title: Isothermal titration calorimetric study defines the substrate binding residues of calreticulin.
abstract: Earlier we established using modeling studies the residues in calreticulin (CRT) important for sugar-binding (M. Kapoor, H. Srinivas, K. Eaazhisai, E. Gemma, L. Ellgaard, S. Oscarson, A. Helenius, A. Surolia, Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone, J. Biol. Chem. 278 (8) (2003) 6194-6200). Here, we discuss the relative roles of Trp-319, Asp-317, and Asp-160 for sugar-binding by using site-directed mutagenesis and isothermal titration calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in CNX play important role towards sugar-binding. From the present study we demonstrate that the residue Asp-160 is not involved in sugar-binding, while Asp-317 plays a crucial role. Further, it is also validated that cation-pi interactions of the sugar with Trp-319 dictate sugar-binding in CRT. This study not only defines further the binding site of CRT but also highlights its subtle differences with that of calnexin.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Jayashree
authors.initials: J
authors.lastname: Gopalakrishnapai
authors.forename: Garima
authors.initials: G
authors.lastname: Gupta
authors.forename: T
authors.initials: T
authors.lastname: Karthikeyan
authors.forename: Sharmistha
authors.initials: S
authors.lastname: Sinha
authors.forename: Eaazhisai
authors.initials: E
authors.lastname: Kandiah
authors.forename: Emiliano
authors.initials: E
authors.lastname: Gemma
authors.forename: Stefan
authors.initials: S
authors.lastname: Oscarson
authors.forename: Avadhesha
authors.initials: A
authors.lastname: Surolia
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D001665:Binding Sites; D002151:Calorimetry; D037282:Calreticulin / Q000737:chemistry* / Q000648:ultrastructure*; D002942:Circular Dichroism; D003198:Computer Simulation; D008358:Mannose; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013329:Structure-Activity Relationship; D017186:Titrimetry
chemical_list: D000596:Amino Acids; D037282:Calreticulin; D008358:Mannose