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Genetic mutations in fibrillin microfibrils cause serious inherited diseases, such as Marfan syndrome and Weill–Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. Here we describe a detailed structural analysis of native fibrillin microfibrils from mammalian tissue by cryogenic electron microscopy. The major bead region showed pseudo eightfold symmetry where the amino and carboxy termini reside. On the basis of this structure, we show that a WMS deletion mutation leads to the induction of a structural rearrangement that blocks interaction with latent TGFβ-binding protein-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. The integrin αvβ3-binding site was also mapped onto the microfibril structure. These results establish that in complex extracellular assemblies, such as fibrillin microfibrils, mutations may have long-range structural consequences leading to the disruption of growth factor signaling and the development of disease.

The ADAMTS superfamily is composed of secreted metalloproteases and structurally related non-catalytic ADAMTS-like proteins. A subset of this superfamily, including ADAMTS6, ADAMTS10 and ADAMTSL2, are involved in elastic fiber assembly and bind to fibrillin and other matrix molecules that regulate the extracellular bioavailability of the potent growth factor TGFβ. Fibrillinopathies, that can also result from mutation of these ADAMTS/L proteins, have been linked to disrupted TGFβ homeostasis. ADAMTS6 and ADAMTS10 are homologous metalloproteases with poorly characterized substrates where ADAMTS10 is thought to process fibrillin-2 and ADAMTS6 latent TGFβ-binding protein (LTBP)-1. In order to understand the contribution of ADAMTS6, and these other members of the ADAMTS/L family, to TGFβ homeostasis, we have analyzed the effects of ADAMTS6, ADAMTS10 and ADAMTSL2 expression on TGFβ activation. We found that their expression increases TGFβ activation in a dose dependent manner, following stimulation with mature TGFβ1. For ADAMTS6, the catalytically active protease is required for effective TGFβ activation, where ADAMTS6 cleaves LTBP3 as well as LTBP1, and binds to the large latent TGFβ complexes of LTBP1 and LTBP3. Furthermore, ADAMTS6 expression increases the mechanotension of cells which results in inactivation of the Hippo Pathway, resulting in an increased translocation of YAP/TAZ complex to the nucleus. Together these findings suggest that when the balance of TGFβ is perturbed ADAMTS6 can influence TGFβ activation via two mechanisms. It directly cleaves the latent TGFβ complexes and also acts indirectly, along with ADAMTS10 and ADAMTSL2, by altering the mechanotension of cells. Together this increases activation of TGFβ from large latent complexes which may contribute to disease pathogenesis.

The formation of multinucleated contractile myofibers from muscle stem cells during myogenesis is indispensable for skeletal muscle formation. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD. However, very few MYOD- regulated effector proteins were shown to be sufficient to promote myogenesis. Here, we identified an unexpected role for the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as a rheostat for Wnt signaling during myogenesis downstream of MYOD. ADAMTSL2 expression was induced during myoblast differentiation and ADAMTSL2 was required for myoblast differentiation. ADAMTSL2 ablation in myogenic precursor cells resulted in aberrant muscle architecture in vivo. The pro-myogenic ADAMTSL2 function was dependent on WNT ligands. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. Finally, we identified a WNT-binding ADAMTSL2 peptide that was sufficient to promote myogenesis. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling node that could integrate and fine-tune WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

Genetic mutations in fibrillin microfibrils cause serious inherited diseases such as Marfan syndrome (MFS) and Weill-Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. In this study, we reveal the detailed cryo-EM structure of native fibrillin microfibrils from mammalian tissue. The major bead region showed pseudo 8-fold symmetry where the N-and C-termini reside. Based on this structure, we show a WMS-deletion mutant induces a rearrangement blocking interaction with latent TGFβ-binding protein (LTBP)-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. The integrin αVβ3-binding site was also mapped onto the microfibril structure. These results establish that in complex extracellular assemblies, such as in fibrillin, mutations may have long-range structural consequences to disrupt growth factor signalling and cause disease.

Genetic mutations in fibrillin microfibrils cause a range of serious inherited diseases such as Marfan syndrome (MFS) and Weill-Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. In this study we reveal the detailed cryo-EM structure of native fibrillin microfibrils from mammalian tissue. The major bead region showed pseudo 8-fold symmetry and a buried protease resistant N-terminal core. Based on this structure, we show a WMS deletion mutant induces a rearrangement with long-range effects blocking interaction with latent TGFβ-binding protein (LTBP)-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. These results establish that in complex extracellular protein assemblies, such as in fibrillin, mutations may have long-range structural consequences to disrupt growth factor signalling and cause disease.

