Elastin architecture

Accepted Manuscript Elastin architecture

Howard Vindin, Suzanne M. Mithieux, Anthony S. Weiss PII: DOI: Reference:

S0945-053X(19)30165-9 https://doi.org/10.1016/j.matbio.2019.07.005 MATBIO 1583

To appear in:

Matrix Biology

Received date: Revised date: Accepted date:

6 April 2019 8 July 2019 8 July 2019

Please cite this article as: H. Vindin, S.M. Mithieux and A.S. Weiss, Elastin architecture, Matrix Biology, https://doi.org/10.1016/j.matbio.2019.07.005

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ACCEPTED MANUSCRIPT Elastin Architecture Howard Vindin1,2, Suzanne M. Mithieux1,2 and Anthony S. Weiss1,2,3,4

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1. Charles Perkins Centre, the University of Sydney, The University of Sydney, 2006,

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Sydney, NSW, Australia

2. School of Life and Environmental Sciences, The University of Sydney, 2006, Sydney,

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NSW, Australia

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3. Bosch Institute, The University of Sydney, 2006, Sydney, NSW, Australia

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4. Sydney Nano Institute, The University of Sydney, 2006, Sydney, NSW, Australia

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* Corresponding author [email protected]

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Introduction Elastic fibers are integral components of resilient extracellular matrix (ECM) in all vertebrates. They provide the structural support and elastic recoil required for the continuous

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mechanical stretching and recovery of soft force-bearing tissues with durability and persistence. The major component of these fibers is elastin, an insoluble polymer that is very

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persistent, due to extensive cross-linking and is normally a metabolically stable unit over the

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human lifespan [1].

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Tropoelastin is the soluble monomer precursor of elastin that is secreted as a 60-70 kDa mature protein through variable splicing by diverse elastogenic cell types including

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fibroblasts, endothelial, smooth muscle, and airway epithelial cells in addition to chondrocytes and keratinocytes [2-7]. Mutations in the tropoelastin gene contribute to the

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genetic diseases supravalvar aortic stenosis and occasionally cutis laxa [8]. Tropoelastin is

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characterized by lysine-rich crosslinking domains, which are interspersed with hydrophobic domains that consist primarily of non-polar aliphatic amino acids [9] that often comprise

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repeating motifs [10-13]. Following translation, tropoelastin is chaperoned to the cell surface by a partly characterized pathway that includes the elastin binding protein (EBP), after which

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the tropoelastin is secreted [14-17]. Tropoelastin self-aggregates on the cell surface before being deposited onto fibrillin microfibrils and crosslinked to form elastic fibers, in a complex multi-step process collectively referred to as elastogenesis [18-22]. Tropoelastin possesses a structure that is organized enough for assembly but flexible enough to confer elasticity, which has made investigations into aspects of its structure challenging. On this basis it is incorrect to refer to tropoelastin as a disordered structure. Knowledge of tropoelastin’s structure has progressed through gene studies, analysis of its encoded domains,

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ACCEPTED MANUSCRIPT and culminated in the realization that structural appreciation can be drawn from holistically examining the structural features of the full-length molecule.

Tropoelastin gene, expression and alternative splicing

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A single gene encodes tropoelastin in nearly all species, while teleosts and amphibians are

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exceptions with two functional genes [23-25]. In humans, tropoelastin is localized to

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chromosome 7q11.1 and typically expresses 34 out of 36 exons, plus an occasionally used exon 26A in humans [23, 26-28]. The hydrophilic crosslinking domains and hydrophobic

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domains are encoded by distinct alternating exons that preserve the reading frame and allow

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for alternative splicing in a cassette-like manner [29].

Mammalian tropoelastin reveals marked heterogeneity due to alternative splicing [30-32].

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Clear sequence elements associated with alternative splicing were identified in the rat [33]. In

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humans, exons 22, 23, 24, 26A, 32 and 33 are consistently identified as being subject to alternative splicing [32, 34, 35]. Neither specific roles for various isoforms of tropoelastin or tissue specificity have been established, although it is appreciated that alternative splicing is

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developmentally regulated and age-related changes in both isoform ratio and splice site usage

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have been found in the rat, cow, and chick [36-38]. Keeley and colleagues recently demonstrated that known splice variants and single nucleotide polymorphisms in human tropoelastin confer defined physical properties including elasticity [39].

