Characterizing environmental stress responses of aposymbiotic Astrangia poculata to divergent thermal challenges.

Anthropogenic climate change threatens corals globally and both high and low temperatures are known to induce coral bleaching. However, coral stress responses across wide thermal breadths remain understudied. Disentangling the role of symbiosis on the stress response in obligately symbiotic corals is challenging because this response is inherently coupled with nutritional stress. Here, we leverage aposymbiotic colonies of the facultatively symbiotic coral, Astrangia poculata, which lives naturally with and without its algal symbionts, to examine how broad thermal challenges influence coral hosts in the absence of symbiosis. A. poculata were collected from their northern range limit and thermally challenged in two independent 16-day common garden experiments (heat and cold challenge) and behavioural responses to food stimuli and genome-wide gene expression profiling (TagSeq) were performed. Both thermal challenges elicited significant reductions in polyp extension. However, there were five times as many differentially expressed genes (DEGs) under cold challenge compared to heat challenge. Despite an overall stronger response to cold challenge, there was significant overlap in DEGs between thermal challenges. We contrasted these responses to a previously identified module of genes associated with the environmental stress response (ESR) in tropical reef-building corals. Cold challenged corals exhibited a pattern consistent with more severe stressors while the heat challenge response was consistent with lower intensity stressors. Given that these responses were observed in aposymbiotic colonies, many genes previously implicated in ESRs in tropical symbiotic species may represent the coral host's stress response in or out of symbiosis.

Americas Antibody Congress 2009. 21-23 September 2009, Washington DC, USA.

The Americas Antibody Congress 2009, held in Washington DC, included topics covering new state-of-the-art advances in antibody therapeutics. This conference report highlights selected presentations on enhanced antibody functionality, bispecific antibodies and antibody-drug conjugates. Technological approaches discussed include Potelligent technology from BioWa Inc, stability-engineered antibodies and BiTE antibodies.

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

Flexibility in the ABC transporter MsbA: Alternating access with a twist.

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. Here we compare four x-ray structures of the bacterial ABC lipid flippase, MsbA, trapped in different conformations, two nucleotide-bound structures and two in the absence of nucleotide. Comparison of the nucleotide-free conformations of MsbA reveals a flexible hinge formed by extracellular loops 2 and 3. This hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of the nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by two different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.

X-ray structures of the signal recognition particle receptor reveal targeting cycle intermediates.

The signal recognition particle (SRP) and its conjugate receptor (SR) mediate cotranslational targeting of a subclass of proteins destined for secretion to the endoplasmic reticulum membrane in eukaryotes or to the plasma membrane in prokaryotes. Conserved active site residues in the GTPase domains of both SRP and SR mediate discrete conformational changes during formation and dissociation of the SRP.SR complex. Here, we describe structures of the prokaryotic SR, FtsY, as an apo protein and in two different complexes with a non-hydrolysable GTP analog (GMPPNP). These structures reveal intermediate conformations of FtsY containing GMPPNP and explain how the conserved active site residues position the nucleotide into a non-catalytic conformation. The basis for the lower specificity of binding of nucleotide in FtsY prior to heterodimerization with the SRP conjugate Ffh is also shown. We propose that these structural changes represent discrete conformational states assumed by FtsY during targeting complex formation and dissociation.

The structures of MsbA: Insight into ABC transporter-mediated multidrug efflux.

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP hydrolysis to the transport of various molecules across cellular membranes. Found in both prokaryotes and eukaryotes, a sub-group of these transporters are involved in the efflux of hydrophobic drugs and lipids, causing anti-microbial and chemotherapeutic multidrug resistance. In this review, we examine recent structural and functional analysis of the ABC transporter MsbA and implications on the mechanism of multidrug efflux.

Structure of the ABC transporter MsbA in complex with ADP.vanadate and lipopolysaccharide.

Select members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter family couple ATP binding and hydrolysis to substrate efflux and confer multidrug resistance. We have determined the x-ray structure of MsbA in complex with magnesium, adenosine diphosphate, and inorganic vanadate (Mg.ADP.Vi) and the rough-chemotype lipopolysaccharide, Ra LPS. The structure supports a model involving a rigid-body torque of the two transmembrane domains during ATP hydrolysis and suggests a mechanism by which the nucleotide-binding domain communicates with the transmembrane domain. We propose a lipid "flip-flop" mechanism in which the sugar groups are sequestered in the chamber while the hydrophobic tails are dragged through the lipid bilayer.

Lipopolysaccharide stabilizes the crystal packing of the ABC transporter MsbA.

The ABC transporter MsbA is an integral membrane protein involved in the transport of lipid A and lipopolysaccharides to the outer leaflet of the inner membrane in bacteria. Here, the critical role of the natural substrate lipopolysaccharide in the crystallization and diffraction quality of MsbA crystals is reported. Initial crystals grown in complex with ATP-vanadate alone diffracted to approximately 9 A. Screening of the natural substrate lipopolysaccharides led to the crystallization of MsbA in complex with ADP-vanadate and Ra lipopolysaccharide. The increased order within the crystal lattice allowed structure determination to 4.2 A.