Search

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.

Elastic tissues owe their functional properties to the composition of their extracellular matrices, particularly the range of extracellular, multidomain extensible elastic fibre and microfibrillar proteins. These proteins include elastin, fibrillin, latent TGFβ binding proteins (LTBPs) and collagens, where their biophysical and biochemical properties not only give the matrix structural integrity, but also play a vital role in the mechanisms that underlie tissue homeostasis. Thus far structural information regarding the structure and hierarchical assembly of these molecules has been challenging and the resolution has been limited due to post-translational modification and their multidomain nature leading to flexibility, which together result in conformational and structural heterogeneity. In this review, we describe some of the matrix proteins found in elastic fibres and the new emerging techniques that can shed light on their structure and dynamic properties.

Latent TGFβ binding protein-4 (LTBP4) is a multi-domain glycoprotein, essential for regulating the extracellular bioavailability of TGFβ and assembly of elastic fibre proteins, fibrillin-1 and tropoelastin. LTBP4 mutations are linked to autosomal recessive cutis laxa type 1C (ARCL1C), a rare congenital disease characterised by high mortality and severely disrupted connective tissues. Despite the importance of LTBP4, the structure and molecular consequences of disease mutations are unknown. Therefore, we analysed the structural and functional consequences of three ARCL1C causing point mutations which effect highly conserved cysteine residues. Our structural and biophysical data show that the LTBP4 N- and C-terminal regions are monomeric in solution and adopt extended conformations with the mutations resulting in subtle changes to their conformation. Similar to LTBP1, the N-terminal region is relatively inflexible, whereas the C-terminal region is flexible. Interaction studies show that one C-terminal mutation slightly decreases binding to fibrillin-1. We also found that the LTBP4 C-terminal region directly interacts with tropoelastin which is perturbed by both C-terminal ARCL1C mutations, whereas an N-terminal mutation increased binding to fibulin-4 but did not affect the interaction with heparan sulphate. Our results suggest that LTBP4 mutations contribute to ARCL1C by disrupting the structure and interactions of LTBP4 which are essential for elastogenesis in a range of mammalian connective tissues.

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.

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.

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.

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.

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.

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.

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.

Control of the bioavailability of the growth factor TGFbeta is essential for tissue formation and homeostasis, yet precisely how latent TGFbeta is incorporated into the extracellular matrix is unknown. Here, we show that deposition of a large latent TGFbeta complex (LLC), which contains latent TGFbeta-binding protein 1 (LTBP-1), is directly dependent on the pericellular assembly of fibrillin microfibrils, which interact with fibronectin during higher-order fibrillogenesis. LTBP-1 formed pericellular arrays that colocalized with microfibrils, whereas fibrillin knockdown inhibited fibrillar LTBP-1 and/or LLC deposition. Blocking alpha5beta1 integrin or supplementing cultures with heparin, which both inhibited microfibril assembly, disrupted LTBP-1 deposition and enhanced Smad2 phosphorylation. Full-length LTBP-1 bound only weakly to N-terminal pro-fibrillin-1, but this association was strongly enhanced by heparin. The microfibril-associated glycoprotein MAGP-1 (MFAP-2) inhibited LTBP-1 binding to fibrillin-1 and stimulated Smad2 phosphorylation. By contrast, fibulin-4, which interacted strongly with full-length LTBP-1, did not induce Smad2 phosphorylation. Thus, LTBP-1 and/or LLC deposition is dependent on pericellular microfibril assembly and is governed by complex interactions between LTBP-1, heparan sulfate, fibrillin-1 and microfibril-associated molecules. In this way, microfibrils control TGFbeta bioavailability.

Bone morphogenetic protein-1 (BMP-1)/tolloid proteinases are fundamental to regulating dorsal ventral patterning and extracellular matrix deposition. In mammals there are four proteinases, the splice variants BMP-1 and mammalian tolloid (mTLD), and tolloid like-1 and -2 (TLL-1/2). BMP-1 has the highest catalytic activity and lacks three non-catalytic domains. We demonstrate that TLL-1, which has intermediate activity, forms a calcium-ion dependent dimer with monomers stacked side-by-side. In contrast, truncated TLL-1 molecules having the same shorter structure as BMP-1 are monomers and have improved activity towards their substrate chordin. The increased activity exceeds not only that of full-length TLL-1 but also BMP-1.Structured summaryMINT-7386098: BMP-1 (uniprotkb:P13497) cleaves (MI:0194) Chordin (uniprotkb:Q9H2X0) by protease assay (MI:0435)