Structure elements in tropoelastin Tropoelastin possesses a unique structure possessing a mosaic of domains in various states of order. Unlike structured proteins which can have a funnel-shaped energy minimum

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ACCEPTED MANUSCRIPT representing the native folded structure (Figure 1A) [40], the free energy landscape of tropoelastin encompasses multiple energy minima with no sizable barriers between them (Figure 1B). The molecule transitions easily between these low energy minima, giving rise to a conformational ensemble that comprises a wide array of structurally related but dissimilar

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states. These feastures of tropoelastin contrast with those of elastin derived peptides (EDPs), such as

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those containing a repeating VPGXG motif and intrinsically disordered proteins (IDPs). In

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contrast to the greater complexity of tropoelastin, these are simpler repetitive sequences [41,

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42] and undergo phase separation during association [43-45].

For many years, structural studies of tropoelastin were delayed by the assumption that the

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molecule is an IDP based on its occasional use of repetitive elements, and similarities in the free energy landscapes in contrast to those of typical globular proteins (Figure 1) [46-48].

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However, the first clues that tropoelastin was not an IDP came from an appreciation of its

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substantial departure in amino acid composition. A key feature of IDPs is the depletion in order-promoting residues tyrosine, phenylalanine, tryptophan, isoleucine, leucine, methionine

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and valine [40]. Tropoelastin is enriched for aliphatic amino acids, such as valine, which is distributed evenly throughout the monomer, while IDPs are enriched in disorder-promoting

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residues including aspartate, glutamate, serine, proline, histidine, glutamine and lysine, many but not all of which are depleted in tropoelastin. Indeed, arising from gene duplication, the second tropoelastin gene present in frogs (XTR-eln2) has a much lower valine content than other tropoelastin genes and is enriched in glutamine and therefore could be classified as an IDP; an enrichment for serine also seen in this gene [25] may make it more susceptible to modification such as during metamorphosis. Further studies comparing XTR-eln1 and XTReln2 may shed light on the functional value of these sequence differences. In contrast,

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ACCEPTED MANUSCRIPT researchers began to appreciate that primarily tropoelastin needs sufficient structure to organize assembly into elastin, yet flexibility to retain elasticity.

Early research into tropoelastin structure

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Early research into the structure of tropoelastin was hampered by the insolubility of highly

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crosslinked elastic fibers, the lack of information afforded by primary sequence analysis due

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to enrichment for few amino acids (glycine, proline, leucine, alanine and valine), its alternative splicing and its relatively large size (~60-72 kDa) [49, 50]. As a result, researchers

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focused on simpler soluble elastin-like products that often either comprised repeated elements of less than 2% of the sequence of tropoelastin, or α- or κ-elastin obtained by partial

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hydrolysis of elastin using oxalic acid and ethanolic potassium hydroxide respectively [5153]. Early work included circular dichroism (CD) studies on α-elastin, NMR using elastin-

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based polypeptides, and sequence-based structure prediction algorithms which suggested that

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elastin had a predominately disordered structure [54-56]. In contrast to tropoelastin, molecular dynamics (MD) simulations of elastin-like peptides

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reveal structural similarities to amyloids in both monomers and aggregates. An amyloid

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propensity for these peptides is likely to be due to their departure from tropoelastin in both backbone hydration and conformational disorder, particularly where these are modulated through enrichment of proline and glycine residues [57]. While these peptides mirror the inverse temperature transition of tropoelastin in coacervation [49, 58, 59], proteomic and peptide studies demonstrate that proline and glycine residues help hydrate the molecule by keeping the peptide backbone hydrated, and so favor a disordered conformation leading to temperature-dependent self-aggregation into insoluble aggregates that is consistent with solid state NMR data [57, 60]. The role of liquid-liquid phase separation in the assembly and

