ACCELERATED COMMUNICATION T-shaped arrangement of the recombinant agrin G3 – IgG Fc protein Trushar R. Patel, 1 Markus Meier, 1 Jianhua Li, 2 Gordon Morris, 3,4 Arthur J. Rowe, 3 and Jo ¨ rg Stetefeld 1 * 1 Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 2 Rigaku Americas, The Woodlands, Texas 77381-5209 3 NCMH, School of Biosciences, University of Nottingham, Sutton Bonington, Leicestershire LE12 5RD, United Kingdom 4 School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, United Kingdom Received 7 February 2011; Revised 14 March 2011; Accepted 16 March 2011 DOI: 10.1002/pro.628 Published online 29 March 2011 proteinscience.org Abstract: Agrin is a large heparin sulphate proteoglycan with multiple domains, which is located in the extracellular matrix. The C-terminal G3 domain of agrin is functionally one of the most important domains. It harbors an a-dystroglycan binding site and carries out acetylcholine receptor clustering activities. In the present study, we have fused the G3 domain of agrin to an IgG Fc domain to produce a G3-Fc fusion protein that we intend to use as a tool to investigate new binding partners of agrin. As a first step of the study, we have characterized the recombinant fusion protein using a multidisciplinary approach using dynamic light scattering, analytical ultracentrifugation and small angle X-ray scattering (SAXS). Interestingly, our SAXS analysis using the high-resolution structures of G3 and Fc domain as models indicates that the G3-Fc protein forms a T-shaped molecule with the G3 domains extruding perpendicularly from the Fc scaffold. To validate our models, we have used the program HYDROPRO to calculate the hydrodynamic properties of the solution models. The calculated values are in excellent agreement with those determined experimentally. Keywords: agrin; analytical ultracentrifugation; dynamic light scattering; small angle X-ray scattering; splice insert Introduction Agrin is a heparin sulphate proteoglycan that is pre- dominantly located in the extracellular matrix. 1 It is synthesized by motor neuron cells, transported along their axons and then secreted from their terminals to accumulate in the basal lamina, which occupies the synaptic cleft at the neuromuscular junction (NMJ). Agrin plays a crucial role during the aggre- gation of the acetylcholine receptor (AChR) and the clustering of other postsynaptic molecules at the NMJ. 2–5 It is responsible for the generation of syn- apses at the NMJ and their maintenance. Agrin is vital for the formation of immunological synapses 6 and synapses in the brain. 7,8 It mediates transcyto- sis of HIV-1 across epithelial cell monolayers during the formation of virological synapses. 9 It is essential to generate functional postsynaptic structures in skeletal muscle. Very recently, it has been shown Grant sponsor: CIHR-Regional Partnership Program and the Manitoba Institute for Child Health. *Correspondence to: Jo ¨ rg Stetefeld, Department of Chemistry, 144 Dysart Road, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. E-mail: stetefel@cc.umanitoba.ca Published by Wiley-Blackwell. V C 2011 The Protein Society PROTEIN SCIENCE 2011 VOL 20:931—940 931