104 105 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Soft CondensedMatter Group Beamline B21 Priming human elastic proteins for assembly into elastic tissues Related publication: Lockhart-Cairns M. P., Newandee H., Thomson J.,Weiss A. S., Baldock C. &Tarakanova A. Transglutaminase-Mediated Cross- Linking of Tropoelastin to Fibrillin Stabilises the Elastin Precursor Prior to Elastic Fibre Assembly. J. Mol. Biol. 432 , 5736–5751 (2020). DOI: 10.1016/j.jmb.2020.08.023 Publication keywords: Tropoelastin; Elastin; Fibrillin; Tissue transglutaminase; Elastic fibres; Coacervation E lastic fibres are the main elastic component of mammalian elastic tissues such as major arteries, the lungs and skin. They provide resilience and recoil, enabling these tissues to expand and contract up to two billion times over a person’s lifetime. The two most abundant components of elastic fibres are tropoelastin and fibrillin. The process by which elastic fibres assemble is not well understood. This information is needed in regenerativemedicine, which aims to replace or regenerate cells, tissues or organs to restore or establish normal function. We know that the enzyme transglutaminase-2 covalently links tropoelastin and fibrillin and that this interaction between tropoelastin and fibrillin enhances tropoelastin assembly. However, we donot understand the importance of the linkage between tropoelastin andfibrillin and why this supports elastic fibre assembly. As part of a long-term collaboration, researchers from the University of Manchester, the University of Sydney and the University of Connecticut used the BioSAXS beamline B21 to collect Small-Angle X-ray Scattering (SAXS) data. They used the data to generatemodels of tropoelastin and fibrillin, and a tropoelastin-fibrillin complex. Thesemodels were used to understand what effect cross-linking had on the dynamics of these proteins. The models suggest that tropoelastin and fibrillin interact in an end-to-end manner and that cross-linking these two proteins together reduces their molecular motions, suggesting a stabilising effect due to this interaction. These findings suggest that the cross-link formation between tropoelastin and fibrillin stabilises the elastin precursor so that it is primed for elastic fibre assembly. Elastic fibres are the main elastic component of mammalian elastic tissues such as major arteries, the lungs and skin, where they provide resilience and recoil. These tissues expand and contract up to two billion times over a person’s lifetime. This extraordinary resilience is needed to carry out essential elasticity to ensure viability. The two most abundant components of elastic fibres are elastin and fibrillin microfibrils: tropoelastin (TE) is assembled to make elastin, and fibrillin microfibrils are assembled from the glycoprotein fibrillin-1. The assembly of elastic fibres occurs outside of the cell and is a highly organised and multistep process.TE can spontaneously and rapidly self-assemble, or coacervate, from a monomer to a multimer which is deposited onto a scaffold of fibrillin microfibrils 1 . Once deposited on fibrillin microfibrils, the TE multimers coalesce to form stable, insoluble elastic fibres.The enzyme transglutaminase-2 catalyses a transamidation reaction that covalently connects lysine residues to glutamine residues and forms a covalent intermolecular cross-link betweenTE and fibrillin 2 . The initial deposition of TE multimers onto fibrillin microfibrils is thought to be the primary step required for further assembly andTE recruitment to form larger elastic fibres, but the molecular details of this process are unknown. In order to understand the impact of cross-link formation on the structure and properties of TE and fibrillin, the structure and dynamics of TE and fibrillin were analysed individually and within the cross-linkedTE-fibrillin complex. Due to their size and flexibility, TE and fibrillin are not suitable for structural determination techniques such as X-ray crystallography or NMR so we had previously worked as a team to obtain the structure of the TE monomer in solution, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). SAXS and SANS can provide shape information on proteins in solution without the need for crystallisation or labelling. We found that it has an extended, asymmetrical shape ~20 nm in length 3 . The region of fibrillin involved in the TE cross-link has also been analysed by SAXS 4 and this region of fibrillin was used for further analysis (referred to as fibrillin). After purifying a cross-linked TE-fibrillin complex, it was analysed by multi-angle light scattering and analytical ultracentrifugation to determine its size, shape and the way that it moves in solution. The structural parameters were then analysed by SAXS on beamline B21. SAXS data for the complex and both individual components were collected and parameters including its overall size, shape and dimensions were compared. From these SAXS data, bead modelling was performed. The resulting model of the complex was L-shaped, comprising a thin, elongated region connected perpendicularly to a shorter, wider region. Comparison of the models of the fibrillin, TE and the TE-fibrillin complex showed that the model of the complex had elements of both fibrillin and TE combined, and displayed dimensions consistent with the addition of these components (Fig. 1). Moreover, docking the N-terminal region of TE to the rounded end of fibrillin gave a shape entirely consistent with the model