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ACCEPTED MANUSCRIPT function of elastin and other ECM proteins has been reviewed elsewhere [61]. Until the advent of tropoelastin conformational studies, an assumption of molecular disorganization had historically dominated models of elastin structure and function. Unfolded proteins tend to aggregate, leading to highly ordered amyloid fibrils that are characterized by extensive secondary structure and a water-excluding hydrophobic core which do not adequately

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describe elastin [62]. Rather, on the path to forming elastin, tropoelastin maintains a high

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level of hydration that allows it to adopt a conformationally defined ensemble that confers an

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ability to assemble into elastin then persistently undergo extension and elastic recoil. CD analysis on the full human tropoelastin protein demonstrated that it is primarily

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composed of β-turns (21%), β-sheets (41%) and undefined structure (33%) with only a small proportion from α-helices (3%) [63]. Similar conclusions were found using Raman

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spectroscopy showing the molecule was composed of 8% α-helices, 36% β-strands, and 56% undefined structure [64]. Knowledge of the coding sequence for full-length human

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tropoelastin sequence and the subsequent development of an inexpensive, highly effective expression system that produces large quantities of protein have provided opportunities to

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explore full-length tropoelastin’s unique structural properties [65, 66].

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Domain structure of tropoelastin Exon 1 encodes a signal sequence that is cleaved from tropoelastin during transit through the rough endoplasmic reticulum [67, 68]. Domains encoded by the remaining exons can be broadly classified based on the observation that the cassette-like organization alternates between cross-linking domains and hydrophobic domains. The secondary structure adopted by these different domain types are generally observed in water, and trifluoroethanol (TFE) which favors intramolecular hydrogen bonding and promotes β-turns and β-helices by

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ACCEPTED MANUSCRIPT stabilizing the hydrophobic collapse of apolar side chains [69]. Water and TFE represent two extremes and in these studies often provided representative environments used to probe changes in peptide structure. These features are summarized here. Glycine-rich hydrophobic domains give rise to high conformational flexibility due to the

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paucity of interactions arising from no side chain. Exons 3, 5, 7, 11, 28, 30, and 32 encode for glycine-rich domains. These peptides have propensity to form polyproline II (PPII)

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structures, defined by the absence of intra- and intermolecular hydrogen bonds, and are easily

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able to convert between conformations to either β-turns or β-sheets [50]. In the presence of TFE, glycine-rich polypeptides show a predominance of type I/II β-sheets along with

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unordered conformations [52].

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Exons 2, 9, 16, 18, 20, 22, 24, 26, and 33 code for polypeptides with a large number of proline residues and regardless of the number of glycine residues are classified as proline-

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rich. Due to its constrained imino group, repeats comprising proline can form defined

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elements and in combination with glycine residues, these contribute to a variety of conformational structures. These regions display temperature-dependent conformational

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properties and typically exist as a mixture of folded type II β-sheets and unordered conformations [70].

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The crosslinking domains in elastin are classically either alanine rich (KA) or proline rich (KP). Both contain lysine residues that have the potential to contribute to intrachain and interchain crosslinks. In human tropoelastin exons 4, 8, 10, and 12 encode for KP, while KA is encoded by exons 6, 15, 17, 19, 21, 23, 25, 27, 29, and 31 [71]. These regions facilitate the formation of lysinonorleucine, allysine aldol and cyclic crosslinks including desmosine and isodesmosine within and between tropoelastin molecules, and so contribute essentially to the formation of stable elastin.

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Structural and functional regions in tropoelastin Over recent decades, a large body of research focused on methodologically deconstructing tropoelastin down to its individual domain or groups of domains to provide insight into the

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defining features of tropoelastin and how these might contribute to its overall structure and biological function. However, these approaches did not shed appreciable light on the intact

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molecule. A breakthrough came by deciphering the nanostructure of tropoelastin using SAXS

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and neutron scattering data, which elucidated a defined solution, envelope shape and

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identified discrete regions in the molecule [72]. These regions were defined structurally and functionally as the N-terminal coil, hinge region, bridge and the foot which extends to the C-

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terminus (Figure 2A).