Deciphering interacting networks of the extracellular matrix is a major challenge. We describe an affinity purification and mass spectrometry strategy that has provided new insights into the molecular interactions of elastic fibers, essential extracellular assemblies that provide elastic recoil in dynamic tissues. Using cell culture models, we defined primary and secondary elastic fiber interaction networks by identifying molecular interactions with the elastic fiber molecules fibrillin-1, MAGP-1, fibulin-5, and lysyl oxidase. The sensitivity and validity of our method was confirmed by identification of known interactions with the bait proteins. Our study revealed novel extracellular protein interactions with elastic fiber molecules and delineated secondary interacting networks with fibronectin and heparan sulfate-associated molecules. This strategy is a novel approach to define the macromolecular interactions that sustain complex extracellular matrix assemblies and to gain insights into how they are integrated into their surrounding matrix.

The bone morphogenetic protein (BMP)-1/tolloid metalloproteinases are evolutionarily conserved enzymes that are fundamental to dorsal–ventral patterning and tissue morphogenesis. The lack of knowledge regarding how these proteinases recognize and cleave their substrates represents a major hurdle to understanding tissue assembly and embryonic patterning. Although BMP-1 and mammalian tolloid (mTLD) are splice variants, it is puzzling why BMP-1, which lacks 3 of the 7 noncatalytic domains present in all other family members, is the most effective proteinase. Using a combination of single-particle electron microscopy, small-angle X-ray scattering, and other biophysical measurements in solution, we show that mTLD, but not BMP-1, forms a calcium-ion-dependent dimer under physiological conditions. Using a domain deletion approach, we provide evidence that EGF2, which is absent in BMP-1, is critical to the formation of the dimer. Based on a combination of structural and functional data, we propose that mTLD activity is regulated by a substrate exclusion mechanism. These results provide a mechanistic insight into how alternative splicing of the Bmp1 gene produces 2 proteinases with differing biological activities and have broad implications for regulation of BMP-1/mTLD and related proteinases during BMP signaling and tissue assembly.

Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype–phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype–phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity.

Collagens are a large family of triple helical proteins that are widespread throughout the body and are important for a broad range of functions, including tissue scaffolding, cell adhesion, cell migration, cancer, angiogenesis, tissue morphogenesis and tissue repair. Collagen is best known as the

The central region of tropoelastin including domains 19–25 of human tropoelastin forms a hot-spot for contacts during the inter-molecular association of tropoelastin by coacervation [Wise, S.G., Mithieux, S.M., Raftery, M.J. and Weiss, A.S (2005). “Specificity in the coacervation of tropoelastin: solvent exposed lysines.” Journal of Structural Biology 149: 273-81.]. We explored the physical properties of this central region using a sub-fragment bordered by domains 17–27 of human tropoelastin (SHEL 17–27) and identified the intra- and inter-molecular contacts it forms during coacervation. A homobifunctional amine reactive crosslinker (with a maximum reach of 11 A, corresponding to ∼ 7 residues in an extended polypeptide chain) was used to capture these contacts and crosslinked regions were identified after protease cleavage and mass spectrometry (MS) with MS/MS verification. An intermolecular crosslink formed between the lysines at positions 353 of each strand of tropoelastin at the lowest of crosslinker concentrations and was observed in all samples tested, suggesting that this residue forms an important initial contact during coacervation. At higher crosslinker concentrations, residues K425 and K437 showed the highest levels of involvement in crosslinks. An intramolecular crosslink between these K425 and K437, separated by 11 residues, indicated that a structural bend must serve to bring these residues into close proximity. These studies were complemented by small angle X-ray scattering studies that confirmed a bend in this important subfragment of the tropoelastin molecule.

Chordin-mediated regulation of bone morphogenetic protein (BMP) family growth factors is essential in early embryogenesis and adult homoeostasis. Chordin binds to BMPs through cysteine-rich von Willebrand factor type C (vWC) homology domains and blocks them from interacting with their cell surface receptors. These domains also self-associate and enable chordin to target related proteins to fine-tune BMP regulation. The chordin–BMP inhibitory complex is strengthened by the secreted glycoprotein twisted gastrulation (Tsg); however, inhibition is relieved by cleavage of chordin at two specific sites by tolloid family metalloproteases. As Tsg enhances this cleavage process, it serves a dual role as both promoter and inhibitor of BMP signalling. Recent developments in chordin research suggest that rather than simply being by-products, the cleavage fragments of chordin continue to play a role in BMP regulation. In particular, chordin cleavage at the C-terminus potentiates its anti-BMP activity in a type-specific manner.