of the complex which supports an interaction between the N-terminal region ofTE and the terminal domain of fibrillin. To computationally simulate the dynamics of TE, fibrillin and the TE-fibrillin complex, bead-spring elastic network models were developed. Bead spacing was assigned based on a regular lattice derived from the SAXS bead models 5 , for the complex and both individual components. Beads were interconnected through elastic springs. Normal mode analysis was performed to extract each of the structures’least energetically expensive, biologically accessible oscillating modes of motion e.g. the molecule’s natural fluctuations.The oscillating motions that characterise each mode within TE, fibrillin and the TE-fibrillin complex were analysed for their dynamic patterns. These data showed that specific movement patterns found in the TE and fibrillin structures individually were not translated to the complex. TE exhibited its characteristic scissor-twist motion, as described previously 5 . For fibrillin, there was an overall bending motion perpendicular to the length of the molecule and a pivot between the upper and lower regions. However, the multiple twisting motions shown in TE were not seen in the TE- fibrillin complex, which had a stationary base with a mild twist in the lower region (Fig. 2). Therefore, cross-linking of TE and fibrillin points to a stabilising effect on the base of the complex, where the cross-linking site is expected to reside. In this research, we describe the low-resolution structure, hydrodynamic properties and simulated dynamics of a transglutaminase cross-linked complex of TE and fibrillin. A previous study showed that a transglutaminase cross-link between fibrillin andTE could be formed which facilitated tropoelastin assembly, but the mechanism by which cross-linking supported coacervation was unknown. Hydrodynamic data on the TE-fibrillin complex suggests that fibrillin andTE interact in an end-to-endmanner which is supported by the SAXS data on the complex showing an elongated L-shaped molecule which retained elements of the individual molecules’ shapes. Our data show that the cross-link between fibrillin and tropoelastin restricts their molecular movement, therefore we propose that the microfibril-elastin interface similarly displays reduced motion (Fig. 3). Moreover, dynamic analysis of elastic network models of the complex supportsourtheoryofapronouncedstabilisingeffect. Inthismodel,thestiffened interface helps to dissipate substantial amounts of elastic energy by structural damping. Furthermore, this stabilisation of tropoelastin by binding to fibrillin microfibrils likely contributes to the mechanical response of mature elastic fibres. References: 1. Godwin A. R. F. et al. The role of fibrillin andmicrofibril binding proteins in elastin and elastic fibre assembly. Matrix Biol. 84 , 17–30 (2019). DOI: 10.1016/j.matbio.2019.06.006 2. Clarke A.W. et al. Coacervation is promoted by molecular interactions between the PF2 segment of fibrillin-1 and the domain 4 region of tropoelastin. Biochemistry 44 , 10271–10281 (2005). DOI: 10.1021/ bi050530d 3. Baldock C. et al. Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity. Proc. Natl. Acad. Sci. U. S. A. 108 , 4322–4327 (2011). DOI: 10.1073/pnas.1014280108 4. Baldock C. et al. Nanostructure of fibrillin-1 reveals compact conformation of EGF arrays andmechanism for extensibility. Proc. Natl. Acad. Sci. U. S. A. 103 , 11922–11927 (2006). DOI: 10.1073/pnas.0601609103 5. Yeo G. C. et al. Biomolecules: Subtle balance of tropoelastinmolecular shape and flexibility regulates dynamics and hierarchical assembly. Sci. Adv. 2 , e1501145 (2016). DOI: 10.1126/sciadv.1501145 Funding acknowledgement: TheWellcome Centre for Cell-Matrix Research is supported by funding from Wellcome (203128/Z/16/Z). CB gratefully acknowledges BBSRC funding (Ref: BB/N015398/1, BB/R008221/1 and BB/S015779/1).This work utilised the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1053575. XSEDE resources Stampede 2 and Ranch at theTexas Advanced Computing Center and Comet at the San Diego Supercomputing Center through allocationTG- MCB180008 were used. Corresponding author: Prof. Clair Baldock, University of Manchester,
[email protected] Figure 1: Models of the region of fibrillin (orange) that interacts with TE (blue) calculated from experimental SAXS data are shown in two orthogonal orientations. The TE-fibrillin complex has an elongated L-shape which supports an interaction (shown in orange/blue) between the N-terminal region of TE and fibrillin. Figure 2: Normal mode analysis of beads of the elastic network model of combination of modes 1-6 of TE, showing a bending coil; the dashed line and pivot indicates the axis about which bending occurs; the TE-binding region of fibrillin, showing an overall bending motion; the dashed line and pivot indicates the axis about which bending occurs. Upon formation of a transglutaminase-2 (TG2) cross-link, the TE-fibrillin complex shows a twisting extension and twisting base; colours correspond to a mobility scale for low mobility (blue), moderate mobility (white) and high mobility (red). Figure 3: In the process of elastic fibre assembly, tropoelastin molecules, which are inherently flexible, are deposited onto fibrillin microfibrils. The enzyme transglutaminase-2 (TG2) forms a covalent bond between fibrillin and tropoelastin which reduces their molecular movement and stabilises the microfibril-elastin interface, to facilitate elastic fibre assembly.