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a. N-terminal coil region (domains 2-20) Tropoelastin’s N-terminal region comprises a narrow-elongated segment ~3 nm in diameter

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that runs for ~14 nm before extending and bifurcating into bridge and foot segments [72]. By comparing the full-length protein with truncated variants, domains 2-18 were mapped onto a

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spring-like nano-coil that contributes to tropoelastin elasticity. This region and the rest of the molecule collectively confer substantial elasticity on this uncrosslinked monomer such that it extends up to eight times its resting length [72]. Recognizing that the structure of tropoelastin collectively relies on interactions and contributions by its constituent domains, it is salient to contrast this with structural properties of tropoelastin’s separate elements. CD analysis of isolated domain 2 predicts a dynamic equilibrium between disordered and poly-proline II helical structure which has been

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ACCEPTED MANUSCRIPT subsequently confirmed by MD [52, 73]. This domain is adjacent to domains 3-5 whose MD simulations reveal short highly transient α- and 310-helical regions. Domains 3-5, 7-9, 11-14, and 18-20 present a predominantly disordered structure composed of coils, bends and turns as evidenced by CD [52, 70, 73, 74]. Domain 6 is mainly unstructured with some PPII also as evidenced by CD data [70]. Domain 10 is primarily composed of coils and bends and a PPII

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helix giving it a highly flexible structure [73]. This may help facilitate cross-linking to

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domains 19 and 25 which possess a relatively more stable α-helical structure. Domains 15

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and 17 are helical domains that have been proposed to stabilize the coil region [72, 73]. These coils are in close association pushing domain 16 out and exposing it to the environment. This

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may be to facilitate interactions with cells, help facilitate coacervation by covering an otherwise lysine rich hydrophilic region, or to partially protect these lysines from

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crosslinking during elastic fiber formation to ensure the stability of the coil region [73]. The role played by the N-terminal region in elastic fiber assembly is yet to be fully

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established. This region is likely important in regulating the overall structure of the tropoelastin coacervate, as suggested by the conformational changes displayed by a peptide containing domains 2-7 following an increase in solvent hydrophobicity [75]. The N-terminus

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may also facilitate the integration of tropoelastin into microfibrils through the interactions

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between domain 4 and fibrillin-1 [76]. The removal of domains 16 and 17 results in aberrant coacervation and severely impaired elastic fiber assembly [77]. There are three or more intermolecular crosslinks between the part of tropoelastin that spans domain 6-15, and a number of additional intermolecular crosslinks to the C-terminal region of another tropoelastin molecule making it highly likely that this region plays a key role in elastogenesis [78, 79]. Domain 10 is a major crosslinking site where this region bridges the desmosinelinked domains 19 and 25 of a second molecule [69]. The likely importance of the N-

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ACCEPTED MANUSCRIPT terminus in supporting elastic fiber formation is furthered by the association of a mutation in domain 12 with a phenotype of cutis laxa marked by deficient dermal elastic fibers [80]. The sole negatively charged residue in the first half of human tropoelastin is aspartate at position 72 (Asp-72) which is located on domain 6. As this same region shares 17 positively

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charged amino acids, it seemed likely this single negatively charged residue has some functional significance; to investigate this hypothesis, Asp-72 was mutated to alanine to give

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a D72A construct [81]. D72A displayed an impaired ability to self-associate, which was

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surprisingly dominant because the ability of tropoelastin to coacervate had been conventionally correlated with overall protein hydrophobicity, and this slight change should

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not have had such an effect [82, 83]. This supported a model where the N-terminal part of the

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molecule contributes to association of tropoelastin by coacervation. These effects were attributed to an altered organizational makeup of the mutant [81]. In addition, this mutant displayed aberrant incorporation into elastic fibers and differential binding to a domain 6-

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targeted antibody that was not seen using those that target other domains. These results collectively demonstrated the critical role of Asp-72 in maintaining the normal structure of the N-terminal region that is required for both the assembly of tropoelastin into elastic fibers

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and to support its normal function by associating with other tropoelastin molecule(s) [81].

