Calretinin and calbindin D28k have different domain organizations.
The domain organization of calretinin (CR) was predicted to involve all six EF-hand motifs (labeled I to VI) condensed into a single domain, as characterized for calbindin D28k (Calb), the closest homolog of calretinin. Unperturbed (1)H,(15)N HSQC NMR spectra of a (15)N-labeled calretinin fragment (CR III-VI, residues 100-271) in the presence of the unlabeled complimentary fragment (CR I-II, residues 1-100) show that these fragments do not interact. Size exclusion chromatography and affinity chromatography data support this conclusion. The HSQC spectrum of (15)N-labeled CR is similar to the overlaid spectra of individual (15)N-labeled CR fragments (CR I-II and CR III-VI), also suggesting that these regions do not interact within intact CR. In contrast to these observations, but in accordance with the Calb studies, we observed interactions between other CR fragments: CR I (1-60) with CR II-VI (61-271), and CR I-III (1-142) with CR IV-VI (145-271). We conclude that CR is formed from at least two independent domains consisting of CR I-II and CR III-VI. The differences in domain organization of Calb and CR may explain the specific target interaction of Calb with caspase-3. Most importantly, the comparison of CR and Calb domain organizations questions the value of homologous modeling of EF-hand proteins, and perhaps of other protein families.
Palczewska
Małgorzata
M
Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland. bromba@nencki.gov.pl
Groves
Patrick
P
Batta
Gyula
G
Heise
Bert
B
Kuźnicki
Jacek
J
D000818:Animals; D064032:Calbindin 2; D064026:Calbindins; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D020832:EF Hand Motifs / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D064030:S100 Calcium Binding Protein G / Q000737:chemistry*
D064032:Calbindin 2; D064026:Calbindins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G