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The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand’s ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling–dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand’s ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling–dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

The human FBN1 gene encodes fibrillin-1 (FBN1); the main component of the 10–12 nm diameter extracellular matrix microfibrils. Marfan syndrome (MFS) is a common inherited connective tissue disorder, caused by FBN1 mutations. It features a wide spectrum of disease severity, from mild cases to the lethal neonatal form (nMFS), that is yet to be explained at the molecular level. Mutations associated with nMFS generally affect a region of FBN1 between domains TB3-cbEGF18—the "neonatal region". To gain insight into the process of fibril assembly and increase our understanding of the mechanisms determining disease severity in MFS, we compared the secretion and assembly properties of FBN1 variants containing nMFS-associated substitutions with variants associated with milder, classical MFS (cMFS). In the majority of cases, both nMFS- and cMFS-associated neonatal region variants were secreted at levels comparable to wild type. Microfibril incorporation by the nMFS variants was greatly reduced or absent compared to the cMFS forms, however, suggesting that nMFS substitutions disrupt a previously undefined site of microfibril assembly. Additional analysis of a domain deletion variant caused by exon skipping also indicates that register in the neonatal region is likely to be critical for assembly. These data demonstrate for the first time new requirements for microfibril biogenesis and identify at least two distinct molecular mechanisms associated with disease substitutions in the TB3-cbEGF18 region; incorporation of mutant FBN1 into microfibrils changing their integral properties (cMFS) or the blocking of wild type FBN1 assembly by mutant molecules that prevents late-stage lateral assembly (nMFS).

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases., AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.

Fibrillins serve as scaffolds for the assembly of elastic fibers that contribute to the maintenance of tissue homeostasis and regulate growth factor signaling in the extracellular space. Fibrillin-1 is a modular glycoprotein that includes 7 latent transforming growth factor β (TGFβ)-binding protein-like (TB) domains and mediates cell adhesion through integrin binding to the RGD motif in its 4th TB domain. A subset of missense mutations within TB4 cause stiff skin syndrome (SSS), a rare autosomal dominant form of scleroderma. The fibrotic phenotype is thought to be regulated by changes in the ability of fibrillin-1 to mediate integrin binding. We characterized the ability of each RGD-binding integrin to mediate cell adhesion to fibrillin-1 or a disease-causing variant. Our data show that 7 of the 8 RGD-binding integrins can mediate adhesion to fibrillin-1. A single amino acid substitution responsible for SSS (W1570C) markedly inhibited adhesion mediated by integrins α5β1, αvβ5, and αvβ6, partially inhibited adhesion mediated by αvβ1, and did not inhibit adhesion mediated by α8β1 or αIIbβ3. Adhesion mediated by integrin αvβ3 depended on the cell surface expression level. In the SSS mutant background, the presence of a cysteine residue in place of highly conserved tryptophan 1570 alters the conformation of the region containing the exposed RGD sequence within the same domain to differentially affect fibrillin's interactions with distinct RGD-binding integrins.

Summary Fibrillin-1 (FBN1) mutations associated with Marfan syndrome lead to an increase in transforming growth factor β (TGF-β) activation in connective tissues resulting in pathogenic changes including aortic dilatation and dissection. Since FBN1 binds latent TGF-β binding proteins (LTBPs), the major reservoir of TGF-β in the extracellular matrix (ECM), we investigated the structural basis for the FBN1/LTBP1 interaction. We present the structure of a four-domain FBN1 fragment, EGF2-EGF3-Hyb1-cbEGF1 (FBN1E2cbEGF1), which reveals a near-linear domain organization. Binding studies demonstrate a bipartite interaction between a C-terminal LTBP1 fragment and FBN1E2cbEGF1, which lies adjacent to the latency-associated propeptide (LAP)/TGF-β binding site of LTBP1. Modeling of the binding interface suggests that, rather than interacting along the longitudinal axis, LTBP1 anchors itself to FBN1 using two independent epitopes. As part of this mechanism, a flexible pivot adjacent to the FBN1/LTBP1 binding site allows LTBP1 to make contacts with different ECM networks while presumably facilitating a force-induced/traction-based TGF-β activation mechanism., Graphical Abstract, Highlights • The structure of the FBN1 N-terminal region shows a near-linear domain organization • LTBP1 binds to FBN1 via a bipartite mode of interaction involving two discreet sites • This allows LTBP1 to connect 10–12 nm FBN1 microfibrils to other ECM networks • This may facilitate force-induced/traction-based activation of TGF-β via integrins, Improving our knowledge of TGF-β regulation by matrix biomechanics is vital for understanding the biology of this enigmatic growth factor. Robertson et al. present a bipartite model for the structure of the fibrillin-1-LTBP1 interaction that functions as a holdfast for TGF-β in the matrix.