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b. Hinge region (domains 21-24) Exon 22 theoretically encodes a short hydrophobic sequence in the central hinge region of tropoelastin, however its coding region is uniquely and universally spliced out of all human tropoelastin [84, 85]. As a result, domains 21 and 23 are contiguous [86, 87] and contribute to the hinge region. The hinge region is flexible [86, 88] and makes a substantial contribution to the mechanics of the molecule [89]. Domain 21 contains over 80% helical structures, while domain 23 contains approximately 60%; this is followed by a less structured domain 24

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ACCEPTED MANUSCRIPT which provides further leverage to the elastic core [73]. MD simulations show that the hinge region can either adopt an open structure, where the neighboring regions are open and parallel, or a closed anti-parallel hairpin conformation [90]. This is consistent with the wide range of structural distributions available to tropoelastin peptides [91]. Alanine substitution(s) of the central hinge in the tropoelastin peptide corresponding with domains 21-23 increase

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rigidity and decrease mobility [92]. The hinge region also contributes to cross-linking in

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elastin assembly as evidenced by mass spectrometry (MS) analysis and MD simulations [93].

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In a recent study, the structural and functional consequences of disrupting the hinge region were assessed by restoring exon 22 (WT+22) to the full-length protein [87]. SAXS analysis

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of the modified protein revealed an altered central region and consequential changes in the

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protein structure. Antibody binding showed no differences in the accessibility of domain 6. However, an antibody targeted to domain 24 showed reduced binding, indicating that the Cterminus was partially inaccessible [87]. This was accompanied by differences in the

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formation and appearance of hydrogels made by crosslinking the modified protein, where WT+22 hydrogels generated substantially compact structures and significantly reduced porosity compared with the wildtype. In vitro, WT+22 showed substantially reduced

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elastogenic properties [87]. These findings demonstrate a tight coupling between hierarchies

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of structure and dynamics and the functional properties of tropoelastin that can be influenced by a relatively small change in the molecule [87]. In accord with the concept that local changes generate global modifications in tropoelastin, sequential alanine replacement of the only two glutamates in domains 19 (E345A) and domain 21 (E414A) and the double mutation (E345A + E414A) led to impaired coacervation, hydrogel formation, and caused profound disruption in elastic fiber formation [94]. These residues are likely to stabilize the hinge and bridge regions through charge interactions, so their removal would have resulted in aberrant intramolecular interactions that led to globally 11

ACCEPTED MANUSCRIPT altered average molecular structures [94]. While the double mutant showed some incorporation into elastic fibers, the absence of elastic fibers for E345A mutant is consistent with a significant role of this part of the molecule in average molecular structure and consequentially functional assembly in elastic fiber formation [94].

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c. Bridge (domains 25-26) This part of the molecule is exposed and highly flexible. The exposed bridge is marked by a

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protease-susceptible arginine 515 (R515) at the junction of domains 25 and 26 that is

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conserved in mammals [95, 96]. The hydrophilic domain 25 forms a desmosine cross-link

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intramolecularly with domain 19 and intermolecularly with domain 10 [69]. Peptide studies point to the value of this region in elastic fiber assembly, as the long hydrophobic domain 26

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is important for coacervation and colocates with other large hydrophobic domains involved in this process [95, 97]. Domain 26 appears to serve as an intramolecular elastic drawbridge that

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functionally connects two broad segments that comprise tropoelastin. It is is a highly flexible

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structure that is dominated by three tandem nonapeptide repeats that facilitate the dynamics of tropoelastin [73]. It also marks the boundary downstream of which disease-associated

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functional mutations of tropoelastin are usually found. The flexibilty of the bridge region, was demonstrated by mutating R515 to alanine (R515A)

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and comparing its behavior with the wild-type protein. This resulted in a higher coacervation temperature for R515A and smaller coacervates than the wild-type, pointing to conformational changes that affected the association of tropoelastin molecules by coacervation [98]. The top surfaces of the hydrogels made by crosslinked coacervated R515A, as well as a construct that spanned from the N terminus to terminate at R515, consisted of ~5um spherules interlinked with fibers and demonstrating that these constructs were unable to form mature cross-linked structures [98]. More revealing information came in

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ACCEPTED MANUSCRIPT the form of SAXS structural data which revealed conformational variability around the bridge and C-terminal regions; the averaged model of the R515A mutant displayed significant dislocation of the C-terminal foot towards the molecule’s center [98], and revealed a crucial role for R515 in maintaining the orientation of the bridge and the downstream foot region.

