Parallel actin monomers in the 8S complex of actin-INF2.

Polymerization and depolymerization of actin play an essential role in eukaryotic cells. Actin exists in cells in both monomeric (G-actin) and filamentous (polymer, F-actin) forms. Actin binding proteins (ABPs) facilitate the transition between these two states, and their interactions with these two states of actin are critical for actin-based cellular processes. Rapid depolymerization of actin is assisted in the brain and/or other cells by its oxidation by the enzyme Mical (yielding Mox-actin), and/or by the binding of Inverted Formin 2 (INF2) - which can also accelerate filaments formation. At their stoichiometric molar ratio INF2 and actin yield the 8S complex (consisting of 4 actin monomers: 2 INF2 dimer molecules). Using biochemical and biophysical methods, we investigate the structural arrangement of actin in the 8S particles and the interaction of INF2 with actin and Mox-actin. To that end, we show 2 D class averages of 8S particles obtained by negative staining electron microscopy. We also show that: (i) 8S particles can seed rapid actin assembly; (ii) Mox-actin and INF2 form 8S particles at proteins ratios similar to those of unoxidized actin; (iii) chemical crosslinkings suggest that actin monomers are in a parallel orientation in the 8S particles of both actin and Mox-actin; and (iv) INF2 accelerates the disassembly of Mox-F-actin. Our results provide better understanding of actin's arrangement in the 8S particles formed during actin depolymerization and in the early polymerization stages of both actin and Mox-actin.Communicated by Ramaswamy H. Sarma.

Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA.

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 ß-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 neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongation.

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.

t-Darpp is an elongated monomer that binds calcium and is phosphorylated by cyclin-dependent kinases 1 and 5.

t-Darpp (truncated isoform of dopamine- and cAMP-regulated phosphoprotein) is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t-Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells, t-Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t-Darpp function, we studied its secondary structure, oligomerization status, metal-binding properties, and phosphorylation by cyclin-dependent kinases 1 and 5. t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn, and 35% random coil structures. It binds calcium, but not other metals commonly found in biological systems. The T39 site, critical for t-Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 and cyclin-dependent kinase 5. Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde-mediated cross-linking experiments demonstrate that the majority of t-Darpp exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.

Inhibition by small-molecule ligands of formation of amyloid fibrils of an immunoglobulin light chain variable domain.

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.

Excessive centrifugal fields damage high density lipoprotein.

HDL is typically isolated ultracentrifugally at 40,000 rpm or greater, however, such high centrifugal forces are responsible for altering the recovered HDL particle. We demonstrate that this damage to HDL begins at approximately 30,000 rpm and the magnitude of loss increases in a rotor speed-dependent manner. The HDL is affected by elevated ultracentrifugal fields resulting in a lower particle density due to the shedding of associated proteins. To circumvent the alteration of the recovered HDL, we utilize a KBr-containing density gradient and a lowered rotor speed of 15,000 rpm to separate the lipoproteins using a single 96 h centrifugation step. This recovers the HDL at two density ranges; the bulk of the material has a density of about 1.115 g/ml, while lessor amounts of material are recovered at >1.2 g/ml. Thus, demonstrating the isolation of intact HDL is possible utilizing lower centrifuge rotor speeds.

Formation of amyloid fibers by monomeric light chain variable domains.

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.

Solution structure of the sortase required for efficient production of infectious Bacillus anthracis spores.

Bacillus anthracis forms metabolically dormant endospores that upon germination can cause lethal anthrax disease in humans. Efficient sporulation requires the activity of the SrtC sortase (BaSrtC), a cysteine transpeptidase that covalently attaches the BasH and BasI proteins to the peptidoglycan of the forespore and predivisional cell, respectively. To gain insight into the molecular basis of protein display, we used nuclear magnetic resonance to determine the structure and backbone dynamics of the catalytic domain of BaSrtC (residues Ser(56)-Lys(198)). The backbone and heavy atom coordinates of structurally ordered amino acids have coordinate precision of 0.42 ± 0.07 and 0.82 ± 0.05 Å, respectively. BaSrtC(Δ55) adopts an eight-stranded ß-barrel fold that contains two short helices positioned on opposite sides of the protein. Surprisingly, the protein dimerizes and contains an extensive, structurally disordered surface that is positioned adjacent to the active site. The surface is formed by two loops (ß2-ß3 and ß4-H1 loops) that surround the active site histidine, suggesting that they may play a key role in associating BaSrtC with its lipid II substrate. BaSrtC anchors proteins bearing a noncanonical LPNTA sorting signal. Modeling studies suggest that the enzyme recognizes this substrate using a rigid binding pocket and reveals the presence of a conserved subsite for the signal. This first structure of a class D member of the sortase superfamily unveils class-specific features that may facilitate ongoing efforts to discover sortase inhibitors for the treatment of bacterial infections.