The assembly of the tropoelastin monomer into elastin is vital for conferring elasticity on blood vessels, skin, and lungs. Tropoelastin has dual needs for flexibility and structure in self-assembly. We explore the structure-dynamics-function interplay, consider the duality of molecular order and disorder, and identify equally significant functional contributions by local and global structures. To study these organizational stratifications, we perturb a key hinge region by expressing an exon that is universally spliced out in human tropoelastins. We find a herniated nanostructure with a displaced C terminus and explain by molecular modeling that flexible helices are replaced with substantial β sheets. We see atypical higher-order cross-linking and inefficient assembly into discontinuous, thick elastic fibers. We explain this dysfunction by correlating local and global structural effects with changes in the molecule’s assembly dynamics. This work has general implications for our understanding of elastomeric proteins, which balance disordered regions with defined structural modules at multiple scales for functional assembly., United States. Office of Naval Research (Presidential Early Career Award for Scientists and Engineers), National Institutes of Health (U.S.) (U01 EB014976)

Fibrillin microfibrils endow mammalian connective tissues with elasticity and play a fundamental role in the deposition of elastin. The microfibrils are 57 nm periodic supramolecular protein polymers with a mass of 2.5 MDa per repeat. The organisation of molecules within a microfibril is still open to debate and structural studies are only just starting to unravel this issue. The contribution of microfibril associated proteins to microfibril ultrastructure and whether there are any tissue specific differences in microfibril structure is still unknown. Therefore, we have used low dose electron microscopy, single particle image analysis and atomic force microscopy to study the structure of fibrillin microfibrils from different tissues. EM images of microfibrils from aorta, ciliary zonules and vitreous humor were collected and more than 500 microfibril repeats from each sample were subjected to averaging. Averages from each sample were analysed using axial stain exclusion patterns and difference images to detect any variations between them. The overall morphology of fibrillin microfibrils was conserved between tissues and there were only very minor differences in the bead and shoulder region of microfibrils. These data suggest that the structure of isolated microfibrils represents the fibrillin scaffold, and either microfibril associated molecules are lost on purification or play only a minor role in microfibril structure.

Fibrillin microfibrils endow mammalian connective tissues with elasticity and are fundamental for the deposition of elastin. The microfibrils are 57nm periodic supramolecular protein polymers with a mass of 2.4MDa per repeat. The detailed structure and organisation of most matrix assemblies is poorly understood due to their large size and complexity and it has proved a major challenge to define their structural organisation. Therefore, we have used low dose electron microscopy and single particle image analysis to study the structure of fibrillin microfibrils. Three novel features were detected: a globular feature that bridges the "arm" region, a double band of density crossing the microfibril and stain penetrating holes present in the interbead region, possibly produced by the removal of microfibril associated proteins in the purification procedure. Fine filaments of approximately 2.4nm diameter are resolved in the interbead region, which correspond to the reported diameter of the fibrillin molecule. Comparison of the stain exclusion pattern of microfibrils with the theoretical stain exclusion pattern of fibrillin packing models indicates that the intramolecular pleating model, where each fibrillin molecule is pleated within one microfibril period allowing extensibility by unpleating, has the best fit to the data.

