Structure and laminin-binding specificity of the NtA domain expressed in eukaryotic cells.
Agrin is a key organizer for postsynaptic differentiation at the neuromuscular junction (NMJ). This activity requires the binding of agrin to the synaptic basal lamina via its N-terminal (NtA) domain. It has been suggested that this binding is mediated by conserved amino acids in the gamma 1 chain of laminin. Here, we report the crystal structure of chicken NtA expressed in eukaryotic HEK293 cells. In contrast to the previously published structure [Stetefeld, J., Jenny, M., Schulthess, T., Landwehr, R., Schumacher, B., Frank, S., Ruegg, M.A., Engel, J., Kammerer, R.A., 2001. The laminin-binding domain of agrin is structurally related to N-TIMP-1. Nat. Struct. Biol., 8, 705-709.], which was derived from the NtA domain expressed in E. coli, the new data show that the N-terminal tail region (amino acid residues Asn1-Arg5) is highly structured. Moreover, the disulfide bridge between Cys2 and Cys74 was also present. In addition, we show that the binding of NtA requires the gamma 1 chain of laminin and is not greatly affected by the composition of beta chains. These results confirm a model of the NtA-laminin complex where conserved amino acids in the gamma 1 chain are prerequisite for the binding to agrin and they further emphasize that the source of protein can be critical in structure determination.
Mascarenhas
Joseph B
JB
Biozentrum Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Rüegg
Markus A
MA
Sasaki
Takako
T
Eble
Johannes A
JA
Engel
Jürgen
J
Stetefeld
Jörg
J
D018171:Agrin / Q000737:chemistry; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002645:Chickens; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D009469:Neuromuscular Junction / Q000166:cytology; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
D018171:Agrin; D004220:Disulfides; D007797:Laminin; D020033:Protein Isoforms; D011994:Recombinant Proteins; C121346:laminin 1; C095567:laminin alpha 3; D003545:Cysteine