The structure of gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).

We had previously identified the WW domain as a novel globular domain that is composed of 38 -40 semiconserved amino acids and is involved in mediating protein-protein interaction. The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals. Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands. By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of Yes kinase-associated protein which we named WW domainbinding proteins 1 and 2. These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine. Herein, we report the cDNA cloning and characterization of the human orthologs of WW domain-binding proteins 1 and 2. The products encoded by these cDNA clones represent novel proteins with no known function. Furthermore, these proteins show no homology to each other except for a proline-rich motif. By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for WW domain-binding proteins 1 and 2 to chromosomes 2p12 and 17q25, respectively. In addition, using sitedirected mutagenesis, we determined which residues in the WW domain of Yes kinase-associated protein are critical for binding. Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif.The Src homology (SH) 1 2 and SH3 domains have assumed essential roles in furthering the understanding of how an extracellular signal is transmitted from the cellular membrane, through the cytoplasm, and finally into the nucleus where the signal is interpreted through the process of gene-specific transcription. The SH2 domain has been shown to interact specifically with sequences containing a phosphotyrosine residue, whereas the SH3 domain mediates binding to proline-rich sequences with the minimal consensus of PXXP (P represents proline, and X designates any amino acid) (1, 2). The SH2 and SH3 domains thus consist of a common binding core that recognizes phosphotyrosine-or proline-rich motifs, respectively, and which achieve binding specificity through unique flanking sequences (3-5). As a result, these domains determine which proteins can interact, and equally important, in what order the interaction occurs in the closely regulated pathways of signal transduction. Recently, two other important signaling modules were characterized: the pleckstrin homology domain and the protein interaction domain/phosphotyrosine binding domain (6 -10). These modular repeats represent true protein domains in that they constitute structurally distinct three-dimensional units that can properly fold and function in the context of other proteins or in isolation (11,12).We have previously identified a Yes ki...

Twelve analogues of diclofenac (1), a nonsteroidal antiinflammatory drug and known inhibitor of transthyretin (TTR) amyloid formation, were prepared and evaluated as TTR amyloid formation inhibitors. High activity was exhibited by five of the compounds. Structure-activity relationships reveal that a carboxylic acid is required for activity, but changes in its position as well as the positions of other substituents are tolerated. High-resolution X-ray crystal structures of four of the active compounds bound to TTR were obtained. These demonstrate the significant flexibility with which TTR can accommodate ligands within its two binding sites.

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The objective of this study was to evaluate the suitability of the WW domain as a desirable model system to understand the folding and stability of an isolated three-stranded antiparallel b-sheet structure. The WW domain was subjected to thermal and chaotropic denaturation0reconstitution utilizing a variety of biophysical methods. This three-stranded sheet folds reversibly and cooperatively utilizing both urea and GdnHCl as denaturants; however, the denatured state retains structure in the form of a hydrophobic cluster involving at least one aromatic side chain. In contrast to chaotropic denaturation, thermal denaturation appears to be more complete and may be a two state process. The suitability of the WW domain for future studies aimed at understanding the kinetics and thermodynamics of antiparallel b-sheet folding clearly emerges from this initial study. The most exciting and significant result in this manuscript is the finding that the chaotropic denatured state of WW has a hydrophobic cluster as discerned by near-UV CD evidence. The role that the denatured state plays in the folding and stability of a three-stranded b-sheets, and its capacity for preventing aggregation may be particularly important and is the subject of ongoing studies.Keywords: b-sheet folding; hydrophobic cluster; reversible folding; WW The WW domain is a recently discovered protein module consisting of a three-stranded antiparallel b-sheet structure that is named for the two highly conserved Trp residues separated in the sequence by