Multiple epiphyseal dysplasia (MED) is a relatively mild and clinically variable osteochondrodysplasia in which the hip and knee joints are most frequently affected. Both autosomal dominant and autosomal recessive forms of MED are recognised. The more severe forms of MED are often described as the “Fairbank type”, whereas the milder cases are known as the “Ribbing type”. However, this classification belies a much greater clinical spectrum in which characteristics such as radiographic features, age of onset, degree of lower limb deformity, stature, and long term morbidity such as osteoarthritis are extremely variable.1–4 It is therefore not surprising that MED is genetically heterogeneous, and to date mutations in six different genes have been shown to cause MED.5,6 Mutations in the genes encoding cartilage oligomeric matrix protein ( COMP ), the α1, α2, and α3 chains of type IX collagen ( COL9A1, COL9A2 , and COL9A3 ) and matrilin-3 ( MATN3 ) all result in autosomal dominant MED,7–11 whereas specific mutations in the sulphate transporter 26A2 ( SLC26A2 /DTDST ) have been shown to result in an autosomal recessive form of MED.12,13 Preliminary genotype–phenotype correlations have suggested that the more severe forms of autosomal dominant MED often result from COMP mutations, while the milder forms are more probably caused by mutations in the genes encoding type IX collagen or matrilin-3.14,15 However, the number of patients available for comparative study, particularly those with type IX collagen and matrilin-3 defects, has remained too limited to date to allow any in depth correlations to be derived. The matrilins are a four member family of extracellular matrix proteins; matrilin-1 and -3 are specifically expressed in cartilaginous tissues, while matrilin-2 and -4 have a wider pattern of expression in a variety of extracellular matrices including non-skeletal tissues.16,17 Each member of the family comprise …

eLife 4, e05508(2015). doi:10.7554/eLife.05508, Members of the Tolloid family of metalloproteinases liberate BMPs from inhibitory complexes to regulate BMP gradient formation during embryonic dorsal-ventral axis patterning. Here, we determine mechanistically how Tolloid activity is regulated by its non-catalytic CUB domains in the Drosophila embryo. We show that Tolloid, via its N-terminal CUB domains, interacts with Collagen IV, which enhances Tolloid activity towards its substrate Sog, and facilitates Tsg-dependent stimulation of cleavage. In contrast, the two most C-terminal Tld CUB domains mediate Sog interaction to facilitate its processing as, based on our structural data, Tolloid curvature positions bound Sog in proximity to the protease domain. Having ascribed functions to the Tolloid non-catalytic domains, we recapitulate embryonic BMP gradient formation in their absence, by artificially tethering the Tld protease domain to Sog. Our studies highlight how the bipartite function of Tolloid CUB domains, in substrate and ECM interactions, fine-tune protease activity to a particular developmental context., Published by eLife Sciences Publications, Cambridge

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the frontline anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-Å resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of 140 nm are stable, but periodicities of ∼100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

Diazaborine and isoniazid are, at first sight, unrelated anti-bacterial agents that inhibit the enoyl-ACP reductase (ENR) of Escherichia coli and Mycobacterium tuberculosis respectively. The crystal structures of these enzymes including that of the diazaborine-inhibited E. coli ENR have been obtained at high resolution. Site-directed mutagenesis was used to study the importance of amino acid residues in diazaborine susceptibility and enzyme function. The results show that drug binding and inhibition require the presence of a glycine residue at position 93 of E. coli ENR or at the structurally equivalent position in the plant homologue, which is naturally resistant to the drug. The data confirm the hypothesis that any amino acid side-chain other than hydrogen at this position within the three-dimensional structure of these enzymes will affect diazaborine resistance by encroaching into the drug binding site. Substitutions of Gly-93 by amino acids with small side-chains, such as serine, alanine, cysteine and valine, hardly affected the catalytic parameters and rendered the bacterial host resistant to the drug, larger amino acid side-chains, such as that of arginine, histidine, lysine acid glutamine, completely inactivated the activity of the enzyme.

Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel β-sheet flanked on each side by three α-helices with a further helix lying at the C terminus of the β-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate.

The diazaborine family of compounds have antibacterial properties against a range of gram-negative bacteria. Initially, this was thought to be due to the prevention of lipopolysaccharide synthesis. More recently, the molecular target of diazaborines has been identified as the NAD(P)H-dependent enoyl acyl carrier protein reductase (ENR), which catalyses the last reductive step of fatty acid synthase. ENR from Mycobacterium tuberculosis is the target for the front-line antituberculosis drug isoniazid. The emergence of isoniazid resistance strains of M. tuberculosis, a chronic infectious disease that already kills more people than any other infection, is currently causing great concern over the prospects for its future treatment, and it has reawakened interest in the mechanism of diazaborine action. Diazaborines only inhibit ENR in the presence of the nucleotide cofactor, and this has been explained through the analysis of the x-ray crystallographic structures of a number of Escherichia coli ENR-NAD+-diazaborine complexes that showed the formation of a covalent bond between the boron atom in the diazaborines and the 2'-hydroxyl of the nicotinamide ribose moiety that generates a noncovalently bound bisubstrate analogue. The similarities in catalytic chemistry and in the conformation of the nucleotide cofactor across the wider family of NAD(P)-dependent oxidoreductases suggest that there are generic opportunities to mimic the interactions seen here in the rational design of bisubstrate analogue inhibitors for other NAD(P)H-dependent oxidoreductases.

