2,4,6-Trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin.
The role of the primary amino groups of lysine sidechains in Ca2+ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca2+ and/or Mg2+ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca2+ and/or Mg2+. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that the C-domain and N-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca2+, TNBS binding to the C-domain was significantly reduced, whereas modification of the N-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca2+ binding to the low affinity/high capacity Ca2+ binding site(s) which are localized to the C-domain but had no effect on the high affinity/low capacity Ca2+ binding localized to the N domain. In the C-domain of calreticulin, which contains the low affinity/high capacity Ca2+ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in the C-domain of calreticulin have a role in the low affinity/high capacity Ca2+ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca2+ store.
Breier
A
A
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Michalak
M
M
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D037282:Calreticulin; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D014302:Trinitrobenzenesulfonic Acid
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D014302:Trinitrobenzenesulfonic Acid; D008239:Lysine; D002118:Calcium