Elastic fibres are essential components of all mammalian elastic tissues such as blood vessels, lung and skin, and are critically important for the mechanical properties they endow. The main components of elastic fibres are elastin and fibrillin, where correct formation of elastic fibres requires a fibrillin microfibril scaffold for the deposition of elastin. It has been demonstrated previously that the interaction between fibrillin and tropoelastin, the elastin precursor, increases the rate of assembly of tropoelastin. Furthermore, tropoelastin and fibrillin can be cross-linked by transglutaminase-2, but the function of cross-linking on their elastic properties is yet to be elucidated. Here we show that transglutaminase cross-linking supports formation of a 1:1 stoichiometric fibrillin–tropoelastin complex. SAXS data show that the complex retains features of the individual proteins but is elongated supporting end-to-end assembly. Elastic network models were constructed to compare the dynamics of tropoelastin and fibrillin individually as well as in the cross-linked complex. Normal mode analysis was performed to determine the structures' most energetically favourable, biologically accessible motions which show that within the complex, tropoelastin is less mobile and this molecular stabilisation extends along the length of the tropoelastin molecule to regions remote from the cross-linking site. Together, these data suggest a long-range stabilising effect of cross-linking that occurs due to the covalent linkage of fibrillin to tropoelastin. This work provides insight into the interactions of tropoelastin and fibrillin and how cross-link formation stabilises the elastin precursor so it is primed for elastic fibre assembly., Graphical abstract Unlabelled Image, Highlights • Elastic fibres, essential for mammalian tissue elasticity, contain elastin and fibrillin. • Tissue transglutaminase cross-links fibrillin and tropoelastin to form a complex. • The complex has an elongated structure that supports end-to-end assembly. • Cross-linking restricts the molecular motions of tropoelastin and fibrillin. • Stabilisation may provide an initiating molecular mechanism for elastic fibre assembly.

BackgroundAlthough carpal tunnel syndrome (CTS) is the most common form of peripheral entrapment neuropathy, its pathogenesis remains largely unknown. An estimated heritability index of 0.46 and an increased familial occurrence indicate that genetic factors must play a role in the pathogenesis.Methods and resultsWe report on a family in which CTS occurred in subsequent generations at an unusually young age. Additional clinical features included brachydactyly and short Achilles tendons resulting in toe walking in childhood. Using exome sequencing, we identified a heterozygous variant (c.5009T>G; p.Phe1670Cys) in the fibrillin-2 (FBN2) gene that co‐segregated with the phenotype in the family. Functional assays showed that the missense variant impaired integrin-mediated cell adhesion and migration. Moreover, we observed an increased transforming growth factor-β signalling and fibrosis in the carpal tissues of affected individuals. A variant burden test in a large cohort of patients with CTS revealed a significantly increased frequency of rare (6.7% vs 2.5%–3.4%, pFBN2 variants in patient alleles compared with controls.ConclusionThe identification of a novel FBN2 variant (p.Phe1670Cys) in a unique family with early onset CTS, together with the observed increased frequency of rare and high-impact FBN2 variants in patients with sporadic CTS, strongly suggest a role of FBN2 in the pathogenesis of CTS.

TGFβ superfamily members are potent growth factors in the extracellular matrix with essential roles in all aspects of cellular behaviour. Latent TGFβ binding proteins (LTBPs) are co-expressed with TGFβ, essential for correct folding and secretion of the growth factor, to form large latent complexes. These large latent complexes bind extracellular proteins such as fibrillin for sequestration of TGFβ in the matrix, essential for normal tissue function, and dysregulated TGFβ signalling is a hallmark of many fibrillinopathies. Transglutaminase-2 (TG2) cross-linking of LTBPs is known to play a role in TGFβ activation but the underlying molecular mechanisms are not resolved. Here we show that fibrillin is a matrix substrate for TG2 and that TG2 cross-linked complexes can be formed between fibrillin and LTBP-1 and -3, and their latent TGFβ complexes. The structure of the fibrillin-LTBP1 complex shows that the two elongated proteins interact in a perpendicular arrangement which would allow them to form distal interactions between the matrix and the cell surface. Formation of the cross-link with fibrillin does not change the interaction between latent TGFβ and integrin αVβ6 but does increase TGFβ activation in cell-based assays. The activating effect may be due to direction of the latent complexes to the cell surface by fibrillin, as competition with heparan sulphate can ameliorate the activating effect. Together, these data support that TGFβ activation can be enhanced by covalent tethering of LTBPs to the matrix via fibrillin.