Summary The human extracellular matrix glycoprotein fibrillin-1 is the primary component of the 10- to 12-nm-diameter microfibrils, which perform key structural and regulatory roles in connective tissues. Relatively little is known about the molecular mechanisms of fibrillin assembly into microfibrils. Studies using recombinant fibrillin fragments indicate that an interaction between the N- and C-terminal regions drives head-to-tail assembly. Here, we present the structure of a fibrillin N-terminal fragment comprising the fibrillin unique N-terminal (FUN) and the first three epidermal growth factor (EGF)-like domains (FUN-EGF3). Two rod-like domain pairs are separated by a short, flexible linker between the EGF1 and EGF2 domains. We also show that the binding site for the C-terminal region spans multiple domains and overlaps with a heparin interaction site. These data suggest that heparan sulfate may sequester fibrillin at the cell surface via FUN-EGF3 prior to aggregation of the C terminus, thereby regulating microfibril assembly., Highlights • The fibrillin unique N-terminal (FUN) domain adopts a novel fold • The binding site for the fibrillin C terminus spans multiple domains • The heparin interaction site overlaps with the C-terminal binding region • Detailed molecular insights into an interaction between fibrillin molecules, Fibrillin microfibrils are key structural and regulatory components of connective tissue, but their assembly is poorly understood. Yadin et al. report a structure of the fibrillin N-terminal domains and propose that heparan sulphate regulates end-to-end assembly.

Human fibrillin-1 is the major structural protein of extracellular matrix 10–12 nm microfibrils. It has a disulfide-rich modular organization which consists primarily of cbEGF (Ca2+-binding epidermal growth factor-like) domains and TB (transforming growth factor β-binding protein-like) domains. TB4 contains an RGD (Arg-Gly-Asp) integrin-binding motif. The atomic structure of this region has been solved by X-ray crystallography and shows the TB4 and flanking cbEGF domains to be arranged as a tetragonal pyramid with N- and C-termini exposed at opposite ends of the fragment. The RGD integrin-binding motif is located within a flexible loop. We have used a variety of biophysical, biochemical and cell biology methods to investigate the molecular properties of integrin–fibrillin-1 interactions and have demonstrated that recombinant fibrillin-1 domain fragments mediate binding to integrins αVβ3, α5β1 and αVβ6. Integrin αVβ3 is a high-affinity fibrillin-1 receptor ( K d ∼40 nM), whereas integrins αVβ6 and α5β1 show moderate-affinity ( K d ∼450 nM) and low-affinity ( K d >1 μM) binding respectively. Different patterns of α5β1 distribution are seen when human keratinocytes and fibroblasts are plated on to fibrillin domain fragments compared with those seen for fibronectin, suggesting that fibrillin may cause a lesser degree or different type of intracellular signalling. A number of disease-causing mutations which affect the TB4 domain have been identified. These are being investigated for their effects on integrin binding and/or changes in intramolecular structure. Abbreviations: BHK, baby-hamster kidney; cbEGF, Ca2+-binding epidermal growth factor-like; EGF, epidermal growth factor; EM, electron microscopy; LTBP, latent transforming growth factor β-binding protein; SPR, surface plasmon resonance; TB, transforming growth factor β-binding protein-like

