SummaryIt is generally believed that cholesterol homoeostasis in the brain is both linked to and impacted by Alzheimer's disease (AD). For example, elevated levels of cholesterol in neuronal plasma and endosome membranes appears to be a pro-amyloidogenic factor. The recent observation that the Cterminal transmembrane domain (C99, also known as the β-CTF) of the amyloid precursor protein (APP) specifically binds cholesterol helps to tie together previously loose ends in the web of our understanding of Alzheimer's-cholesterol relationships. In particular, binding of cholesterol to C99 appears to favor the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts. In turn, the products of this pathway-amyloid-β and the intracellular domain of the APP (AICD) -may down-regulate ApoE-mediated cholesterol uptake and cholesterol biosynthesis. If confirmed, this negative-feedback loop for membrane cholesterol levels has implications for understanding the function of the APP and for devising anti-amyloidogenic preventive strategies in AD.

CD44 is the main cell surface receptor for hyaluronic acid (HA) and contains a functional HA-binding domain (HABD) composed of a Link module with N-and C-terminal extensions. The contact residues of human CD44 HABD for HA have been determined by cross-saturation experiments and mapped on the topology of CD44 HABD, which we elucidated by NMR. The contact residues are distributed in both the consensus fold for the Link module superfamily and the additional structural elements consisting of the flanking regions. Interestingly, the contact residues exhibit small changes in chemical shift upon HA binding. In contrast, the residues with large chemical shift changes are localized in the C-terminal extension and the first ␣-helix and are generally inconsistent with the contact residues. These results suggest that, upon ligand binding, the C-terminal extension and the first ␣-helix undergo significant conformational changes, which may account for the broad ligand specificity of CD44 HABD.CD44 is a type I transmembrane glycoprotein with diverse functions and is expressed on the surface of many cell types (1, 2). CD44 reportedly recognizes a variety of ligands (1, 2). The most investigated aspect of CD44 function is its ability to bind hyaluronic acid (HA), 1 a major component of the extracellular matrix (1-3). HA is a very high molecular mass glycosaminoglycan, composed of a repeating disaccharide, D-glucuronic acid (␤133) N-acetyl-D-glucosamine (␤134) (4). The binding of CD44 to HA has been implicated in both cell adhesion to the extracellular matrix components and cellular signaling cascades (1, 2, 5). While HA exists as a high molecular mass polymer, HA fragments of various molecular sizes can be generated in vivo by a variety of mechanisms, and they exhibit different biological activities (6, 7). In addition to HA, CD44 interacts with the chondroitin sulfate (ChS) proteoglycans, serglycin (8 -10), versican (11), and aggrecan (12). The CD44-binding elements on these proteoglycans are ChS side chains. These ChS proteoglycans may be involved in the adherence and activation of CD44-expressing cells.CD44 has an N-terminal functional domain that interacts with the HA and ChS chains (11)(12)(13). This ligand recognition domain contains a homology region, termed the Link module (14,15). Link modules are found in extracellular matrix molecules (link protein, aggrecan, versican, neurocan, and brevican) and the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6). The HA-binding domains from Link modulecontaining proteins are divided into three subgroups as described in Ref. 16. Briefly, a single Link module, a Link module with N-and C-terminal extensions, and a pair of Link modules, which are sufficient for a high affinity interaction with HA, are classified as Types A, B, and C, respectively. In addition, a single Link module from TSG-6, belonging to Type A, interacts specifically with chondroitin 4-sulfate (Ch4S) but not with chondroitin 6-sulfate (Ch6S) (17). Bovine link protein, which contains the Type C Link ...

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the ␤-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

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The nature of the supramolecular complex between fibrillar collagen and collagen-binding proteins (CBPs) has hindered detailed X-ray and NMR analyses of the ligand-recognition mechanism at atomic resolution because of the lack of appropriate approaches for studying large heterogeneous supramolecular complexes. Recently, we proposed an NMR method, termed transferred cross-saturation (TCS), that enables the rigorous identification of contact residues in a huge protein complex. Here we used TCS to study the supramolecular complex between the A3 domain of von Willebrand factor and fibrillar collagen, which allowed the successful determination of the ligand-binding site of the A3 domain. The binding site of the A3 domain was located at its hydrophobic 'front' surface and was completely different from that of the I domain from the a2 subunit of integrin (alpha2-I domain), which was reported to be the hydrophilic 'top' surface of alpha2-I, although the A3 domain and the alpha2-I domain share a similar fold and possess the identical function of collagen binding.