The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.

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.

PURPOSE: AMD has a complex etiology with environmental and genetic risk factors. Ten fibulin 5 sequence variants have been associated with AMD and two other fibulin 5 mutations cause autosomal-recessive cutis laxa. Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Biophysical techniques were used to detect structural changes in the fibulin 5 mutants and to determine whether changes are predictive of pathogenicity. METHODS: Native PAGE, nonreduced SDS-PAGE, size-exclusion column multiangle laser light scattering, sedimentation velocity, and circular dichroism (CD) were used to investigate the mobility, hydrodynamic radii, folding, and oligomeric states of the fibulin 5 mutants in the absence and presence of Ca(2+). RESULTS: CD showed that all mutants are folded, although perturbations to secondary structure contents were detected. Both cutis laxa mutants increased dimerization. Most other mutants slightly increased self-association in the absence of Ca(2+) but this was also demonstrated by G202R, a polymorphism detected in a control individual. The AMD-associated mutant G412E showed lower-than-expected mobility during native-PAGE, the largest hydrodynamic radius for the monomer form and the highest levels of aggregation in both the absence and presence of Ca(2+). CONCLUSIONS: The results identified structural differences for the disease-causing cutis laxa mutants and for one AMD variant (G412E), suggesting that this may also be pathogenic. Although the other AMD-associated mutants showed no gross structural differences, they cannot be excluded as pathogenic by differences outside the scope of this study-for example, disruption of heterointeractions.

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca2+ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin.

The interactions underlying the cooperativity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes during neurotransmission are not known. Here, we provide a molecular characterization of a dimer formed between the cytoplasmic portions of neuronal SNARE complexes. Dimerization generates a two-winged structure in which the C termini of cytosolic SNARE complexes are in apposition, and it involves residues from the vesicle-associated SNARE synaptobrevin 2 that lie close to the cytosol-membrane interface within the full-length protein. Mutation of these residues reduces stability of dimers formed between SNARE complexes, without affecting the stability of each individual SNARE complex. These mutations also cause a corresponding decrease in the ability of botulinum toxin-resistant synaptobrevin 2 to rescue regulated exocytosis in toxin-treated neuroendocrine cells. Moreover, such synaptobrevin 2 mutants give rise to a dominant-negative inhibition of exocytosis. These data are consistent with an important role for SNARE complex dimers in neurosecretion. © 2008 by The American Society for Cell Biology.

Mutations in fibrillin-1 result in Marfan syndrome, which affects the cardiovascular, skeletal and ocular systems. The multiorgan involvement and wide spectrum of associated phenotypes highlights the complex pathogenesis underlying Marfan syndrome. To elucidate the genotype to phenotype correlations, we engineered four Marfan syndrome causing mutations into a fibrillin-1 fragment encoded by exons 18-25, a region known to interact with tropoelastin. Biophysical and biochemical approaches, including small angle x-ray scattering, analytical ultracentrifugation, and circular dichroism, were used to study the impact of these mutations upon the structure and function of the protein. Mutations G880S, C862R, and C908R, situated within the second hybrid domain, disrupted the ratio of alpha-helix to beta-sheet leading to a more compact conformation. These data clearly demonstrate the importance of the previously uncharacterized hybrid domain in fibrillin-1 structure. In contrast, mutation K1023N situated within the linker region between the third eight cysteine motif and cbEGF 11 markedly extended the length of the fragment. However, none of the mutations affected tropoelastin binding. The profound effects of all four mutations on fragment conformation suggest that they contribute to the pathogenesis of Marfan syndrome by disrupting protein folding and its assembly into fibrillin-rich microfibrils.

We have defined the homotypic interactions of fibrillin-1 to obtain new insights into microfibril assembly. Dose-dependent saturable high affinity binding was demonstrated between N-terminal fragments, between furin processed C-terminal fragments, and between these N- and C-terminal fragments. The N terminus also interacted with a downstream fragment. A post-furin cleavage site C-terminal sequence also interacted with the N terminus, with itself and with the furin-processed fragment. No other homotypic fibrillin-1 interactions were detected. Some terminal homotypic interactions were inhibited by other terminal sequences, and were strongly calcium-dependent. Treatment of an N-terminal fragment with N-ethylmaleimide reduced homotypic binding. Microfibril-associated glycoprotein-1 inhibited N- to C-terminal interactions but not homotypic N-terminal interactions. These fibrillin-1 interactions are likely to regulate pericellular fibrillin-1 microfibril assembly.