Force-bearing tissues such as blood vessels, lungs, and ligaments depend on the properties of elasticity and flexibility. The 10 to 12 nm diameter fibrillin microfibrils play vital roles in maintaining the structural integrity of these highly dynamic tissues and in regulating extracellular growth factors. In humans, defective microfibril function results in several diseases affecting the skin, cardiovascular, skeletal, and ocular systems. Despite the discovery of fibrillin-1 having occurred more than two decades ago, the structure and organization of fibrillin monomers within the microfibrils are still controversial. Recent structural data have revealed strategies by which fibrillin is able to maintain its architecture in dynamic tissues without compromising its ability to interact with itself and other cell matrix components. This review summarizes our current knowledge of microfibril structure, from individual fibrillin domains and the calcium-dependent tuning of pairwise interdomain interactions to microfibril dynamics, and how this relates to microfibril function in health and disease.

Fibrillins and LTBPs [latent TGFβ (transforming growth factor β)-binding proteins] perform vital and complex roles in the extracellular matrix and are relevant to a wide range of human diseases. These proteins share a signature ‘eight cysteine’ or ‘TB (TGFβ-binding protein-like)’ domain that is found nowhere else in the human proteome, and which has been shown to mediate a variety of protein–protein interactions. These include covalent binding of the TGFβ propeptide, and RGD-directed interactions with a repertoire of integrins. TB domains are found interspersed with long arrays of EGF (epidermal growth factor)-like domains, which occur more widely in extracellular proteins, and also mediate binding to a large number of proteins and proteoglycans. In the present paper, newly available protein sequence information from a variety of sources is reviewed and related to published findings on the structure and function of fibrillins and LTBPs. These sequences give valuable insight into the evolution of TB domain proteins and suggest that the fibrillin domain organization emerged first, over 600 million years ago, prior to the divergence of Cnidaria and Bilateria, after which it has remained remarkably unchanged. Comparison of sequence features and domain organization in such a diverse group of organisms also provides important insights into how fibrillins and LTBPs might perform their roles in the extracellular matrix.

The fibrillins and latent transforming growth factor-beta binding proteins (LTBPs) form a superfamily of structurally-related proteins consisting of calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with 8-cysteine-containing transforming growth factor beta-binding protein-like (TB) and hybrid (hyb) domains. Fibrillins are the major components of the extracellular 10-12 nm diameter microfibrils, which mediate a variety of cell-matrix interactions. Here we present the crystal structure of a fibrillin-1 cbEGF9-hyb2-cbEGF10 fragment, solved to 1.8 A resolution. The hybrid domain fold is similar, but not identical, to the TB domain fold seen in previous fibrillin-1 and LTBP-1 fragments. Pairwise interactions with neighboring cbEGF domains demonstrate extensive interfaces, with the hyb2-cbEGF10 interface dependent on Ca(2+) binding. These observations provide accurate constraints for models of fibrillin organization within the 10-12 nm microfibrils and provide further molecular insights into how Ca(2+) binding influences the intermolecular interactions and biomechanical properties of fibrillin-1.

The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1(DSL-EGF3)). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1(DSL-EGF3) binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

Fibrillin microfibrils are 10–12 nm diameter, extracellular matrix assemblies that provide dynamic tissues of metazoan species with many of their biomechanical properties as well as sequestering growth factors and cytokines. Assembly of fibrillin monomers into microfibrils is thought to occur at the cell surface, with initial steps including proprotein processing, multimerization driven by the C terminus, and the head-to-tail alignment of adjacent molecules. At present the mechanisms that regulate microfibril assembly are still to be elucidated. We have used structure-informed protein engineering to create a recombinant, GFP-tagged version of fibrillin-1 (GFP-Fbn) to study this process. Using HEK293T cells transiently transfected with GFP-Fbn constructs, we show that (i) the C-terminal propeptide is an essential requirement for the secretion of full-length fibrillin-1 from cells; (ii) failure to cleave off the C-terminal propeptide blocks the assembly of fibrillin-1 into microfibrils produced by dermal fibroblasts; and (iii) the requirement of the propeptide for secretion is linked to the presence of domains cbEGF41-43, because either deletion or exchange of domains in this region leads to cellular retention. Collectively, these data suggest a mechanism in which the propeptide blocks a key site at the C terminus to prevent premature microfibril assembly.