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d. Foot region (domains 27-36) The foot region extends from domain 27 to the end of domain 36 at tropoelastin’s C-

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terminus, which comprises a cell interacting region [99, 100]. MD simulations indicate that

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the large hydrophobic domains 28, 30, 32 and 33 comprise coils, bends and turns in

transiently assume coiled structures [73].

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agreement with CD data [70, 73, 101], while the crosslinking domains 27, 29, and 31

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The hydrophobic domain 30 plays a role in both elastic fiber assembly and elasticity. Replacing domain 30 with poly-GA stiffens the molecule and increases its stress relaxation,

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while augmentation of prolines to prevent β-sheet formation lowers the elastic modulus and

material [102, 103].

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stress relaxation, compared to unmodified tropoelastin, giving rise to a more extensible

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Tropoelastin has only two cysteines, which are spaced just 5 amino acids apart in domain 36,

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followed by GRKRK which terminates the molecule in a highly positively charged tract that is well conserved across species. The region is exposed in the monomer but crosslinked in elastin. Treatment of tropoelastin with bifunctional cross-linkers results in high frequency crosslinking that utilizes C-terminal lysines [79]. Mass spectrometry of proteolytic digests from elastic fibers in bovine and human samples do not identify the RKRK motif [104, 105]; on this basis, bovine elastin contains only up to ~0.2% unmodified C-terminal epitope and provides biochemical evidence for crosslinking in this region [106]. This part of bovine tropoelastin is essential for association with the ECM [107]. Removal of bovine domain 36

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ACCEPTED MANUSCRIPT substantially impairs assembly of tropoelastin into the elastic fiber [108]. A similar effect is seen by deleting just the final RKRK sequence and in cysteine point mutations [108]. Domain 36, and in particular the C-terminal RKRK motif, are considered essential for classical elastic fiber formation.

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While it has been well established that elastin and its degradation products bind to cells through elastin binding protein (EBP) [109-111], much less is known about its other

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biological functions. The first evidence of additional binding sites was from a study showing

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that the C-terminal domain interacts with cell surface glycosaminoglycans (GAGs), where

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further experiments narrowed the binding site to the last 17 amino acids [100]. Domain 36 also facilitates binding between tropoelastin and integrin αvβ3 [112], with

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subsequent work identifying the C-terminal GRKRK motif as the integrin binding site required for cell adhesion [99]. Oxidation of the C-terminal domain with peroxynitrite

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(ONOO-) inhibits cell binding by modifying cysteine [113] suggesting that the introduction of

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negatively charged residues near the positively charged RKRK cluster is adverse [113]. A contribution by the RKRK motif in the binding of integrin αvβ3 to tropoelastin and its role in

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subsequent crosslinking in the mature elastic fiber all support a model where this interaction plays a crucial role in elastic fiber formation.

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Specific integrin interactions are likely to involve coordinated contributions from multiple motifs across tropoelastin. A binding site for integrin αvβ5 is localized to domains 17 and 18 near the central region of the molecule [114] and encompasses binding to both GAGs and integrin αvβ5 [115]. This interplay of GAG and integrin binding is a common feature of tropoelastin; substitution of specific lysines result in the abolishment of cell attachment highlighting the role for GAGs, while integrin blocking antibodies cause a marked reduction in cell spreading [115]. These results lead to a proposed model whereby GAGs interact with

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ACCEPTED MANUSCRIPT tropoelastin to facilitate cell attachment which is then followed by integrin-mediated cell spreading [115]. Integrin interactions assist tropoelastin in exerting biological effects on surfaces and in solution. Tropoelastin can exceed the pro-proliferative effect of insulin-like growth factor-1

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and basic fibroblast growth factor on mesenchymal stem cells and allows for a greater reduction in serum than either growth factor or fibronectin [116]. This effect is at least

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partially driven by integrins αvβ3 and αvβ5 as demonstrated by a ~60% decrease in cell

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expansion following the addition of blocking antibodies to these integrins, similar to the

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decrease seen using a pan anti-αv antibody [116], so other contributing interactions are likely.