As essential components of our connective tissues, elastic fibres give tissues such as major blood vessels, skin and the lungs their elasticity. Their formation is complex and co-ordinately regulated by multiple factors. In this review, we describe key players in elastogenesis: fibrillin-1, tropoelastin, latent TGFβ binding protein-4, and fibulin-4 and -5. We summarise their roles in elastogenesis, discuss the effect of their mutations on relevant diseases, and describe their interactions involved in forming the elastic fibre network. Moreover, we look into their roles in wound repair for a better understanding of their potential application in tissue regeneration.

Asprosin, the C-terminal furin cleavage product of profibrillin-1, was reported to act as a hormone that circulates at nanomolar levels and is recruited to the liver where it induces G protein-coupled activation of the cAMP-PKA pathway and stimulates rapid glucose release into the circulation. Although derived upon C-terminal cleavage of fibrillin-1, a multidomain extracellular matrix glycoprotein with a ubiquitous distribution in connective tissues, little is known about the mechanisms controlling the bioavailability of asprosin in tissues. In the current view, asprosin is mainly produced by white adipose tissue from where it is released into the blood in monomeric form. Here, by employing newly generated specific asprosin antibodies we monitored the distribution pattern of asprosin in human and murine connective tissues such as placenta, and muscle. Thereby we detected the presence of asprosin positive extracellular fibers. Further, by screening established cell lines for asprosin synthesis we found that most cells derived from musculoskeletal tissues render asprosin into an oligomerized form. Our analyses show that asprosin already multimerizes intracellularly, but that stable multimerization via covalent bonds is facilitated by transglutaminase activity. Further, asprosin fiber formation requires an intact fibrillin-1 fiber network for proper linear deposition. Our data suggest a new extracellular storage mechanism of asprosin in an oligomerized form which may regulate its cellular bioavailability in tissues.

Since their discovery as pluripotent cytokines extractable from bone matrix, it has been speculated how bone morphogenetic proteins (BMPs) become released and activated from the extracellular matrix (ECM). In contrast to TGF-βs, most investigated BMPs are secreted as bioactive prodomain (PD)-growth factor (GF) complexes (CPLXs). Recently, we demonstrated that PD-dependent targeting of BMP-7 CPLXs to the extracellular fibrillin microfibril (FMF) components fibrillin-1 and -2 represents a BMP sequestration mechanism by rendering the GF latent. Understanding how BMPs become activated from ECM scaffolds such as FMF is crucial to elucidate pathomechanisms characterized by aberrant BMP activation and ECM destruction. Here, we describe a new MMP-dependent BMP-7 activation mechanism from ECM-targeted pools via specific PD degradation. Using Edman sequencing and mutagenesis, we identified a new and conserved MMP-13 cleavage site within the BMP-7 PD. A degradation screen with different BMP family PDs and representative MMP family members suggested utilization of the identified site in a general MMP-driven BMP activation mechanism. Furthermore, sandwich ELISA and solid phase cleavage studies in combination with bioactivity assays, single particle TEM, and in silico molecular docking experiments provided evidence that PD cleavage by MMP-13 leads to BMP-7 CPLX disintegration and bioactive GF release.

The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet specific transition mechanisms ensuring ECM proteostasis and their regulatory impact are poorly defined. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of mouse Adamts6 and Adamts10 mutant embryos, lacking these homologous secreted metalloproteases individually and in combination, along with in vitro analysis of microfibrils, measurement of ADAMTS6-fibrillin affinities and N-terminomics identification of ADAMTS6-cleaved sites, demonstrates a transcriptionally adapted system for fibrillin-2 proteolysis that contributes to postnatal fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development resulting from a drastic reduction of aggrecan, cartilage link protein and impaired BMP, but not TGFβ signaling in cartilage. Although ADAMTS6 cleaves fibrillin-1 and fibrillin-2 as well as fibronectin, which provides the initial scaffold for microfibril assembly, primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by reversal of these defects in Adamts6-/- embryos by genetic reduction of Fbn2, but not Fbn1.

Elastic fibers are an important component of the extracellular matrix, providing stretch, resilience, and cell interactivity to a broad range of elastic tissues. Elastin makes up the majority of elastic fibers and is formed by the hierarchical assembly of its monomer, tropoelastin. Our understanding of key aspects of the assembly process have been unclear due to the intrinsic properties of elastin and tropoelastin that render them difficult to study. This review focuses on recent developments that have shaped our current knowledge of elastin assembly through understanding the relationship between tropoelastin’s structure and function.

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprisingaggregated oligomeric structures of POR–Pchlide–NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a ‘lid’ to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR ‘octamer’ has been isolated and its structure investigated by cryo-electron microscopy at 7.7 Å resolution.This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORsdemonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation – a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.

