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Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and β-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.

The formin Delphilin binds the glutamate receptor, GluRδ2, in dendritic spines of Purkinje cells. Both proteins play a role in learning. To understand how Delphilin functions in neurons, we studied the actin assembly properties of this formin. Formins have a conserved formin homology 2 domain, which nucleates and associates with the fast-growing end of actin filaments, influencing filament growth together with the formin homology 1 (FH1) domain. The strength of nucleation and elongation varies widely across formins. Additionally, most formins have conserved domains that regulate actin assembly through an intramolecular interaction. Delphilin is distinct from other formins in several ways: its expression is limited to Purkinje cells, it lacks classical autoinhibitory domains, and its FH1 domain has minimal proline-rich sequence. We found that Delphilin is an actin nucleator that does not accelerate elongation, although it binds to the barbed end of filaments. In addition, Delphilin exhibits a preference for actin isoforms, nucleating nonmuscle actin but not muscle actin, which has not been described or systematically studied in other formins. Finally, Delphilin is the first formin studied that is not regulated by intramolecular interactions. We speculate how the activity we observe is consistent with its localization in the small dendritic spines.

The formin Delphilin binds the glutamate receptor, GluRδ2, in dendritic spines of Purkinje cells. Both proteins play a role in learning. To understand how Delphilin functions in neurons, we studied the actin assembly properties of this formin. Formins have a conserved formin homology 2 domain, which nucleates and associates with the fast-growing end of actin filaments, influencing filament growth together with the formin homology 1 (FH1) domain. The strength of nucleation and elongation varies widely across formins. Additionally, most formins have conserved domains that regulate actin assembly through an intramolecular interaction. Delphilin is distinct from other formins in several ways: its expression is limited to Purkinje cells, it lacks classical autoinhibitory domains, and its FH1 domain has minimal proline-rich sequence. We found that Delphilin is an actin nucleator that does not accelerate elongation, although it binds to the barbed end of filaments. In addition, Delphilin exhibits a preference for actin isoforms, nucleating nonmuscle actin but not muscle actin, which has not been described or systematically studied in other formins. Finally, Delphilin is the first formin studied that is not regulated by intramolecular interactions. We speculate how the activity we observe is consistent with its localization in the small dendritic spines.

Overproduction of immunoglobulin light chains leads to systemic amyloidosis, a lethal disease characterized by the formation of amyloid fibrils in patients' tissues. Excess light chains are in equilibrium between dimers and less stable monomers which can undergo irreversible aggregation to the amyloid state. The dimers therefore must disassociate into monomers prior to forming amyloid fibrils. Here we identify ligands that inhibit amyloid formation by stabilizing the Mcg light chain variable domain dimer and shifting the equilibrium away from the amyloid-prone monomer.

Systemic light chain amyloidosis is a lethal disease characterized by excess immunoglobulin light chains and light chain fragments composed of variable domains, which aggregate into amyloid fibers. These fibers accumulate and damage organs. Some light chains induce formation of amyloid fibers, whereas others do not, making it unclear what distinguishes amyloid formers from non-formers. One mechanism by which sequence variation may reduce propensity to form amyloid fibers is by shifting the equilibrium toward an amyloid-resistant quaternary structure. Here we identify the monomeric form of the Mcg immunoglobulin light chain variable domain as the quaternary unit required for amyloid fiber assembly. Dimers of Mcg variable domains remain stable and soluble, yet become prone to assemble into amyloid fibers upon disassociation into monomers.

Formins are a conserved family of proteins known to enhance actin polymerization. Most formins are regulated by an intramolecular interaction. The Drosophila formin, Cappuccino (Capu), was believed to be an exception. Capu does not contain conserved autoinhibitory domains and can be regulated by a second protein, Spire. We report here that Capu is, in fact, autoinhibited. The N-terminal half of Capu (Capu-NT) potently inhibits nucleation and binding to the barbed end of elongating filaments by the C-terminal half of Capu (Capu-CT). Hydrodynamic analysis indicates that Capu-NT is a dimer, similar to the N-termini of other formins. These data, combined with those from circular dichroism, suggest, however, that it is structurally distinct from previously described formin inhibitory domains. Finally, we find that Capu-NT binds to a site within Capu-CT that overlaps with the Spire-binding site, the Capu-tail. We propose models for the interaction between Spire and Capu in light of the fact that Capu can be regulated by autoinhibition.

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.

A study of the Treponema palladium outer membrane protein 1 (Tromp1) properties is presented. Results indicate that they can be replicated by renaturation of recombinant Tromp1.

Monoclonal antibody complexes formed with only one antibody and complexes formed using two antibodies were investigated using dynamic light scattering and sedimentation velocity techniques. Findings show that the largest complex formed with a single monoclonal antibody was that of an low-density lipoprotein dimer. With two antibodies, a variety of complexes was observed. Monoclonal antibodies MB47 and 4G3, which bound about 55 degrees apart, formed a very stable dimer. Monoclonal antibodies MB47 and 4G3, which bound 136 degrees apart, formed a very stable tetramer.

Recombinant apolipoprotein(a) was studied by hydrodynamic and electron microscopy techniques. Recombinant apo(a) forms a tight, noncovalent complex with human low density lipoprotein (LDL). Hydrodynamic modeling suggests that it made contact with the LDL through a few kringles, with the remainder of the molecule extending into solution. This complex dissociates in the presence of 50 mM 6-aminohexanoic acid.

Study is presented to show that the myelin protein zero transmembrane (MPZ TM) domain forms stable dimers that are mediated by the glycine zipper motif. Based on this result a new mode is proposed for the MPZ adhesion structure in which dimers of tetramers form a complementary lattice that bonds adjacent layers of the myelin sheath.

The tertiary structure of retrocyclin-2 is determined in a membrane-mimicking environment and a well-defined [beta]-hairpin structure is present in the presence of SDS micelles. The ability to self-associate has contributed to the high-affinity binding of retrocyclins for glycoproteins by increasing the valency and increasing the ability of retrocyclins to cross-link cell surface glycoproteins.

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.