Fibrillins form the structural framework of a unique and essential class of extracellular microfibrils that endow dynamic connective tissues with long–range elasticity. Their biological importance is emphasized by the linkage of fibrillin mutations to Marfan syndrome and related connective tissue disorders, which are associated with severe cardiovascular, ocular and skeletal defects. These microfibrils have a complex ultrastructure and it has proved a major challenge both to define their structural organization and to relate it to their biological function. However, new approaches have at last begun to reveal important insights into their molecular assembly, structural organization and biomechanical properties. This paper describes the current understanding of the molecular assembly of fibrillin molecules, the alignment of fibrillin molecules within microfibrils and the unique elastomeric properties of microfibrils.

Fibrillin-rich microfibrils have endowed tissues with elasticity throughout multicellular evolution. We have used molecular combing techniques to determine Young's modulus for individual microfibrils and X-ray diffraction of zonular filaments of the eye to establish the linearity of microfibril periodic extension. Microfibril periodicity is not altered at physiological zonular tissue extensions and Young's modulus is between 78 MPa and 96 MPa, which is two orders of magnitude stiffer than elastin. We conclude that elasticity in microfibril-containing tissues arises primarily from reversible alterations in supra-microfibrillar arrangements rather than from intrinsic elastic properties of individual microfibrils which, instead, act as reinforcing fibres in fibrous composite tissues.

Acyl Carrier Protein [ACP] is a small acidic protein which is an essential component of the type 2, dissociable fatty acid synthetase [1]. ACP is synthesised as an apo-protein and posttranslationally modified, by the addition of a 4’ phosphopantetheine [4’PANT] prosthetic group, which is linked to a serine residue by a phosphodiester bond. The amino acid sequence around the modified serine residue is highly conserved [2]. The post—translational modification is catalysed by holo-ACP synthetase [HAS], and this enzyme can also catalyse the direct conversion of apo-ACP to acyl-ACP using acyl-CoA as a source of the both the 4’ PANT and the acyl group [3]. Apart from the role of ACP in fatty acid biosynthesis it is an important component of a number of other reactions which require acyl-transferase steps, these include: membrane derived oligosaccharides, polyketide antibiotics, biotin precursor, and acyl-transfer to glycerol-3-phosphate [E.coli and plants] and lysophosphatidic acid. Additionally acyl-ACPs are the substrates for both thioesterases and soluble desaturases. NMR structures exist for holo-ACP and acyl-ACPs [4] and recently a X-ray crystallographic structure has been reported for a HAS-ACP complex from Bacillus subtilis [5]. In this study we are interested in determining the x-ray structure for an acyl-ACP and have used a combination of recombinant and non-recombinant proteins. Our eventual aim is to obtain structural data on a variety of different acyl-ACPs as well as acyl-ACP’ s complexed to enzymes of lipid metabolism.

Acyl carrier protein (ACP) is an essential cofactor in biosynthesis of fatty acids and many other reactions that require acyl transfer steps. We have determined the first crystal structures of an acylated form of ACP from E. coli, that of butyryl-ACP. Our analysis of the molecular surface of ACP reveals a plastic hydrophobic cavity in the vicinity of the phosphopantethylated Ser36 residue that is expanded and occupied by the butyryl and β-mercaptoethylamine moieties of the acylated 4′-phosphopantetheine group in one of our crystal forms. In the other form, the cavity is contracted, and we propose that the protein has adopted the conformation after delivery of substrate into the active site of a partner enzyme.

