Summary Immune recognition of protein antigens relies upon the combined interaction of multiple antibody loops, which provides a fairly large footprint and constrains the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of antibody C05 that neutralizes strains from multiple subtypes of influenza A viruses, including H1, H2, and H3. Crystal and EM structures show that C5 recognizes conserved elements of the receptor binding site on the hemagglutinin (HA) surface glycoprotein. Recognition of the HA receptor binding site is dominated by a single HCDR3 loop, with minor contacts from HCDR1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved, functional sites on otherwise hypervariable antigens.

Previous work on the responses of mitogen-activated protein (MAP) kinase cascade components in a Xenopus oocyte extract system demonstrated that p42 MAP kinase (MAPK) exhibits a sharp, sigmoidal stimulus/response curve, rather than a more typical hyperbolic curve. One plausible explanation for this behavior requires the assumption that MAP kinase kinase (MAPKK) carries out its dual phosphorylation of p42 MAPK by a distributive mechanism, where MAPKK dissociates from MAPK between the first and second phosphorylations, rather than a processive mechanism, where MAPKK carries out both phosphorylations before dissociating. Here we have investigated the mechanism through which a constitutively active form of human MAPKK-1 (denoted MAPKK-1 R4F or MAPKK-1*) phosphorylates Xenopus p42 MAPK in vitro. We found that the amount of monophosphorylated MAPK formed during the phosphorylation reaction exceeded the amount of MAPKK-1* present, which would not be possible if the phosphorylation occurred exclusively by a processive mechanism. The monophosphorylated MAPK was phosphorylated predominantly on tyrosine, but a small proportion was phosphorylated on threonine, indicating that the first phosphorylation is usually, but not invariably, the tyrosine phosphorylation. We also found that the rate at which pulse-labeled monophosphorylated MAPK became bisphosphorylated depended on the MAPKK-1* concentration, behavior that is predicted by the distributive model but incompatible with the processive model. These findings indicate that MAPKK-1* phosphorylates p42 MAPK by a two-collision, distributive mechanism rather than a single-collision, processive mechanism, and provide a mechanistic basis for understanding how MAP kinase can convert graded inputs into switch-like outputs.The MAP 1 kinases (MAPKs) are a family of protein kinases conserved across the eukaryotic kingdoms and implicated in diverse biological processes, ranging from mitogenesis, cell fate induction, and stress responses in mammalian cells through cell wall formation and mating pheromone responses in yeast (1-6). MAPKs are activated by phosphorylation of two sites, threonine and tyrosine residues within the kinase's activation loop (7,8). In rat Erk2 these residues are threonine 183 and tyrosine 185, and they lie within the sequence TEY. Dephosphorylation of either residue inactivates the kinase, and mutant kinases with either residue replaced by a non-phosphorylatable residue are inactive or minimally active (9 -13).Examples of MAP kinases with this TEY motif, the TEY subgroup of MAP kinases, have been identified in animals, plants, fungi, and protists (6). Other MAP kinase subgroups have slightly different phosphorylation site signatures as follows: TPY for the stress activated protein kinases or Jnk proteins, TGY for the Hog1-like MAPKs, and TNY for the budding yeast Smk1 protein. However, in all cases examined, phosphorylation of the threonine and tyrosine residues has proven to be essential for activation. Thus the requirement for dual phosphorylation is conserved ...

We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 1012 random decapeptides was incubated in an Escherichia coil S30 coupled transcription/translation system. Polysomes were isolated and screened by afflnity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems.Peptide libraries derived from the cloning and expression of random-sequence oligonucleotides provide a rich source of ligands. These libraries, which often contain >108 recombinants, are characterized by the physical linkage of each peptide to its encoding DNA. This feature permits the affinity purification of peptides and associated DNA on an immobilized receptor and the DNA is sequenced to identify the peptide. This general approach provides a powerful tool for discovering ligands.A widely used version of this strategy is the display of peptides on the outer surface of filamentous phage particles. The peptide sequences are encoded by random oligonucleotides inserted into the 5' region of the genes encoding the capsid proteins pIII or pVIII (1-3). The peptides are expressed on the phage fused to the N terminus of the coat proteins. Several rounds of screening and amplification result in the enrichment of phage expressing peptides that bind to the receptor. Another system employs peptides fused to the C terminus of the lac repressor Lacd (4). The repressor protein physically links the peptides to the plasmid encoding them by binding to the lac operator sequences on the plasmid. The peptide-LacI-plasmid complexes are screened in a manner analogous to the phage system. These library approaches to ligand discovery often rely on the generation of huge numbers of peptides with the expectation that those of the appropriate structure will be rare. The size of a cell-based library is limited by the number of recombinants recovered from the transformation step. To create a peptide display system that avoids this limitation and is capable of screening much larger libraries, we developed the in vitro polysome system describe...

The widespread incidence of H5N1 influenza viruses in bird populations poses risks to human health. Although the virus has not yet adapted for facile transmission between humans, it can cause severe disease and often death. Here we report the generation of combinatorial antibody libraries from the bone marrow of five survivors of the recent H5N1 avian influenza outbreak in Turkey. To date, these libraries have yielded >300 unique antibodies against H5N1 viral antigens. Among these antibodies, we have identified several broadly reactive neutralizing antibodies that could be used for passive immunization against H5N1 virus or as guides for vaccine design. The large number of antibodies obtained from these survivors provide a detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 and H5 subtype influenza viruses.

Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a "cytostatic factor" arrest, the arrest responsible for preventing the parthenogenetic activation of unfertilized eggs. The protein kinase p90 Rsk is a substrate of p42 MAPK; thus, the role of p90 Rsk in p42 MAPK-induced mitotic arrest was examined. Xenopus laevis egg extracts immunodepleted of Rsk lost their capacity to undergo mitotic arrest in response to activation of the Mos-MEK-1-p42 MAPK cascade of protein kinases. Replenishing Rsk-depleted extracts with catalytically competent Rsk protein restored the ability of the extracts to undergo mitotic arrest. Rsk appears to be essential for cytostatic factor arrest.