Crystal Structure of the Agrin-responsive Immunoglobulin-like Domains 1 and 2 of the Receptor Tyrosine Kinase MuSK

Abstract

Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed exclusively in skeletal muscle, where it is required for formation of the neuromuscular junction. MuSK is activated by agrin, a neuron-derived heparan sulfate proteoglycan. Here, we report the crystal structure of the agrin-responsive first and second immunoglobulin-like domains (Ig1 and Ig2) of the MuSK ectodomain at 2.2 A resolution. The structure reveals that MuSK Ig1 and Ig2 are Ig-like domains of the I-set subfamily, which are configured in a linear, semi-rigid arrangement. In addition to the canonical internal disulfide bridge, Ig1 contains a second, solvent-exposed disulfide bridge, which our biochemical data indicate is critical for proper folding of Ig1 and processing of MuSK. Two Ig1-2 molecules form a non-crystallographic dimer that is mediated by a unique hydrophobic patch on the surface of Ig1. Biochemical analyses of MuSK mutants introduced into MuSK(-/-) myotubes demonstrate that residues in this hydrophobic patch are critical for agrin-induced MuSK activation.

Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling

“…3A). The first half of the A strand forms a hydrogen bond with the B strand and the A′ strand forms hydrogen bonds with the G strand, similar to the structure of Ig-like domain Ig1 and Ig2 from the extracellular region at RTK MuSK (18). The crossover connection between strand βE and βF includes a single helical turn at residues 729-731.…”

“…The asymmetric unit contains one molecule with solvent content of 64.1%. The structure was solved by molecular replacement with MolRep using models based on the structures of Ig domains from MUSK (2IEP) (18), telokin (1TLK) (17), and D4 of KIT (2E9W) (8) as search models. The structure refinement and model-building cycles were performed using RefMac (27) and COOT (28).…”

Thermodynamic and Structural Studies of Carbohydrate Binding by the Agrin-G3 Domain

“…The classical biology approaches used to identify the molecular components of neuromuscular junctions (1,11,13,14,17,21) have recently begun to be complemented by biophysical and structural studies which provide mechanistic information about molecular function (25,46,(48)(49)(50)(51)(52)(53). In the case of the agrin-G3 domain, it has been established that the domain adopts an LNS β-jellyroll fold structure (8), that the insert loops required for AChR clustering activity occur in a dynamically flexible region of the protein between strands β2 and β3 (8), and that the agrin-G3 domain binds Ca 2+ (22,28) which reduces but does not eliminate the mobility of the surface loops that mediate the domain's activities (8).…”

MuSK IgG4 autoantibodies cause myasthenia gravis by inhibiting binding between MuSK and Lrp4

“…The first Ig-like domain in MuSK is required for MuSK to bind Lrp4. Mutation of a single residue, I96, on a solvent-exposed surface of the first Ig-like domain, prevents MuSK from binding Lrp4 and responding to Agrin (20,24). A hydrophobic surface on the opposite side of the first Ig-like domain mediates MuSK homodimerization, which is essential for MuSK transphosphorylation (24).…”

“…Mutation of a single residue, I96, on a solvent-exposed surface of the first Ig-like domain, prevents MuSK from binding Lrp4 and responding to Agrin (20,24). A hydrophobic surface on the opposite side of the first Ig-like domain mediates MuSK homodimerization, which is essential for MuSK transphosphorylation (24). Although MuSK is expressed by muscle and not by motor neurons, MuSK is critical for presynaptic as well as postsynaptic differentiation (9).…”