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.AFM | SAXS | atomic force microscopy A ll mammals rely on elastin to convey extensional elasticity to their tissues. Elastin dominates the mass of the aorta where it encounters the peaks and troughs of systole and diastole over the course of two billion heartbeats in a lifetime (1). The lung expands with each intake of breath and elastically contracts on exhalation. The function of these tissues benefits from minimal energy loss during elastic return in each cycle of expansion and contraction. Additionally, elastin is required to function in an environment that relies on cellular contact (2-4) without compromising persistent elasticity. This high level of physical performance demanded of elastin vastly exceeds and indeed outlasts all human-made elastomers (5).Elastin is constructed by the hierarchical assembly and crosslinking of many tropoelastin monomers that accumulate on a microfibrillar skeleton. Tropoelastin is encoded by a single gene in humans and predominantly laid down in utero and early childhood, providing a durable resource that is intended to elastically serve until old age. This exquisite assembly helps to generate elastic tissues as diverse as artery, lung, and skin (4). Consequences of elastolytic damage in aortic aneurysms, emphysema, and solar elastosis confirm the key roles of elastin in structure and cellular interactions (6-8). These tissues rely on this paradoxical combination of organized tissue structures built from an intrinsically unstructured protein. Tropoelastin serves as a component of rigidly organized assemblies, yet enables the formation of dynamically distensible, elastic tissues.Tropoelastin is frequently described in the literature as an unstructured protein, mainly because models of elasticity invoke an element of disorder within the structure (4, 9, 10). While this concept appears to be the case at the fine, more subtle intramolecular leve...

Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection.

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The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the ␣5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease. IntroductionThe Lutheran glycoprotein (Lu gp) cellular adhesion molecule is widely expressed in human tissues 1 and is known for carrying antigens of the Lutheran blood group system. On erythrocytes, Lu gp is expressed as 2 isoforms of 78 and 85 kDa. 2 Both share a common extracellular portion that has previously been predicted to comprise 5 immunoglobulin superfamily (IgSF) domains. 1 The 78-kDa isoform (also known as BCAM 3 or Lu[v13] 4 ) results from alternative splicing and lacks 40 C-terminal amino acids within the cytoplasmic domain that contain an SH3-binding motif, a dileucine motif responsible for basolateral targeting, 5 and 5 potential phosphorylation sites. 1 Lu gp binds specifically and with high affinity to the extracellular matrix (ECM) protein Laminin (Ln) containing the ␣5 subunit 6-9 (Laminin 511 and Laminin 521 (Ln511/521) (numbering as in Aumailley et al 10 ). This interaction plays a direct role in the pathophysiology of sickle cell disease by mediating adhesion of sickle cells, via Lu gp, to exposed Ln511/521 of inflamed or damaged vascular endothelium. 9,11,12 Recent studies have shown that higher than normal intracellular levels of cAMP in sickle erythrocytes influence a protein kinase A-mediated or Rap1-mediated signaling pathway resulting in increased adhesion of sickle cells to the basement membrane glycoproteins Ln511/ 521. 13,14 Furthermore, phosphorylation of the 85-kDa Lu gp at S621 in epinephrine-stimulated K562 cells alters adhesion to Ln511/521. 15 The Ln-binding activity of Lu gp is a property of the amino terminal region of Lu gp, and has been localized to a region predicted to form the first 3 IgSF domains (D1D2D3). 6,16 The complementary binding site on Ln is located within a large carboxyl-terminal globular domain of the ␣5 subunit, known to comprise 5 ...

An X-ray scanning imaging technique using the integrated intensity of the smallangle X-ray scattering (SAXS) signal is presented. The technique is based on two-dimensional scanning of a thin sample section with an X-ray microbeam, collecting SAXS patterns at every scanning step using a two-dimensional detector. The integrated intensity within pre-defined regions of interest of the SAXS patterns is used to image bulk nanostructural features in the specimen with micrometre resolution which are usually not accessible by other methods such as light microscopy or scanning electron microscopy. The possibilities and limitations of the method are discussed with particular emphasis on the sources of contrast in the SAXS region for three biological specimens: cortical bone, eggshell and hair. Two main sources of image contrast are identified in the form of orientation effects for strongly anisotropic systems like cortical bone and differences in the local volume fraction of the scattering entities in eggshell. Moreover, other parameters than the integrated intensity can be quantitatively deduced from the SAXS patterns, for instance, the mean thickness of mineral platelets in bone or the strain distributions in a hair deformed plastically by microindentation.