Biophysical journal 119(3), 667 - 689 (2020). doi:10.1016/j.bpj.2020.06.018, PSD-95 is a member of the membrane-associated guanylate kinase class of proteins that forms scaffolding interactions with partner proteins, including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/disks large/zona occludens (PDZ) domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of this dual domain in both apo- and ligand-bound form: thermodynamic analysis of the ligand association and small-angle x-ray scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a three-dimensional scaffold that can be described by a space group. The concentration of the components in this study is comparable with those found in compartments of excitable cells such as the postsynaptic density and juxtaparanodes of Ranvier. These in vitro experiments inform the basis of the scaffolding function of PSD-95 and provide a detailed model for scaffold formation by the PDZ domains of PSD-95., Published by Soc., Bethesda, Md.

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous "heavy chains" (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering-based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

PSD-95 is a member of Membrane Associated Guanylate Kinase class of proteins which form scaffolding interactions with partner proteins including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/Disks Large/Zona Occludens domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of the dual domain in both in apo and ligand-bound form; thermodynamic analysis of ligand association and Small Angle X-ray Scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a scaffold in the complete sense of the word. The concentration of the components in this study is comparable to those found in compartments of excitable cells such as the postsynaptic density and juxta-paranodes of Ranvier. The properties of the dual domain explain the basis of the scaffolding function of PSD-95, and provide a more detailed understanding of the integration of key components of neuronal specializations involved in nervous signal transmission.

Lentiviral systems have proven advantageous in the delivery and long-term integration of gene sequences into the genome of several cell types in vitro, in vivo, as well as in clinical trials. Here we detail the protocols involved in the molecular cloning of ADAMTSL2 and ADAMTSL4 into the human immunodeficiency virus (HIV)-derived pCDH lentiviral system. We also describe the lentiviral transduction of ADAMTSL2 and ADAMTSL4 into mammalian HEK293-EBNA cells to create stable cell lines, as well as their recombinant expression.

Recombinantly produced proteins are used in many biological disciplines. However, their purity and quality are vital for downstream applications used to determine their structure and functions. Several purification and detection strategies can be used in combination to obtain protein samples with homogeneity and structural conformity. Here we detail the protocols involved in the purification of ADAMTSL2 from mammalian cells. We also describe the protocols used to validate the purity of the protein samples.

Recombinantly produced proteins are used in many biological disciplines. However, their purity and quality are vital for downstream applications used to determine their structure and functions. Several purification and detection strategies can be used in combination to obtain protein samples with homogeneity and structural conformity. Here we detail the protocols involved in the purification of ADAMTSL2 from mammalian cells. We also describe the protocols used to validate the purity of the protein samples.

Lentiviral systems have proven advantageous in the delivery and long-term integration of gene sequences into the genome of several cell types in vitro, in vivo, as well as in clinical trials. Here we detail the protocols involved in the molecular cloning of ADAMTSL2 and ADAMTSL4 into the human immunodeficiency virus (HIV)-derived pCDH lentiviral system. We also describe the lentiviral transduction of ADAMTSL2 and ADAMTSL4 into mammalian HEK293-EBNA cells to create stable cell lines, as well as their recombinant expression.

Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multiangle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.

Inter-α-inhibitor (IαI) is a proteoglycan essential for mammalian reproduction that also plays a less well-characterised role in inflammation. IαI is composed of 2 homologous ‘heavy chains’ (HC1 and HC2) covalently attached to chondroitin sulphate on the bikunin core protein. Prior to ovulation HCs are transferred onto the polysaccharide hyaluronan (HA), thereby stabilising a matrix that is required for fertilisation. Here we show that human HC1 has a structure similar to integrin β-chains and contains a functional MIDAS (metal ion-dependent adhesion site) motif that can mediate self-association of heavy chains, providing a mechanism for matrix crosslinking. Surprisingly, its interaction with RGD-containing integrin ligands, such as vitronectin and the latency-associated peptides of TGFβ, occurs in a MIDAS/cation-independent manner. However, HC1 utilises its MIDAS motif to bind to, and inhibit the cleavage of, complement C3, thus identifying it as a novel regulator of innate immunity through inhibition of the alternative pathway C3 convertase.AbbreviationsADPs, atomic displacement parameter; AUC, analytical ultracentrifugation; CMG2, capillary morphogenesis protein-2; COC, cumulus-oocyte complex; CS, chondroitin sulphate; FB, complement factor B; FnIII; fibronectin type III; HA, hyaluronan; HC, heavy chain; HC•HA, covalent complex of HC with HA; IαI, inter-α-inhibitor; ITGA, integrin α-chain; ITGB, integrin β-chain; LAP, latency associated peptide; LLC, large latent complex; LTBP, latent TGFβ binding protein; MIDAS, metal ion-dependent adhesion site; PαI, pre-α-inhibitor; PTX3, pentraxin-3; rHC1, recombinant HC1; SAXS, small-angle X-ray scattering; SHAP, serum-derived HA binding protein; SLC, small latent complex; TEM8, tumour endothelial marker-8; TGFβ, transforming factor β; TSG-6, tumour necrosis factor-stimulated gene-6; TSG-6•HC, covalent complex of TSG-6 and HC; vWFA domain, von Willebrand Factor A domain.