Elastic fibers consist primarily of an amorphous elastin core associated with microfibrils, 10-12 nm in diameter, containing fibrillins and microfibril-associated glycoproteins (MAGPs). To investigate the interaction of MAGP-1 with tropoelastin and fibrillin-1, we expressed human MAGP-1 as a T7-tag fusion protein in Escherichia coli. Refolding of the purified protein produced a soluble form of MAGP-1 that displayed saturable binding to tropoelastin. Fragments of tropoelastin corresponding to the N-terminal, C-terminal, and central regions of the molecule were used to characterize the MAGP-1 binding site. Cleavage of tropoelastin with kallikrein, which cleaves after Arg(515) in the central region of the molecule, disrupted the interaction, suggesting that the separated N- and C-terminal fragments were insufficient to determine MAGP-1 binding to intact tropoelastin. In addition, no evidence of an interaction was observed between MAGP-1 and a tropoelastin construct consisting of domains 17-27 that brackets the kallikrein cleavage site, suggesting a complex mechanism of interaction between the two molecules. Binding of MAGP-1 was also tested with overlapping recombinant fibrillin-1 fragments. MAGP-1 bound to a region at the N terminus of fibrillin-1 in a calcium-dependent manner. In summary, these results suggest a model for the interaction of elastin with the microfibrillar scaffold.

Coacervation of soluble tropoelastin molecules is characterized by thermodynamically reversible association as temperature is increased under appropriately juxtaposed ionic conditions, protein concentration and pH. Coacervation plays a critical role in the assembly of these elastin precursors in elastic fiber formation. To examine the effect of physiological parameters on the ability of tropoelastin molecules to associate, solutions of recombinant human tropoelastin were monitored spectrophotometrically by light scattering over a broad range of temperatures. Coacervation of recombinant human tropoelastin is strongly influenced by the concentration of protein and NaCl and to a lesser extent on pH. Trends towards maximal association are apparent when each of these parameters is varied. Remarkably, optimal coacervation is found at 37 degrees C, 150 mM NaCl and pH 7-8. Using the data generated by time courses, estimates of thermodynamic parameters were made. These estimates confirm that coacervation is endothermic and is marked by a strong entropic contribution. Circular dichroism of recombinant human tropoelastin revealed that, rather than being random, the structure is compatible with being largely that, of an all-beta protein (with secondary structure estimated to be 3% alpha-helix, 41% beta-sheet, 21% beta-turn and 33% other), exhibiting a spectrum as previously seen for tropoelastin populations and soluble elastin from naturally-derived sources.

Fibrillins are large extracellular glycoproteins that form the principal component of microfibrils. These perform a vital structural function in the extracellular matrix of many tissues. Fibrillins have also been implicated in mediating a number of protein-protein interactions, some of which may be significant in regulating growth factors such as transforming growth factor β. Here we present the backbone and side-chain (1)H, (13)C and (15)N assignments for a 19 kDa protein fragment derived from the N-terminus of human fibrillin-1, encompassing four domains in total. These domains include the second and third epidermal growth factor-like (EGF) domains, the first hybrid domain (hyb1), and the first calcium-binding EGF domain of fibrillin-1. This region of fibrillin-1 is of particular interest as the hyb1 domain has been suggested to play a role in microfibril assembly, as well as several other protein-protein interactions.

International audience; Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFb-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFb signaling were consistent features in GD and AD fibro-blasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFb signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.

