TY - JOUR
T1 - Structure and laminin-binding specificity of the NtA domain expressed in eukaryotic cells
AU - Mascarenhas, Joseph B.
AU - Rüegg, Markus A.
AU - Sasaki, Takako
AU - Eble, Johannes A.
AU - Engel, Jürgen
AU - Stetefeld, Jörg
N1 - Funding Information: The authors are grateful to Dr. Suat Oezbek for his contributions and critical reading of the manuscript. JS thanks in great detail Dr. Sascha Popow from DESY for his excellent support during data collections. This work was supported by the Swiss National Science Foundation, the Kanton of Basel-Stadt (JE and MAR) and the Swiss Foundation for Research on Muscle Diseases (MAR).
PY - 2005/1
Y1 - 2005/1
N2 - 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 γ1 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 γ1 chain of laminin and is not greatly affected by the composition of β chains. These results confirm a model of the NtA-laminin complex where conserved amino acids in the γ1 chain are prerequisite for the binding to agrin and they further emphasize that the source of protein can be critical in structure determination.
AB - 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 γ1 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 γ1 chain of laminin and is not greatly affected by the composition of β chains. These results confirm a model of the NtA-laminin complex where conserved amino acids in the γ1 chain are prerequisite for the binding to agrin and they further emphasize that the source of protein can be critical in structure determination.
KW - Agrin
KW - Agrin-laminin interaction
KW - N-terminal agrin
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U2 - 10.1016/j.matbio.2004.11.003
DO - 10.1016/j.matbio.2004.11.003
M3 - Article
C2 - 15694127
SN - 0945-053X
VL - 23
SP - 507
EP - 513
JO - Matrix Biology
JF - Matrix Biology
IS - 8
ER -