Fibrillin microfibrils are extracellular matrix assemblies that form the template for elastic fibres, endow blood vessels, skin and other elastic tissues with extensible properties. They also regulate the bioavailability of potent growth factors of the TGF-β superfamily. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)10 is an essential factor in fibrillin microfibril function. Mutations in fibrillin-1 or ADAMTS10 cause Weill–Marchesani syndrome (WMS) characterized by short stature, eye defects, hypermuscularity and thickened skin. Despite its importance, there is poor understanding of the role of ADAMTS10 and its function in fibrillin microfibril assembly. We have generated an ADAMTS10 WMS mouse model using Clustered Regularly Spaced Interspaced Short Palindromic Repeats and CRISPR associated protein 9 (CRISPR-Cas9) to introduce a truncation mutation seen in WMS patients. Homozygous WMS mice are smaller and have shorter long bones with perturbation to the zones of the developing growth plate and changes in cell proliferation. Furthermore, there are abnormalities in the ciliary apparatus of the eye with decreased ciliary processes and abundant fibrillin-2 microfibrils suggesting perturbation of a developmental expression switch. WMS mice have increased skeletal muscle mass and more myofibres, which is likely a consequence of an altered skeletal myogenesis. These results correlated with expression data showing down regulation of Growth differentiation factor (GDF8) and Bone Morphogenetic Protein (BMP) growth factor genes. In addition, the mitochondria in skeletal muscle are larger with irregular shape coupled with increased phospho-p38 mitogen-activated protein kinase (MAPK) suggesting muscle remodelling. Our data indicate that decreased SMAD1/5/8 and increased p38/MAPK signalling are associated with ADAMTS10-induced WMS. This model will allow further studies of the disease mechanism to facilitate the development of therapeutic interventions.

Summary Procollagen C-proteinase enhancer-1 (PCPE-1) is a secreted protein that specifically accelerates proteolytic release of the C-propeptides from fibrillar procollagens, a crucial step in fibril assembly. As such, it is a potential therapeutic target to improve tissue repair and prevent fibrosis, a major cause of mortality worldwide. Here we present the crystal structure of the active CUB1CUB2 fragment of PCPE-1 bound to the C-propeptide trimer of procollagen III (CPIII). This shows that the two CUB domains bind to two different chains of CPIII and that the N-terminal region of one CPIII chain, close to the proteolytic cleavage site, lies in the cleft between CUB1 and CUB2. This suggests that enhancing activity involves unraveling of this chain from the rest of the trimer, thus facilitating the action of the proteinase involved. Support for this hypothesis comes from site-directed mutagenesis, enzyme assays, binding studies, and molecular modeling., Graphical Abstract, Highlights • The crystal structure of PCPE-1 bound to the C-propeptides has been determined • The N terminus of one propeptide chain binds to the CUB1CUB2 fragment of PCPE-1 • PCPE-1 seems to unravel the propeptide trimer to enable proteolytic release • Molecular modeling with the proteinase and its substrate supports this hypothesis, Collagens are the most abundant proteins in mammals. Fiber-forming collagens are synthesized as procollagens, where the C-propeptides are released by specific proteinases (BMP-1). C-propeptide release is accelerated by another extracellular matrix protein called PCPE-1. Here, based on structural data, the authors present a mechanism for the acceleration of C-propeptide release.

Tropoelastin is the dominant building block of elastic fibers, which form a major component of the extracellular matrix, providing structural support to tissues and imbuing them with elasticity and resilience. Recently, the atomistic structure of human tropoelastin is described, obtained through accelerated sampling via replica exchange molecular dynamics simulations. Here, principal component analysis is used to consider the ensemble of structures accessible to tropoelastin at body temperature (37 °C) at which tropoelastin naturally self-assembles into aggregated coacervates. These coacervates are relevant because they are an essential intermediate assembly stage, where tropoelastin molecules are then cross-linked at lysine residues and integrated into growing elastic fibers. It is found that the ensemble preserves the canonical tropoelastin structure with an extended molecular body flanked by two protruding legs, and identifies variations in specific domain positioning within this global shape. Furthermore, it is found that lysine residues show a large variation in their location on the tropoelastin molecule compared with other residues. It is hypothesized that this perturbation of the lysines increases their accessibility and enhances cross-linking. Finally, the principal component modes are extracted to describe the range of tropoelastin's conformational fluctuation to validate tropoelastin's scissor-twist motion that was predicted earlier.