Acyl carrier proteins carry the lipid substrate to the enzymes of the fatty acid synthase system. Crystals of Escherichia coli acyl carrier protein to which a butyryl group has been attached via a thioester link to the phosphopantetheine prosthetic arm have been obtained by the hanging-drop vapour-diffusion method. These crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 27.6, b = 41.6, c = 63.7 A. The asymmetric unit appears to contain one subunit, corresponding to a packing density of 2.1 A(3) Da(-1). Crystals of the selenomethionine-substituted (SeMet) protein were obtained using different conditions and belong to space group P6(3), with unit-cell parameters a = b = 63.4, c = 37.0 A, alpha = beta = 90, gamma = 120 degrees and with a monomer in the asymmetric unit (V(M) = 2.5 A(3) Da(-1)). Crystals of a SeMet butyryl-ACP I62M variant were obtained using the conditions for the native protein. Like the native crystals, these belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 27.3, b = 41.9, c = 64.5 A. A data set suitable for MAD phasing was collected from the crystals of the I62M variant to 1.8 A resolution on the ESRF beamline ID14-4.

Fibrillins are large, cysteine-rich glycoproteins that form microfibrils and play a central role in elastic fibrillogenesis. Fibrillin-1 and fibrillin-2, encoded by FBN1 on chromosome 15q21.1 and FBN2 on chromosome 5q23-q31, are highly similar proteins. The finding of mutations in FBN1 and FBN2 in the autosomal dominant microfibrillopathies Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), respectively, has highlighted their essential role in the development and homeostasis of elastic fibres. MFS is characterized by cardiovascular, skeletal and ocular abnormalities, and CCA by long, thin, flexed digits, crumpled ears and mild joint contractures. Although mutations arise throughout FBN1, those clustering within exons 24-32 are associated with the most severe form of MFS, so-called neonatal MFS. All the mutations described in CCA occur in the "neonatal region" of FBN2. Both MFS and CCA are thought to arise via a dominant negative mechanism. The analysis of mouse mutations has demonstrated that fibrillin-1 microfibrils are mainly engaged in tissue homeostasis rather than elastic matrix assembly. In the current investigation, we have analysed the classical mouse mutant shaker-with-syndactylism using a positional candidate approach and demonstrated that loss-of-function mutations outside the "neonatal region" of Fbn2 cause syndactyly in mice. These results suggest that phenotypes distinct from CCA may result in man as a consequence of mutations outside the "neonatal region" of FBN2.

Background: Enoyl acyl carrier protein reductase (ENR) catalyzes the NAD(P)H-dependent reduction of trans - Δ 2-enoyl acyl carrier protein, an essential step in de novo fatty acid biosynthesis. Plants contain both NADH-dependent and separate NADPH-dependent ENR enzymes which form part of the dissociable type II fatty acid synthetase. Highly elevated levels of the NADH-dependent enzyme are found during lipid deposition in maturing seeds of oilseed rape ( Brassica napus ). Results The crystal structure of an ENR–NAD binary complex has been determined at 1.9 a resolution and consists of a homotetramer in which each subunit forms a single domain comprising a seven-stranded parallel β sheet flanked by seven α helices. The subunit has a topology highly reminiscent of a dinucleotide-binding fold. The active site has been located by difference Fourier analysis of data from crystals equilibrated in NADH. Conclusion The structure of ENR shows a striking similarity with the epimerases and short-chain alcohol dehydrogenases, in particular, 3 α ,20 β -hydroxysteroid dehydrogenase (HSD). The similarity with HSD extends to the conservation of a catalytically important lysine that stabilizes the transition state and to the use of a tyrosine as a base — with subtle modifications arising from differing requirements of the reduction chemistry.

The extracellular glycoprotein fibrillin-1 forms microfibrils that act as the template for elastic fibers. Most mutations in fibrillin-1 cause Marfan syndrome with severe cardiovascular and ocular symptoms, and tall stature. This is in contrast to mutations within a heparin-binding TB domain (TB5), which is downstream of the arg-gly-asp cell adhesion domain, which can cause Weill-Marchesani syndrome (WMS) or Acromicric (AD) and Geleophysic Dysplasias (GD). WMS is characterized by short limbs, joint stiffness and ocular defects, whilst fibrillin-1 AD and GD have severe short stature, joint defects and thickened skin. We previously showed that TB5 binds heparin. Here, we show that the corresponding region of fibrillin-2 binds heparin very poorly, highlighting a novel functional difference between the two isoforms. This finding enabled us to map heparin/heparan sulfate binding to two sites on fibrillin-1 TB5 using a mutagenesis approach. Once these sites were mapped, we were able to investigate whether disease-causing mutations in this domain disrupt binding to HS. We show that a WMS deletion mutant, and five AD and GD point mutants all have disrupted heparin binding to TB5. These data provide insights into the biology of fibrillins and the pathologies of WMS, AD and GD.