FBLN5 encodes fibulin-5, an extracellular matrix calcium-binding glycoprotein that is essential for elastic fibre formation. FBLN5 mutations are associated with two distinct human diseases, age-related macular degeneration (AMD) and cutis laxa (CL), but the biochemical basis for the pathogenic effects of these mutations is poorly understood. Two missense mutations found in AMD patients (I169T and G267S) and two missense mutations found in CL patients (G202R and S227P) were analysed in a native-like context in recombinant fibulin-5 fragments. Limited proteolysis, NMR spectroscopy and chromophoric calcium chelation experiments showed that the G267S and S227P substitutions cause long-range structural effects consistent with protein misfolding. Cellular studies using fibroblast cells further demonstrated that these recombinant forms of mutant fibulin-5 were not present in the extracellular medium, consistent with retention. In contrast, no significant effects of I169T and G202R substitutions on protein fold and secretion were identified. These data establish protein misfolding as a causative basis for the effects of G267S and S227P substitutions in AMD and CL, respectively, and raise the possibility that the I169T and G202R substitutions may be polymorphisms or may increase susceptibility to disease.

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common structural motif in extracellular and transmembrane proteins. K(d) values for Ca2+ vary from the millimolar to nanomolar range; however the molecular basis for this variation is poorly understood. We have measured K(d) values for six fibrillin-1 cbEGF domains, each preceded by a transforming growth factor beta-binding protein-like (TB) domain. Using NMR and titration with chromophoric chelators, we found that K(d) values varied by five orders of magnitude. Interdomain hydrophobic contacts between TB-cbEGF domains were studied by site-directed mutagenesis and could be correlated directly with Ca2+ affinity. Furthermore, in TB-cbEGF pairs that displayed high-affinity binding, NMR studies showed that TB-cbEGF interface formation was strongly Ca2+-dependent. We suggest that Ca2+ affinity is a measure of interface formation in both homologous and heterologous cbEGF domain pairs, thus providing a measure of flexibility in proteins with multiple cbEGF domains. These data highlight the versatile role of the cbEGF domain in fine tuning the regional flexibility of proteins and provide new constraints for the organization of fibrillin-1 within 10-12-nm microfibrils of the extracellular matrix.

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.

Tropoelastin is the soluble precursor of elastin, the major component of the extracellular elastic fiber. Tropoelastin undergoes self-association via an inverse temperature transition termed coacervation, which is a crucial step in elastogenesis. Coacervation of tropoelastin takes place through multiple intermolecular interactions of its hydrophobic domains. Previous work has implicated those hydrophobic domains located near the center of the polypeptide as playing a dominant role in coacervation. Short constructs of domains 18, 20, 24, and a mutated form of domain 26 were largely disordered at 20 degrees C but displayed increased order on heating that was consistent with the formation of beta-structures. However, their conformational transitions were not sensitive to physiological temperature in contrast to the observed behavior of the native domain 26. A polypeptide consisting of domains 17-27 of tropoelastin coacervated at temperatures above 60 degrees C, whereas individually expressed hydrophobic regions were not capable of coacervation. We conclude that coacervation depends on the hydrophobicity of the molecule and, by inference, the number of hydrophobic domains. Tropoelastin mutants were constructed to contain a Pro --Ala mutation in domain 26, separate deletions of domains 18 and 26, and a displacement of domain 26. These constructs displayed unequal capacities for coacervation, even when they contained the same number of hydrophobic regions and comparable levels of secondary structure. Thus, the capability for coacervation is determined by contributions from individual hydrophobic domains for which function should be considered in the context of their positions in the intact tropoelastin molecule.

The temperature-dependent association of tropoelastin molecules through coacervation is an essential step in their assembly leading to elastogenesis. The relative contributions of C-terminal hydrophobic domains in coacervation were assessed. Truncated tropoelastins were constructed with N termini positioned variably downstream of domain 25. The purified proteins were assessed for their ability to coacervate. Disruption to domain 26 had a substantial effect and abolished coacervation. Circular dichroism spectroscopy of an isolated peptide comprising domain 26 showed that it undergoes a structural transition to a state of increased order with increasing temperature. Protease mapping demonstrated that domain 26 is flanked by surface sites and is likely to be in an exposed position on the surface of the tropoelastin molecule. These results suggest that the hydrophobic domain 26 is positioned to play a dominant role in the intermolecular interactions that occur during coacervation.