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Chirality is ubiquitous in biology, including in biomineralization, where it is found in many hardened structures of invertebrate marine and terrestrial organisms (for example, spiralling gastropod shells). Here we show that chiral, hierarchically organized architectures for calcium carbonate (vaterite) can be controlled simply by adding chiral acidic amino acids (Asp and Glu). Chiral, vaterite toroidal suprastructure having a ‘right-handed’ (counterclockwise) spiralling morphology is induced by L -enantiomers of Asp and Glu, whereas ‘left-handed’ (clockwise) morphology is induced by D -enantiomers, and sequentially switching between amino-acid enantiomers causes a switch in chirality. Nanoparticle tilting after binding of chiral amino acids is proposed as a chiral growth mechanism, where a ‘mother’ subunit nanoparticle spawns a slightly tilted, consequential ‘daughter’ nanoparticle, which by amplification over various length scales creates oriented mineral platelets and chiral vaterite suprastructures. These findings suggest a molecular mechanism for how biomineralization-related enantiomers might exert hierarchical control to form extended chiral suprastructures. Chiral structures are formed in numerous processes including biomineralization of calcium carbonate. Here, the authors demonstrate that the chiral, hierarchically-organized architecture of the calcium carbonate mineral, vaterite, can be controlled simply by the addition of chiral acidic amino acids.

Since Pasteur first successfully separated right-handed and left-handed tartrate crystals in 1848, the understanding of how homochirality is achieved from enantiomeric mixtures has long been incomplete. Here, we report on a chirality dominance effect where organized, three-dimensional homochiral suprastructures of the biomineral calcium carbonate (vaterite) can be induced from a mixed nonracemic amino acid system. Right-handed (counterclockwise) homochiral vaterite helicoids are induced when the amino acid l -Asp is in the majority, whereas left-handed (clockwise) homochiral morphology is induced when d -Asp is in the majority. Unexpectedly, the Asp that incorporates into the homochiral vaterite helicoids maintains the same enantiomer ratio as that of the initial growth solution, thus showing chirality transfer without chirality amplification. Changes in the degree of chirality of the vaterite helicoids are postulated to result from the extent of majority enantiomer assembly on the mineral surface. These mechanistic insights potentially have major implications for high-level advanced materials synthesis. Induction of complex homochiral architectures by chiral transformation in a mixed enantiomer system has remained largely elusive. Here, the authors report a chirality dominance effect which induces homochiral suprastructures of calcium carbonate by a mixed, heterochiral nonracemic amino acid enantiomer system.

The plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP)-rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore pathological mineralization (calcification). The mutant mouse strain D2,Ahsg-/- combines fetuin-A deficiency with the calcification-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue calcification. Here we studied mechanisms leading to soft tissue calcification, organ damage and death in these mice. We analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice. Fetuin-A-deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature death. Importantly, early-stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the calcification was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis. Collectively, these results demonstrate that soft tissue calcification can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of calcified matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.

Elastogenesis is a hierarchical process by which cells form functional elastic fibers, providing elasticity and the ability to regulate growth factor bioavailability in tissues, including blood vessels, lung, and skin. This process requires accessory proteins, including fibulin-4 and -5, and latent TGF binding protein (LTBP)-4. Our data demonstrate mechanisms in elastogenesis, focusing on the interaction and functional interdependence between fibulin-4 and LTBP-4L and its impact on matrix deposition and function. We show that LTBP-4L is not secreted in the expected extended structure based on its domain composition, but instead adopts a compact conformation. Interaction with fibulin-4 surprisingly induced a conformational switch from the compact to an elongated LTBP-4L structure. This conversion was only induced by fibulin-4 multimers associated with increased avidity for LTBP-4L; fibulin-4 monomers were inactive. The fibulin-4–induced conformational change caused functional consequences in LTBP-4L in terms of binding to other elastogenic proteins, including fibronectin and fibrillin-1, and of LTBP-4L assembly. A transient exposure of LTBP-4L with fibulin-4 was sufficient to stably induce conformational and functional changes; a stable complex was not required. These data define fibulin-4 as a molecular extracellular chaperone for LTBP-4L. The altered LTBP-4L conformation also promoted elastogenesis, but only in the presence of fibulin-4, which is required to escort tropoelastin onto the extended LTBP-4L molecule. Altogether, this study provides a dual mechanism for fibulin-4 in 1) inducing a stable conformational and functional change in LTBP-4L, and 2) promoting deposition of tropoelastin onto the elongated LTBP-4L.

Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells. Microfibrillar-associated protein 4 (MFAP4) is involved in fibrotic and cardiovascular diseases. Wozny et al. reveal structural aspects mediating MFAP4 octamer formation critical for its interaction with elastogenic proteins and cells.

Microfibrils are essential elements in elastic and nonelastic tissues contributing to homeostasis and growth factor regulation. Fibrillins form the core of these multicomponent assemblies. Various human genetic disorders, the fibrillinopathies, arise from mutations in fibrillins and are frequently associated with aberrant microfibril assembly. These disorders include Marfan syndrome, Weill-Marchesani syndrome, Beals syndrome, and others. Although homotypic and heterotypic fibrillin self-interactions are considered to provide critical initial steps, the detailed mechanisms for microfibril assembly are unknown. We show here that the C-terminal recombinant half of fibrillin-1 assembles into disulfide-bonded multimeric globular structures with peripheral arms and a dense core. These globules are similar to the beaded structures observed in microfibrils isolated from tissues. Only these C-terminal fibrillin-1 multimers interacted strongly with the fibrillin-1 N terminus, whereas the monomers showed very little self-interaction activity. The multimers strongly inhibited microfibril formation in cell culture, providing evidence that these recombinant assemblies can also interact with endogenous fibrillin-1. The C-terminal self-interaction site was fine-mapped to the last three calcium-binding EGF domains in fibrillin-1. These results suggest a new mechanism for microfibril formation where fibrillin-1 first oligomerizes via its C terminus before the partially or fully assembled bead-like structures can further interact with other beads via the fibrillin-1 N termini.

Fibronectin (FN) is a ubiquitously expressed cell adhesion protein capable of assembling into large, extended fibrillar networks as part of an extracellular matrix (ECM) that regulates cell behavior. FN is a substrate for certain members of the transglutaminase family of protein-crosslinking enzymes—enzymes which can modify the ability of FN to support cell adhesion. In this study, we have analyzed the thermo-chemical stability of plasma FN in its noncrosslinked form, and after crosslinking by transglutaminase 2 (TG2), using dynamic light scattering. We report that FN is found in a generally globular (8.7 nm hydrodynamic radius), dimerized form in aqueous solutions, but unfolds into a linear arrangement at high ionic (1 M NaCl) and chaotropic (5 M urea) environments. FN conformation remained stable after multiple heating and cooling cycles ranging from 4 to 60 °C. Crosslinking of FN with TG2 formed large, multimeric complexes having high chemical stability in aqueous, high ionic and chaotropic environments, demonstrating that this covalent modification stabilizes FN. Given recent data that substrate (e.g. ECM) rigidity profoundly affects cell differentiation and behavior, we further studied how TG2 crosslinking affects the molecular rigidity of FN by obtaining atomic force microscopy nanoindentation measurements from untreated and crosslinked FN samples embedded in acrylamide gels. We demonstrate that TG2-mediated crosslinking of FN significantly increases Young’s modulus (of elasticity), an observation of increased rigidity having important implications with respect to the biological role of ECM protein-crosslinking in cell signaling and guiding cell differentiation.

