Molecular structure of the third immunoglobulin domain (Ig3) of human Muscle-Specific kinase (MuSK)

• hMuSK-Ig3 crystal structure determined experimentally using long-wavelength (sulfur + potassium) SAD. • Unexpected super-helical assembly of hMuSK-Ig3 domains arranged in a pairwise antiparallel fashion. • K + binding sites shaped by unique features found in hMuSK-Ig3. • Inter-domain contacts involving residues unique to MuSK Ig3, possibly suggestive of MuSK-specific interaction sites with molecular partners. The Muscle-Specific Kinase (MuSK) is a monotopic transmembrane receptor responsible for key signaling events during development and maintenance of neuromuscular junctions. The N-terminal extracellular portion of MuSK is characterized by multiple domains, extensively involved in molecular interactions with co-receptor LRP4 during MuSK activation. The molecular mechanisms underlying MuSK activation through self- and non-self- molecular interactions are still poorly understood. In this work, we have recombinantly produced and characterized the third Ig domain of human MuSK (hMuSK-Ig3) using X-ray crystallography. Long-wavelength experimental phasing serendipitously revealed several potassium ions bound to the ten copies of hMuSK-Ig3 found in the crystallographic asymmetric unit, arranged in a super-helical fashion with paired antiparallel inter-domain molecular contacts involving β-sheets from two neighboring molecules. Collectively, our data highlight unique structural features of this domain, including metal ion binding and surface contact hot-spots possibly suggestive of contact sites relevant for interactions with co-receptor LRP4 and/or other molecular partners involved in MuSK signaling.