chem. 282, 8935–8946. Jensen, S.A., Corbett, A.R., Knott, V., Redfield, C., and Handford, P.A. (2005). J. Biol. Chem. 280, 14076–14084. Jensen, S.A., Iqbal, S., Lowe, E.D., Redfield, C., and Handford, P.A. (2009). Structure 17, this issue, 759–768.
Figure 1. Domain Organization of the Human Fibrillin/LTBP Superfamily Members The domains discussed in this preview are indicated in color. Jensen et al. (2009) solved the structure of the hyb2 domain (H2, blue) in the context of its flanking cbEGF9 and cbEGF10 domains as indicated. The hyb1 domain (H1, blue) of all fibrillins contain an additional unpaired ninth cysteine residue, while the hyb domains in LTBPs do not contain such an extra cysteine. For simplicity, only the longest splice variant of each LTBP is displayed.
bind calcium with very high affinity with dissociation constants in the low nanomolar range, which is again explained by the extensive domain interactions at least observed between hyb2 and cbEGF10. Under physiological calcium concentrations (1.5 mM), the region around hyb2 adopts a near linear shape. Together with previously published structural data available for the TB/8-Cys domains and
several cbEGF domains in their natural domain context, the authors predict the region between cbEGF3 and cbEGF43 (i.e., the majority of the fibrillin-1 molecule) to form an extended structure, as suggested by low resolution methods that include electron microscopy after rotary shadowing (Sakai et al., 1991). This information will be very useful to validate existing models for fibrillin organization in
636 Structure 17, May 13, 2009 ª2009 Elsevier Ltd All rights reserved
Knott, V., Downing, A.K., Cardy, C.M., and Handford, P. (1996). J. Mol. Biol. 255, 22–27. Lee, S.S., Knott, V., Jovanovic, J., Harlos, K., Grimes, J.M., Choulier, L., Mardon, H.J., Stuart, D.I., and Handford, P.A. (2004). Structure 12, 717–729. Ono, R.N., Sengle, G., Charbonneau, N.L., Carlberg, V., Bachinger, H.P., Sasaki, T., LeeArteaga, S., Zilberberg, L., Rifkin, D.B., Ramirez, F., et al. (2009). J. Biol. Chem., in press. Published online April 6, 2009. 10.1074/jbcM809348200. Reinhardt, D.P., Gambee, J.E., Ono, R.N., Ba¨chinger, H.P., and Sakai, L.Y. (2000). J. Biol. Chem. 275, 2205–2210. Sakai, L.Y., Keene, D.R., Glanville, R.W., and Ba¨chinger, H.P. (1991). J. Biol. Chem. 266, 14763–14770.