The interface between nano-crystalline hydroxyapatite (HA) thin films and a titanium alloy (Ti5Al2.5Fe) has been studied by means of Fourier transform infrared spectrophotometry and X-ray diffraction at grazing incidence. The HA thin films were deposited by radio-frequency magnetron sputtering in low pressure dry argon on substrates kept at low temperature or heated at 550 degrees C. The effect of film treatment by sputtering and annealing in humid air, as a simple, effective way of restoring the crystallinity and stoichiometry of the HA bulk, was studied in correlation with the development of a titanium oxide layer at the film-substrate interface. An interfacial TiO(2 )film grew at the interface during annealing in moist air, while a TiO(2) layer diffused into the HA films when directly sputtered at 550 degrees C. The formation of an interfacial titanium oxide layer was inhibited by the insertion of a crystalline TiN buffer interlayer between the substrate and the HA film. Separately, the mechanical characteristics of the different HA films were monitored by nanoindentation to find out how they had been affected.

Microfibrils are essential elements in elastic and nonelastic tissues contributing to homeostasis and growth factor regulation. Fibrillins form the core of these multicomponent assemblies. Various human genetic disorders, the fibrillinopathies, arise from mutations in fibrillins and are frequently associated with aberrant microfibril assembly. These disorders include Marfan syndrome, Weill–Marchesani syndrome, Beals syndrome, and others. Although homotypic and heterotypic fibrillin self-interactions are considered to provide critical initial steps, the detailed mechanisms for microfibril assembly are unknown. We show here that the C-terminal recombinant half of fibrillin-1 assembles into disulfide-bonded multimeric globular structures with peripheral arms and a dense core. These globules are similar to the beaded structures observed in microfibrils isolated from tissues. Only these C-terminal fibrillin-1 multimers interacted strongly with the fibrillin-1 N terminus, whereas the monomers showed very little self-interaction activity. The multimers strongly inhibited microfibril formation in cell culture, providing evidence that these recombinant assemblies can also interact with endogenous fibrillin-1. The C-terminal self-interaction site was fine-mapped to the last three calcium-binding EGF domains in fibrillin-1. These results suggest a new mechanism for microfibril formation where fibrillin-1 first oligomerizes via its C terminus before the partially or fully assembled bead-like structures can further interact with other beads via the fibrillin-1 N termini.

Microfibrillar-associated protein 4 (MFAP4) is a 36-kDa extracellular glycoprotein with critical roles in human pathologies, including fibrosis in several organ systems, chronic obstructive pulmonary disease, and cardiovascular disorders. In elastic tissues such as arteries, lungs, and skin, MFAP4 associates with microfibrils and elastic fibres, which are the central extracellular fibres affected in thoracic aneurysms. MFAP4 directly interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. MFAP4 multimerisation represents a critical hallmark required for its physiological and pathological properties. However, molecular details and functional consequences of MFAP4 multimerisation are lacking. Here we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer with D2 point group symmetry, where two sets of homodimers constitute the top and bottom halves of each octamer. Each of the homodimers is linked together by an inter-molecular disulfide bond. An engineered C34S missense mutation in MFAP4 prevented disulfide-bond formation between monomers, but the mutant formed octamers similar to wild type MFAP4. The atomic model, built into the 3.55 Å cryo-EM map, suggests that several salt-bridges are important for interactions within and between homodimers, while non-polar interactions are important for octamer halves to assemble. In the absence of calcium, MFAP4 dissociates into tetramers, representing the top/bottom halves of the octamers. Binding studies with elastogenic proteins, including fibrillin-1, tropoelastin, LTBP4, and small fibulins showed that MFAP4 has multiple surfaces for protein-protein interactions, which depend upon the higher-order assembly of MFAP4. While the disulfide-bond mediated by C34S contributes little to those protein interactions, it modulated cell interaction. When MFAP4 forms assemblies with fibrillin-1, it abrogates MFAP4 interactions with cells. Overall, the study provides detailed molecular structure-function relationships of MFAP4 interactions with elastogenic proteins and cells.