FibA and PilA act cooperatively during fruiting body formation of Myxococcus xanthus

The extracellular matrix (ECM) of Myxococcus xanthus is essential for social (S-) motility and fruiting body formation. An ECM-bound protein, FibA, is homologous to M4 zinc metalloproteases and is important for stimulation by a phosphatidylethanolamine (PE) chemoattractant and for formation of discrete aggregation foci. In this work, we demonstrate that a correlation exists between a reduced ability to respond to PE and the observed defects in fruiting body morphogenesis. Furthermore, the fibA aggregation defect is accentuated by the absence of either PilA, the structural subunit of type IV pili, or DifD, a chemosensory response regulator. The inability to form fruiting bodies is not due to a loss of S-motility, but rather the loss of PilA and pili as pilT fibA mutants form fruiting bodies. The FibA active site residue E342 is important for fruiting body morphogenesis in the absence of PilA. Mutants exhibiting defects in fruiting body morphogenesis also produce fewer viable spores. It is proposed that FibA and PilA act as extracellular sensors for developmental signals.     

Synthesis, SAR exploration, and X-ray crystal structures of factor XIa inhibitors containing an alpha-ketothiazole arginine

Using an alpha-ketothiazole arginine moiety as a key recognition element, a series of small peptidomimetic molecules was designed and synthesized, and their co-crystal structures with factor XIa were studied in an effort to develop smaller, less peptidic inhibitors as antithrombotic agents.     

Structure and dynamics of dark-state bovine rhodopsin revealed by chemical cross-linking and high-resolution mass spectrometry

Recent work using chemical cross-linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G-protein-coupled receptor bovine rhodopsin in its native membrane using lysine- and cysteine-targeted bifunctional cross-linking reagents. Cross-linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT-ICR mass spectrometry with automated data-reduction and assignment software. Tandem mass spectrometry was used to verify cross-link assignments and locate the exact sites of cross-link attachment. Cross-links were observed to form between 10 pairs of residues in dark-state rhodopsin. For each pair, cross-linkers with a range of linker lengths were tested to determine an experimental distance-of-closest-approach (DCA) between reactive side-chain atoms. In all, 28 cross-links were identified using seven different cross-linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross-link between C316 and K325 in the C-terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross-linking reactions. Biochemical and spectroscopic data from other studies have found that the C-terminal region is highly mobile in solution and not fully represented by X-ray crystallography data. Our results show that chemical cross-linking can provide reliable three-dimensional structural information and insight into local conformational dynamics in a membrane protein.     

Neural cells secrete a unique repertoire of proteins

Proteins that are released from cells consist of those in the extracellular matrix, as well as extracellular signaling and adhesion molecules. The majority of these extracellular proteins are, however, unknown. To determine their identity, we have used a proteomics approach to define proteins released from neurons, astrocytes and neural precursor cells. Using two-dimensional gels and liquid chromatography/mass spectrometry technology, it is shown that while astrocytes release a relatively small number of proteins, neurons and neuronal precursor cells release a larger number of proteins with more functional diversity. Although there is overlap between the different cell types, the exact composition of the extracellular protein pool is unique for each cell population. The various subsets of extracellular neural proteins include those involved in cellular Redox regulation and chaperones. In addition, many proteolytic enzymes are found outside of the cell. These data show that the extracellular space within the nervous system has a more diverse protein composition than previously thought.     

Design and expression of a dimeric form of human immunodeficiency virus type 1 antibody 2G12 with increased neutralization potency

The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.     

Differential targeting of Tetrahymena ORC to ribosomal DNA and non‐rDNA replication origins

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis‐acting replication determinant—the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non‐rDNA fragment containing two closely associated replicators, ARS1‐A (0.8 kb) and ARS1‐B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA‐independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non‐rDNA ARS1 chromosome changed across the cell cycle. In G₂ phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1‐A/B. Remarkably, ORC and Mcm6 associated with just the ARS1‐A replicator in G₁ phase when pre‐replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non‐rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.