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Coacervation of tropoelastin and its subsequent crosslinking into elastin

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The regulation of coacervation and subsequent crosslinking of tropoelastin into elastic fibers

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is a highly complex process involving several key accessory proteins including fibulin-4 , fibulin-5 , latent transforming growth factor β binding protein 4 (LTBP-4), and microfibril associated protein 4 (MFAP4) [117-123]. Due to space limitations, consideration focuses

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here on coacervation and crosslinking of tropoelastin, while comprehensive descriptions of

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the processes driving elastic fiber assembly have been reviewed by several groups [18-22]. Association by coacervation is an entropically driven process that involves interactions between tropoelastin’s hydrophobic domains, with assistance in a less understood process by its hydrophilic sequences and is critical to elastic fiber assembly [124, 125]. Modeling of this process in vitro shows that it consists of a phase separation stage where an increase in temperature monomers causes reversible transition to n-mers that tandemly assemble head-totail (Figure 2B), followed by maturation where tropoelastin coalesces irreversibly into

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ACCEPTED MANUSCRIPT fibrillar structures [126]. Optimal coacervation of human tropoelastin occurs at the physiologically relevant conditions of 37 °C, 150 mM NaCl at pH 7-8 [63]. The process of coacervation has been reviewed in detail [126]. In mature elastic fibers, glycosaminoglycans including heparan sulfate (HS) are found with

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the amorphous elastin core, where they may exert multiple roles within the fiber [127]. Heparin has the highest negative charge density of any known biological molecule making it

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an ideal candidate for mediating the coacervation of positively-charged tropoelastin through

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neutralizing charge interactions [128, 129]. On this basis, chondroitin sulfate B, heparin, and heparan sulfate substantially reduce the critical concentration required for tropoelastin

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coacervation [130].

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Following secretion from the cell, tropoelastin coalesces into ~200 nm particles [131] which subsequently fuse to form stable 1-2 um spherules on the cell surface [132]. As these

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spherules are incorporated into the ECM, they progressively fuse into ~6 um coacervates,

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which are then crosslinked by members of the lysyl oxidase (LOX) and lysyl oxidase like (LOXL) family of copper-dependent amine oxidases [133, 134] to form elastic fibers (Figure

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3). LOX and LOXL1 share the most similarities, suggesting these belong to one LOX subfamily while LOXL2-4 appear to belong to another subfamily [135]. These enzymes

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oxidize peptidyl lysine to the peptidyl aldehyde α-aminoadipic-δ-semialdehyde (allysine) [136], which then either reacts with an unmodified lysine or another allysine through adolcondensation to form the linear crosslinks lysinonorleucine and allysine aldol, culminating in cyclized desmosine and isodesmosine. LOX and LOXL1 are secreted as pro-proteins (proLOX and proLOXL1); their proteolytic cleavage mediated by bone morphogenic protein1 (BMP-1) or tolloid-like-1 (TLL1) which releases the free catalysts [137]. Fibronectin positively regulates LOX activity, both directly and indirectly, through binding to BMP-1 and enhancing its proteolytic activity [138, 139]. Knockout mouse models for LOX resulted in 16

ACCEPTED MANUSCRIPT perinatal death due to diaphragmatic rupture and aortic aneurysms [135, 140]. While most studies on LOX mediated crosslinking have focused on LOX and LOXL1, recent evidence indicates that LOXL2 and other members of the family are also involved in this process [141].

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MD simulations of tropoelastin at 37 °C demonstrates oscillations in tropoelastin’s structure drive a twist in the coil region and scissor-like bending of the hinge and foot regions [142].

