The calcium- and zinc-responsive regions of calreticulin reside strictly in the N-/C-domain.
Calreticulin (CRT) is a chaperone of the endoplasmic reticulum. We dissected CRT into its two structural domains, N-/C-domain and P-domain, to identify its metal ion-responsive region. For this, we constructed bacterial expression systems for the N-/C-domain (1-180 fused by a linker to 290-400) and P-domain (189-280). Circular dichroism (CD) studies showed that calcium ions increased tertiary packing and thermal stability of apo N-/C-domain, whereas zinc ions had a strong destabilizing effect. Interestingly, neither calcium nor zinc ions altered the structural properties of apo P-domain. These results indicate that the calcium- and zinc-responsive regions reside strictly in the N-/C-domain. Analysis of thermal denaturation curves of CRT, N-/C-domain, and P-domain suggested a structural role for the P-domain in CRT. Rotary shadowing electron microscopy (EM) analysis of CRT and calnexin provided convincing evidence for their structural relatedness. This analysis also revealed that apo P-domain adopts various curved shapes suggesting conformational flexibility. EM images of apo N-/C-domain revealed objects having wide gaps suggesting weak interactions between the N- and C-domains. This is consistent with the larger size of apo N-/C-domain on the gel filtration column. Our studies provide a framework for correlating the structural organization of CRT with its metal ion-responsive region.
Tan
Yan
Y
University of Connecticut, School of Pharmacy, 69 North Eagleville Road U-3092, Storrs, CT 06269, USA.
Chen
Mingnan
M
Li
Zhenjie
Z
Mabuchi
Katsuhide
K
Bouvier
Marlene
M
D002118:Calcium / Q000737:chemistry*; D037281:Calnexin / Q000737:chemistry / Q000648:ultrastructure; D037282:Calreticulin / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008854:Microscopy, Electron; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015032:Zinc / Q000737:chemistry*
D037282:Calreticulin; D011994:Recombinant Proteins; D037281:Calnexin; D015032:Zinc; D002118:Calcium