Protein folding poses unique challenges for large, disordered proteins due to the low resolution of structural data accessible in experiment and on the basis of short time scales and limited sampling attainable in computation. Such molecules are uniquely suited to accelerated-sampling molecular dynamics algorithms due to a flat-energy landscape. We apply these methods to report here the folded structure in water from a fully extended chain of tropoelastin, a 698-amino acid molecular precursor to elastic fibers that confer elasticity and recoil to tissues, finding good agreement with experimental data. We then study a series of artificial and disease-related mutations, yielding molecular mechanisms to explain structural differences and variation in hierarchical assembly observed in experiment. The present model builds a framework for studying assembly and disease and yields critical insight into molecular mechanisms behind these processes. These results suggest that proteins with disordered regions are suitable candidates for characterization by this approach.

Bone morphogenetic proteins (BMPs) are essential signalling molecules involved in developmental and pathological processes and are regulated in the matrix by secreted glycoproteins. One such regulator is BMP-binding endothelial cell precursor-derived regulator (BMPER) which can both inhibit and enhance BMP signalling in a context and concentration-dependent manner. Twisted gastrulation (Tsg) can also promote or ablate BMP activity but it is unclear whether Tsg and BMPER directly interact and thereby exert a synergistic function on BMP signalling. Here, we show that human BMPER binds to Tsg through the N-terminal BMP-binding region which alone more potently inhibits BMP-4 signalling than full-length BMPER. Additionally, BMPER and Tsg cooperatively inhibit BMP-4 signalling suggesting a synergistic function to dampen BMP activity. Furthermore, full-length BMPER is targeted to the plasma membrane via binding of its C-terminal region to cell surface heparan sulphate proteoglycans but the active cleavage fragment is diffusible. Small-angle X-ray scattering and electron microscopy show that BMPER has an elongated conformation allowing the N-terminal BMP-binding and C-terminal cell-interactive regions to be spatially separated. To gain insight into the regulation of BMPER bioavailability by internal cleavage, a disease-causing BMPER point mutation, P370L, previously identified in the acid-catalysed cleavage site, was introduced. The mutated protein was secreted but the mutation prevented intracellular cleavage resulting in a lack of bioactive cleavage fragment. Furthermore, mutant BMPER was extracellularly cleaved at a downstream site presumably becoming available due to the mutation. This susceptibility to extracellular proteases and loss of bioactive N-terminal cleavage fragment may result in loss of BMPER function in disease., Highlights • BMPER and Tsg work in concert to increase the inhibition of BMP4 signalling. • The N-terminal BMPER cleavage product is a more potent inhibitor of BMP4 activity. • BMPER is elongated and flexible with distinct BMP- and HS-binding regions. • A disease causing BMPER point mutation prevents internal auto-catalytic cleavage.

CorA channels are responsible for the uptake of essential magnesium ions by bacteria. X-ray crystal structures have been resolved for two full-length CorA channels, each in a non-conducting state with magnesium ions bound to the protein: These structures reveal a homo-pentameric quaternary structure with approximate 5-fold rotational symmetry about a central pore axis. We report the structure of the detergent solubilized Methanocaldococcus jannaschii CorA channel determined by Cryo-Electron Microscopy and Single Particle Averaging, supported by Small Angle X-ray Scattering and X-ray crystallography. This structure also shows a pentameric channel but with a highly asymmetric domain structure. The asymmetry of the domains includes differential separations between the trans-membrane segments, which reflects mechanical coupling of the cytoplasmic domain to the trans-membrane domain. This structure therefore reveals an important aspect of the gating mechanism of CorA channels by providing an indication of how the absence of magnesium ions leads to major structural changes., Graphical abstract, Highlights • CorA channels regulate magnesium ion concentration in the bacterial cytoplasm. • We report the first structural data on CorA when depleted of magnesium ions. • The absence of magnesium ions leads to conformational changes in the channel. • CorA structure is influenced by ionic bonds between channel domains. • CorA structure is influenced by monovalent ions binding to cytoplasmic domains.