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Of particular interest was that these resulted in enhanced fluctuations of specific domains

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and, more specifically, lysine residues within the molecule. High resolution MS on bovine elastin has cataloged a diverse array of intramolecular and intermolecular crosslinks from KA

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and KP domains. Unmodified lysine residues were also found, due to incomplete oxidative

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deamination. These results show that tropeolastin molecules are connected by different crosslinks with remarkable heterogeneity. For example, Lys-275 is optionally unmodified, cross-linked through lysinonorleucine to a KF and more, cross-linked through allysine aldol

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to AKA, and intramolecularly with Lys-252, as well as involved in desmosine/isodesmosine peptides [143]. The diverse array of crosslinks seen in bovine elastin is also seen in human elastin in both the skin and aorta providing a clear demonstration that this is a stochastic

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process [144]. This work also demonstrated for the first time the presence of multiple

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tropoelastin isoforms at the protein level in both tissue types [144]. This heterogeneity in crosslinking is consistent with the existence of a conformational ensemble of tropoelastin molecules that vary their structures and ensuing crosslinks based on the order of lysine oxidation [145]. While significant progress has been made in understanding the structural and functional properties of tropoelastin and defining the mechanistic aspects that drive elastogenesis, there is still much to be discovered. It is interesting to speculate upon some of the uncertainties mentioned herein. While the majority of work to date has focused on a single isoform, the 17

ACCEPTED MANUSCRIPT presence of multiple isoforms at the protein level has now been confirmed. Whether these isoforms define specific fiber populations with unique properties or whether single elastic fibers are made up of multiple isoforms that regulate, for example, branch points or additional protein binding is yet to be explored. In addition, while one of the first and most well-known observations is the highly autofluorescent nature of elastic fibers, research on this

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phenomenon is sparse. Further investigation into the fluorescent properties of tropoelastin

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and elastic fibers will provide further insight into their structural properties and the

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mechanisms driving elastogenesis

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ACCEPTED MANUSCRIPT Figure legends

Figure 1. Models for free energy landscapes typical of folded globular proteins in contrast to intrinsically disordered proteins and tropoelastin. Horizontal and vertical axes represent

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conformational space, while the z-axis represents free energy. (A) Example of a typical folded globular protein shows a dominant funnel-shaped energy minimum that corresponds to

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the protein’s tertiary structure. (B) In contrast the free energy landscape of intrinsically

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disordered proteins and tropoelastin is characterized by multiple local minima, that allow for the co-existence of an ensemble of low energy tertiary structures. These low energy structures

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are accompanied by molecules in the population that occupy additional low abundance higher

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energy states.

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Figure 2. Tropoelastin features and assembly. (A) Tropoelastin is an asymmetric monomer in

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solution. Its major regions are indicated and discussed in the text [72]. (B) Tropoelastin initially assembles in a head-to-tail fashion allowing for tandem propagation. In a

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hypothetical model, in association with microfibrils these linear assemblies are presented as

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linear stylized parallel arrays, that coalesce to form globules.

Figure 3. Simplified schematic of coacervation and cross-linking of tropoelastin. (A) Assembling tropoelastin coalesces into 200-300 nm nanoparticles that remain on the elastogenic cell surface soon after secretion. (B) These nanoparticles fuse to give 1-2 µm spherules that (C) grow and move from the cell surface until they are (D) deposited onto microfibrillar scaffolds and the growing elastic fiber. (E) Lysyl oxidase and lysyl oxidase-

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ACCEPTED MANUSCRIPT like proteins oxidize lysine residues in tropoelastin before and during coacervation, allowing

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for (F) their covalent crosslinking into elastin. Figure not to scale.

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ACCEPTED MANUSCRIPT Acknowledgements H.V. acknowledges an Australian Postgraduate Award and Australian Government Research Training Program Stipend Scholarship. A.S.W. acknowledges funding from the Australian

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Research Council, National Health & Medical Research Council and RSL Australia.

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Elastin is required for elasticity in diverse vertebrate tissues. In mammals, elastin forms a very persistent, elastic crosslinked protein network that is not normally synthesized beyond the first few years of life. Tropoelastin is encoded by one gene in most vertebrates and subject to variable splicing Tropoelastin is crosslinked to form elastin Tropoelastin is a soluble asymmetric protein monomer with a defined set of structures that interconvert between an ensemble of defined, low energy states. Tropoelastin displays distinctive association by coacervation.

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