Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments.

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The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-b4 (Tb4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tb4 (G1-Tb4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tb4:actin complex at 2 Å resolution. The structure reveals that Tb4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tb4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motifcontaining proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.

Gelsolin superfamily members are Ca 21 -dependent, multidomain regulators of the actin cytoskeleton. Calcium binding activates gelsolin by inducing molecular gymnastics (large-scale conformational changes) that expose actin interaction surfaces by releasing a series of latches. A specialized tail latch has distinguished gelsolin within the superfamily. Active gelsolin exhibits actin filament severing and capping, and actin monomer sequestering activities. Here, we analyze a combination of sequence, structural, biophysical and biochemical data to assess whether the molecular plasticity, regulation and actinrelated properties of gelsolin are also present in other superfamily members. We conclude that all members of the superfamily will be able to transition between a compact conformation and a more open form, and that most of these open forms will interact with actin. Supervillin, which lacks the severing domain 1 and the F-actin binding-site on domain 2, is the clear exception. Eight calcium-binding sites are absolutely conserved in gelsolin, adseverin, advillin and villin, and compromised to increasing degrees in CapG, villin-like protein, supervillin and flightless I. Advillin, villin and supervillin each contain a potential tail latch, which is absent from CapG, adseverin and flightless I, and ambiguous in villin-like protein. Thus, calcium regulation will vary across the superfamily. Potential novel isoforms of the superfamily suggest complex regulation at the gene, transcript and protein levels. We review animal, clinical and cellular data that illuminate how the regulation of molecular flexibility in gelsolin-like proteins permits cells to exploit the force generated from actin polymerization to drive processes such as cell movement in health and disease. V C 2013 Wiley Periodicals, Inc.

Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca 2؉ and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca 2؉ locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed.actin ͉ calcium activated ͉ calcium dependent ͉ TIRF

PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A ؉ B 3 A⅐B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex. PDZ4 domains are found in a few hundred human proteins, either as a single domain or in arrays. These domains mediate binding to other proteins and in this way play important roles in scaffolding and signal transduction (1, 2). Structural studies have shown that the PDZ domains usually bind to the C terminus of their target proteins. A number of crystal and NMR structures of PDZ domains have been solved both with and without bound peptide (for example, Refs 3-6) ( Fig. 1). A wealth of data on different peptides binding to different PDZ domains has been obtained by screening (for example, Refs. 7 and 8) and selection (for example, Refs. 9 and 10). Such studies and those using the yeast twohybrid technique (for example, Ref. 11) provide important information on possible cellular targets for distinct PDZ domains as well as the specificity of the interaction. Moreover, theory and NMR experiments have suggested that the dynamics of PDZ domains and the residues outside of the binding pocket influence their interaction with ligands (12, 13). Despite considerable effort to clarify the structural basis for the PDZ-ligand interaction, only a handful of studies have assessed the binding energetics and specificity of PDZ-peptide interactions using proper equilibrium assays in solution (3, 11, 14 -24). Kinetics of chemical reactions not only provide "end point data" such as equilibrium constants but also yield microscopic rate constants and, more importantly, the possibility of elucidating the mechanisms of binding and probing the binding dynamics as well as the properties of the transition state of the reaction. To assess the binding mechanism, we have performed the first kinetic study of PDZ domains in solution using stopped-flow fluorimetry. The PDZ domains chosen were PDZ3 from human PSD-95, one of the most well studied PDZ domains, and the second PDZ domain from mouse protein tyrosine phosphatase-L1 (PTP-BL; also known...

We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25-amino-acid N-terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane-spanning domain of 23 amino acids, and an intracellular C-terminal region of 209 amino acid residues. Three types of cysteine-rich repeats characteristic of the low-density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand-binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor-like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated-pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src-homology 3 recognition motifs, one Src-homology 2 recognition motif for the p8.5 regulatory subunit of phosphatidylinositol 3-kinase, and additional sites for protein kinase C, casein kinase I1 and CAMP-/cCMPdependent protein kinase. There is approximately 77 5% amino acid identity between human and rat gp330 with rninor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca" regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.

BACKGROUND:High-sensitivity cardiac troponin T (cTnT) assays detect small clinically important myocardial infarctions (MI) but also yield higher rates of false-positive results owing to increased concentrations sometimes present in patients without MI. Better understanding is needed of factors influencing the 99th percentile of cTnT concentrations across populations and the frequency of changes in cTnT concentrations Ͼ20% often used in combination with increased cTnT concentrations for diagnosis of MI.