Binding of EphrinA5 to RET receptor tyrosine kinase : An in vitro study

Eph/Ephrin signaling pathways are crucial in regulating a large variety of physiological processes during development, such as cell morphology, proliferation, migration and axonal guidance. EphrinA (efn-A) ligands, in particular, can be activated by EphA receptors at cellcell interfaces and have been proposed to cause reverse signaling via RET receptor tyrosine kinase. Such association has been reported to mediate spinal motor axon navigation, but conservation of the interactive signaling pathway and the molecular mechanism of the interaction are unclear. Here, we found Danio rerio efn-A5b bound to Mus musculus EphA4 with high affinity, revealing structurally and functionally conserved EphA/efn-A signaling. Interestingly, we observed no interaction between efn-A5b and RET from zebrafish, unlike earlier cell-based assays. Their lack of association indicates how complex efn-A signaling is and suggests that there may be other molecules involved in efn-A5-induced RET signaling.Peer reviewe

Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells

Abstract The advent of the multiwavelength anomalousdiffraction phasing methodhas significantlyacceleratedcrystal structuredeterminationand hasbecomethe norm in protein crystallography. This method allowsresearchersto takeadvantageofthe anomalous signal fromdivers eatoms,but thedominantmethod forderivativ epreparation is selenomethionine substitution.S everal generallya pplicable, high-efficiency labeling protocolsh aveb een developed for use in the bacterial, yeast, andbaculovirus/insect cell expressionsystems butnot formammalian tissuec ulture.A sal arge number of proteinso fb iomedical importance cano nlyb ep roducedi ny ields sufficient forX -ray diffraction experimentsi nm ammalian expression systems, it becomesa ll them ore importantt od evelops uchp rotocols.W et herefore evaluateds everal variablest hatp lay roles in determining incorporation levels andr eporth ere as imple protocolf or selenom ethionine modification of proteinsi n mammalian cellsr outinely yielding >90%l abeling efficiency. Keywords: proteinl abeling; proteinc rystallography; selenomethionine; multiplew avelength anomalous diffraction; mammalian cell culture The multiwavelength anomalous diffraction (MAD)phasingm ethod (Hendrickson1 991) hasb ecome them ethod of choice for X-ray phase determination, with >50% of thee xperimentally phased structures deposited in the PDB during thep asty ear beingd etermined by MAD. WhileM AD has allowed researchers to take advantage of thea nomalouss ignal from several diverse heavy atoms, thed ominantm ethodf or heavya tomd erivative preparation is selenomethioninesubstitution. Several factors contribute to the widespread use of selenomethionine substitution, including simplicity, adaptabilitytodifferent expression systems, scalability,a nd, in some cases, an almost quantitative replacement of methionine resulting in ahomogeneousprotein population. This method results in modified proteins withoutsignificantstructural perturbations due to heavy atom incorporation,w hile eliminatingthe difficult and time-consuming screenings forheavy atom derivatives. It is estimated that, foras uccessful MADe xperiment, ones elenomethionine residue is required for every ; 75-100a mino acids (Hendrickson andO gata 1997). This corresponds to ; 80%o fa ll proteins, which have am ethioninecontentof1%ormore (Strub et al.2003). There are twol imitations to the method: First, the calculations abovea ssumeq uantitative( or near-quantitative) methionine substitution, which often is not the case.F or example, as the complexity of the expression system host increases, so does the complexity of the media requiredfor their growth, Article published onlinea head of print. Articlea nd publication date are at http://www.proteinscience.org/cg

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein

Three distinct molecular surfaces in ephrin-A5 are essential for a functional interaction with EphA3

Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2 center dot ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E alpha-helix, the E-F loop, the underlying H beta-strand, as well as the nearby B - C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes

Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines

Henipavirus Mediated Membrane Fusion, Virus Entry and Targeted Therapeutics

The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G) and class I fusion (F) envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins

Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr

Bacteria have developed complex strategies to detoxify and repair damage caused by reactive oxygen species. These compounds, produced during bacterial aerobic respiration as well as by the host immune system cells as a defense mechanism against the pathogenic microorganisms, have the ability to damage nucleic acids, proteins and phospholipid membranes. Here we describe the crystal structure of Pseudomonas aeruginosa Ohr, a member of a recently discovered family of organic hydroperoxide resistance proteins. Ohr is a tightly folded homodimer, with a novel α/β fold, and contains two active sites located at the monomer interface on opposite sides of the molecule. Using in vitro assays, we demonstrate that Ohr functions directly as a hydroperoxide reductase, converting both inorganic and organic hydroperoxides to less toxic metabolites. Site-directed mutagenesis confirms that the two conserved cysteines in each active site are essential for catalytic activity. We propose that the Ohr catalytic mechanism is similar to that of the structurally unrelated peroxiredoxins, directly utilizing highly reactive cysteine thiol groups to elicit hydroperoxide reduction