Excessive ultraviolet radiation (UVR) exposure of the skin is associated with adverse clinical outcomes. Although both exogenous sunscreens and endogenous tissue components (including melanins and tryptophan-derived compounds) reduce UVR penetration, the role of endogenous proteins in absorbing environmental UV wavelengths is poorly defined. Having previously demonstrated that proteins which are rich in UVR-absorbing amino acid residues are readily degraded by broadband UVB-radiation (containing UVA, UVB and UVC wavelengths) here we hypothesised that UV chromophore (Cys, Trp and Tyr) content can predict the susceptibility of structural proteins in skin and the eye to damage by physiologically relevant doses (up to 15.4 J/cm2) of solar UVR (95% UVA, 5% UVB). We show that: i) purified suspensions of UV-chromophore-rich fibronectin dimers, fibrillin microfibrils and β- and γ-lens crystallins undergo solar simulated radiation (SSR)-induced aggregation and/or decomposition and ii) exposure to identical doses of SSR has minimal effect on the size or ultrastructure of UV chromophore-poor tropoelastin, collagen I, collagen VI microfibrils and α-crystallin. If UV chromophore content is a factor in determining protein stability in vivo, we would expect that the tissue distribution of Cys, Trp and Tyr-rich proteins would correlate with regional UVR exposure. From bioinformatic analysis of 244 key structural proteins we identified several biochemically distinct, yet UV chromophore-rich, protein families. The majority of these putative UV-absorbing proteins (including the late cornified envelope proteins, keratin associated proteins, elastic fibre-associated components and β- and γ-crystallins) are localised and/or particularly abundant in tissues that are exposed to the highest doses of environmental UVR, specifically the stratum corneum, hair, papillary dermis and lens. We therefore propose that UV chromophore-rich proteins are localised in regions of high UVR exposure as a consequence of an evolutionary pressure to express sacrificial protein sunscreens which reduce UVR penetration and hence mitigate tissue damage., Graphical abstract, Highlights Major structural proteins such as collagen I and tropoelastin are UVA-resistant. In contrast, proteins which are rich in Cys, Trp and Tyr residues are UV-susceptible. These proteins are concentrated in UV exposed tissues. UV-chromophore (Cys, Trp, Tyr)-rich proteins may act as endogenous sunscreens.

A novel, pure, synthetic material is presented that promotes the repair of full-thickness skin wounds. The active component is tropoelastin and leverages its ability to promote new blood vessel formation and its cell recruiting properties to accelerate wound repair. Key to the technology is the use of a novel heat-based, stabilized form of human tropoelastin which allows for tunable resorption. This implantable material contributes a tailored insert that can be shaped to the wound bed, where it hydrates to form a conformable protein hydrogel. Significant benefits in the extent of wound healing, dermal repair, and regeneration of mature epithelium in healthy pigs are demonstrated. The implant is compatible with initial co-treatment with full- and split-thickness skin grafts. The implant's superiority to sterile bandaging, commercial hydrogel and dermal regeneration template products is shown. On this basis, a new concept for a prefabricated tissue repair material for point-of-care treatment of open wounds is provided.

Predicting the concentrated solution behavior for monoclonal antibodies requires developing and using minimal models to describe their shape and interaction potential. Toward this end, the small-angle X-ray scattering (SAXS) profiles for a monoclonal antibody (COE-03) have been measured under solution conditions chosen to produce weak self-association. The experiments are complemented with molecular simulations of a three-bead antibody model with and without interbead attraction. The scattering profile is extracted directly from the molecular simulation to avoid using the decoupling approximation. We examine the ability of the three-bead model to capture features of the scattering profile and the dependence of compressibilty on protein concentration. The three-bead model is able to reproduce generic features of the experimental structure factor as a function of wave vector S(k) including a well-defined shoulder, which is a consequence of the planar structure of the antibody, and a well-defined minimum in S(k) at k ∼ 0.025 Å

The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.

Bone morphogenetic proteins (BMPs) orchestrate key cellular events, such as proliferation and differentiation, in development and homeostasis. Extracellular antagonists, such as chordin, are essential regulators of BMP signaling. Chordin binds to BMPs blocking interaction with receptors, and cleavage by tolloid proteinases is thought to relieve this inhibition. A model has been previously proposed where chordin adopts a horseshoe-like arrangement enabling BMP binding cooperatively by terminal domains (1). Here, we present the nanoscale structure of human chordin using electron microscopy, small angle X-ray scattering, and solution-based biophysical techniques, which together show that chordin indeed has a compact horseshoe-shaped structure. Chordin variants were used to map domain locations within the chordin molecule. The terminal BMP-binding domains protrude as prongs from the main body of the chordin structure, where they are well positioned to interact with the growth factor. The spacing provided by the chordin domains supports the principle of a cooperative BMP-binding arrangement that the original model implied in which growth factors bind to both an N- and C-terminal von Willebrand factor C domain of chordin. Using binding and bioactivity assays, we compared full-length chordin with two truncated chordin variants, such as those produced by partial tolloid cleavage. Cleavage of either terminal domain has little effect on the affinity of chordin for BMP-4 and BMP-7 but C-terminal cleavage increases the efficacy of chordin as a BMP-4 inhibitor. Together these data suggest that partial tolloid cleavage is insufficient to ablate BMP inhibition and the C-terminal chordin domains play an important role in BMP regulation.

