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.

Summary Mutations in peripheral myelin protein 22 (PMP22) can result in the common peripheral neuropathy, Charcot-Marie-Tooth disease (CMTD). The Leu16Pro mutation in PMP22 results in misassembly of the protein, which causes the Trembler-J (TrJ) disease phenotype. Here we elucidate the structural defects present in a partially folded state of TrJ PMP22 that are decisive in promoting CMTD-causing misfolding. In this state transmembrane helices 2-4 (TM2-4) form a molten-globular bundle while transmembrane helix 1 (TM1) is dissociated from this bundle. The TrJ mutation was seen to profoundly disrupt the TM1 helix, resulting in increased backbone dynamics and changes in the tertiary interactions of TM1 with the PMP22 TM2-4 core in the folded state. Consequently, TM1 undergoes enhanced dissociation from the other transmembrane segments in TrJ PMP22, becoming available for recognition and sequestration by protein folding quality control, leading to loss of function and toxic accumulation of aggregates that results in CMTD.

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe.

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