Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola – one from genetically-humanized mice, and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the SARS-CoV-2 spike protein, yielding a large collection of fully-human antibodies that were characterized for binding, neutralization and three dimensional structure. Based on these criteria, we selected pairs of highly-potent individual antibodies that simultaneously bind the receptor-binding domain of the spike protein, providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single antibody treatment.

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region.

The muscle-specific receptor tyrosine kinase MuSK plays a crucial role in neuromuscular synapse formation. Activation of MuSK is induced by agrin leading to clustering of several proteins, including acetylcholine receptors, at synaptic sites. In a first step to elucidate the signal transduction cascade following MuSK activation and leading to clustering of synaptic proteins, we sought to identify the tyrosine residues that are phosphorylated in activated MuSK. We mapped the tyrosine residues that are phosphorylated in vitro and in vivo using methods that provide high sensitivity and do not require radioactive tracers. We expressed MuSK in insect cells by using a baculovirus expression vector and mapped the tyrosines that are phosphorylated in MuSK in an in vitro kinase assay using matrix-assisted laser desorption ionization MS to sequence tryptic peptides fractionated by HPLC. In addition, we isolated MuSK from Torpedo electric organ and used nanoelectrospray tandem mass spectrometry and parent ion scanning to identify the tyrosine residues that are phosphorylated in activated, endogenous MuSK in vivo. We found that six of the nineteen intracellular tyrosine residues in MuSK are phosphorylated in activated MuSK: the juxtamembrane tyrosine (Y553), the tyrosines within the activation loop (Y750, Y754, and Y755), a tyrosine near the beginning of the kinase domain (Y576), and a tyrosine (Y812) within the C-terminal lobe of the kinase domain. Our biochemical data are consistent with results from functional experiments and establish a good correlation between tyrosine residues that are phosphorylated in activated MuSK and tyrosines that are required for MuSK signaling. neuromuscular synapse ͉ acetylcholine receptor ͉ agrin ͉ synapse formation ͉ mass spectrometry

MuSK, a muscle-specific receptor tyrosine kinase that is activated by agrin, has a critical role in neuromuscular synapse formation. In cultured myotubes, agrin stimulates the rapid phosphorylation of MuSK, leading to MuSK activation and tyrosine phosphorylation and clustering of acetylcholine receptors. Agrin, however, fails to stimulate tyrosine phosphorylation of MuSK that is force-expressed in myoblasts and fibroblasts, indicating that myotubes contain an additional activity that is required for agrin to stimulate MuSK. Certain glycosyltransferases are expressed selectively at synaptic sites in skeletal muscle, raising the possibility that carbohydrate modifications of MuSK, catalyzed by glycosyltransferases expressed selectively in myotubes, may be essential for agrin to bind and activate MuSK. We identifed two N-linked glycosylation sites in MuSK, and we expressed MuSK mutants lacking one or both N-linked sites into MuSK mutant myotubes to determine whether N-linked carbohydrate modifications of MuSK have a role in MuSK activation. We found that N-linked glycosylation restrains ligand-independent tyrosine phosphorylation of MuSK and downstream signaling but is not necessary for agrin to stimulate MuSK.