The transcription factor ICP4 from herpes simplex virus has a central role in regulating the gene expression cascade which controls viral infection. Here we present the crystal structure of the functionally essential ICP4 DNA binding domain in complex with a segment from its own promoter, revealing a novel homo-dimeric fold. We also studied the complex in solution by small angle X-Ray scattering, nuclear magnetic resonance and surface-plasmon resonance which indicated that, in addition to the globular domain, a flanking intrinsically disordered region also recognizes DNA. Together the data provides a rationale for the bi-partite nature of the ICP4 DNA recognition consensus sequence as the globular and disordered regions bind synergistically to adjacent DNA motifs. Therefore in common with its eukaryotic host, the viral transcription factor ICP4 utilizes disordered regions to enhance the affinity and tune the specificity of DNA interactions in tandem with a globular domain.

Elastin enables the reversible deformation of elastic tissues and can withstand decades of repetitive forces. Tropoelastin is the soluble precursor to elastin, the main elastic protein found in mammals. Little is known of the shape and mechanism of assembly of tropoelastin as its unique composition and propensity to self-associate has hampered structural studies. In this study, we solve the nanostructure of full-length and corresponding overlapping fragments of tropoelastin using small angle X-ray and neutron scattering, allowing us to identify discrete regions of the molecule. Tropoelastin is an asymmetric coil, with a protruding foot that encompasses the C-terminal cell interaction motif. We show that individual tropoelastin molecules are highly extensible yet elastic without hysteresis to perform as highly efficient molecular nanosprings. Our findings shed light on how biology uses this single protein to build durable elastic structures that allow for cell attachment to an appended foot. We present a unique model for head-to-tail assembly which allows for the propagation of the molecule’s asymmetric coil through a stacked spring design.

The structure of α-crustacyanin has been determined to 30 Å resolution using negative-stain electron microscopy (EM) single-particle averaging and modelling with the β-crustacyanin dimer from the crystal structure (Protein Data Bank code 1gka), guided by PISA protein subunit interface calculations for 1gka, and compared with the protein arrangements observed in the crystal lattice of 1gka. This α-crustacyanin EM model has been checked against SAXS experimental data, including comparison with rigid-body models calculated from the SAXS data, and finally with analytical ultracentrifugation measurements., The low-resolution structure of α-crustacyanin has been determined to 30 Å resolution using negative-stain electron microscopy (EM) with single-particle averaging. The protein, which is an assembly of eight β-crustacyanin dimers, appears asymmetrical and rather open in layout. A model was built to the EM map using the X-ray crystallographic structure of β-crustacyanin guided by PISA interface analyses. The model has a theoretical sedimentation coefficient that matches well with the experimentally derived value from sedimentation velocity analytical ultracentrifugation. Additionally, the EM model has similarities to models calculated independently by rigid-body modelling to small-angle X-ray scattering (SAXS) data and extracted in silico from the β-crustacyanin crystal lattice. Theoretical X-ray scattering from each of these models is in reasonable agreement with the experimental SAXS data and together suggest an overall design for the α-crustacyanin assembly.

Collagen VI is an extracellular protein that most often contains the three genetically distinct polypeptide chains, α1(VI), α2(VI), and α3(VI), although three recently identified chains, α4(VI), α5(VI), and α6(VI), may replace α3(VI) in some situations. Each chain has a triple helix flanked by N- and C-terminal globular domains that share homology with the von Willebrand factor type A (VWA) domains. During biosynthesis, the three chains come together to form triple helical monomers, which then assemble into dimers and tetramers. Tetramers are secreted from the cell and align end-to-end to form microfibrils. The precise molecular mechanisms responsible for assembly are unclear. Mutations in the three collagen VI genes can disrupt collagen VI biosynthesis and matrix organization and are the cause of the inherited disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. We have identified a Ullrich congenital muscular dystrophy patient with compound heterozygous mutations in α2(VI). The first mutation causes skipping of exon 24, and the mRNA is degraded by nonsense-mediated decay. The second mutation is a two-amino acid deletion in the C1 VWA domain. Recombinant C1 domains containing the deletion are insoluble and retained intracellularly, indicating that the mutation has detrimental effects on domain folding and structure. Despite this, mutant α2(VI) chains retain the ability to associate into monomers, dimers, and tetramers. However, we show that secreted mutant tetramers containing structurally abnormal C1 VWA domains are unable to associate further into microfibrils, directly demonstrating the critical importance of a correctly folded α2(VI) C1 domain in microfibril formation.