Autoinhibition of the formin Cappuccino in the absence of canonical autoinhibitory domains.

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.

Multiple forms of Spire-actin complexes and their functional consequences.

Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.

Crystal structure of the central coiled-coil domain from human liprin-ß2.

Liprins are a conserved family of scaffolding proteins important for the proper regulation and development of neuronal synapses. Humans have four liprin-αs and two liprin-ßs which all contain long coiled-coil domains followed by three tandem SAM domains. Complex interactions between the coiled-coil and SAM domains are thought to create liprin scaffolds, but the structural and biochemical properties of these domains remain largely uncharacterized. In this study we find that the human liprin-ß2 coiled-coil forms an extended dimer. Several protease-resistant subdomains within the liprin-ß1 and liprin-ß2 coiled-coils were also identified. A 2.0 Å crystal structure of the central, protease-resistant core of the liprin-ß2 coiled-coil reveals a parallel helix orientation. These studies represent an initial step toward determining the overall architecture of liprin scaffolds and understanding the molecular basis for their synaptic functions.

Atomic force microscopy reveals drebrin induced remodeling of f-actin with subnanometer resolution.

We show by high-resolution atomic force microscopy analysis that drebrin A (a major neuronal actin binding protein) induced F-actin structural and mechanical remodeling involves significant changes in helical twist and filament stiffness (+55% persistence length). These results provide evidence of a unique mechanical role of drebrin in the dendrites, contribute to current molecular-level understanding of the properties of the neuronal cytoskeleton, and reflect the role of biomechanics at the nanoscale, to modulate nanofilament-structure assemblies such as F-actin.

Grifonin-1: a small HIV-1 entry inhibitor derived from the algal lectin, Griffithsin.

BACKGROUND: Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin's sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties. METHODOLOGY/RESULTS: The new peptides derived from a trio of homologous ß-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC50 of 190.8±11.0 nM in in vitro TZM-bl assays and an EC(50) of 546.6±66.1 nM in p24gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular ß-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface. CONCLUSION: Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1.

Identifying and characterizing a functional HIV-1 reverse transcriptase-binding site on integrase.

Integrase (IN) from human immunodeficiency virus, type 1 (HIV-1) exerts pleiotropic effects in the viral replication cycle. Besides integration, IN mutations can impact nuclear import, viral maturation, and reverse transcription. IN and reverse transcriptase (RT) interact in vitro, and the IN C-terminal domain (CTD) is both necessary and sufficient for binding RT. We used nuclear magnetic resonance spectroscopy to identify a putative RT-binding surface on the IN CTD, and surface plasmon resonance to obtain kinetic parameters and the binding affinity for the IN-RT interaction. An IN K258A substitution that disrupts reverse transcription in infected cells is located at the putative RT-binding surface, and we found that this substitution substantially weakens IN CTD-RT interactions. We also identified two additional IN amino acid substitutions located at the putative RT-binding surface (W243E and V250E) that significantly impair viral replication in tissue culture. These results strengthen the notion that IN-RT interactions are biologically relevant during HIV-1 replication and also provide insights into this interaction at the molecular level.

Transmembrane domain of myelin protein zero can form dimers: possible implications for myelin construction.

Myelin protein zero (MPZ) is the major integral membrane protein of peripheral nerve myelin in higher vertebrates, mediating homoadhesion of the multiple, spiraling wraps of the myelin sheath. Previous studies have shown that full-length MPZ can form dimers and tetramers, and biochemical studies on the extracellular domain (ECD) indicate that it can form a tetramer, albeit very weakly. On the basis of cross-linking studies and equilibrium sedimentation of a transmembrane (TM) domain peptide (MPZ-TM), we find that the MPZ-TM can form homodimers. We further characterized the dimer by measuring the effects of alanine and leucine substitutions on the ability of the TM to dimerize in Escherichia coli membranes. Our results indicate that the primary packing interface for the MPZ TM homodimer is a glycine zipper (GxxxGxxxG) motif. We also find that the G134R mutation, which lies within the glycine zipper packing interface and causes Charcot-Marie-Tooth disease type 1B, severely inhibits dimerization, suggesting that dimerization of the TM domain may be important for the normal functioning of MPZ. By combining our new results with prior work, we suggest a new model for an MPZ lattice that may form during the construction of myelin.

Retrocyclin-2: structural analysis of a potent anti-HIV theta-defensin.

Retrocyclins are circular mini-defensins with significant potential as agents against human immunodeficiency virus, influenza A, and herpes simplex virus. Retrocyclins bind carbohydrate-containing surface molecules such as gp120 and CD4 with high affinity (Kd, 10-100 nM), promoting their localization on cell membranes. The structural features important for activity have yet to be fully elucidated, but here, we have determined the first three-dimensional structure of a retrocyclin, namely, one of the most potent forms, retrocyclin-2. In the presence of SDS micelles, a well-defined beta-hairpin braced by three disulfide bonds that defines the cystine ladder motif is present. By contrast, a well-defined structure could not be determined in aqueous solution, suggesting that the presence of SDS micelles stabilizes the extended conformation of retrocyclin-2. Translational diffusion measurements indicate that retrocyclin-2 interacts with the SDS micelles, and such a membrane-like interaction may be an important feature in the mechanism of action of these antimicrobial peptides. Analytical ultracentrifugation and the NMR data indicated that retrocyclin-2 self-associates to form a trimer in a concentration-dependent manner. The ability to self-associate may contribute to the high-affinity binding of retrocyclins for glycoproteins by increasing the valency and enhancing the ability of retrocyclins to cross-link cell surface glycoproteins.

Exon repression by polypyrimidine tract binding protein.

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.

BMP binding peptide: a BMP-2 enhancing factor deduced from the sequence of native bovine bone morphogenetic protein/non-collagenous protein.

Forty years ago, Marshall Urist described a partially purified extract of demineralized bone matrix which induced the formation of ectopic bone. This substance, bone morphogenetic protein/non-collagenous protein (BMP/NCP), was never purified to homogeneity but other investigators used similar starting materials to clone a number of recombinant BMPs. Urist recognized that his material probably contained the BMPs which had been cloned by others but always contended that it contained another, more potent, bone inducing material which differed significantly in its physical and chemical properties from the known BMPs. We have used Urist's protocol to isolate a protein that has the chemical and physical properties of Urist's "BMP". It is an 18.5 kD fragment of the bone matrix protein, SPP-24. This fragment contains the cystatin-like domain of SPP-24. We have located a 19 amino acid region which is similar to the TGF-beta/BMP-binding region of fetuin, a member of the cystatin family of protease inhibitors. A cyclic peptide, which we call BMP binding peptide (BBP) was generated using this sequence. The peptide avidly bound rhBMP-2 with a KD of 3 x 10(-5) M. When implanted alone in mouse muscle, the peptide frequently induced dystrophic calcification. When implanted with rhBMP-2, the peptide enhanced the osteogenic activity of the recombinant molecule. We hypothesize that Urist's "BMP" was a fragment of SPP-24 which influenced bone induction by binding to bone morphogenetic proteins. BBP may be clinically useful because of its effects on other bone-inducing substances.

Variable region domain exchange in human IgGs promotes antibody complex formation with accompanying structural changes and altered effector functions.

Variable region domain exchanged IgG, or "inside-out (io)," molecules, were produced to investigate the effects of domain interactions on antibody structure and function. Studies using ultracentrifugation and electron microscopy showed that variable region domain exchange induces non-covalent multimerization through Fab domains. Surprisingly, variable region exchange also affected Fc-associated functions such as serum half-life and binding to protein G and FcgammaRI. These alterations were not merely a consequence of IgG aggregation. Both the extent of multimerization and alterations in Fc-associated properties depended on the IgG isotype.