Peptide and metal ion-dependent association of isolated helix-loop-helix calcium binding domains: studies of thrombic fragments of calmodulin.
Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium-binding regulatory protein, has been cleaved by thrombin to create two fragments. TM1 (1-106) and TM2 (107-148). NMR and CD results indicate that TMI and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix-loop-helix domains, but rather within an alpha-helix. Cadmium-113 NMR results show that this complex has enhanced metal-ion binding properties when compared to either TM1 or TM2 alone. This complex can bind several CaM-binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl-13C-Met-labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex. Cadmium-113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine. However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM-dependent enzymes. This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage. The results presented here reflect the importance of the existence of helix-loop-helix Ca2+-binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.
Brokx
R D
RD
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Vogel
H J
HJ
D000818:Animals; D002104:Cadmium / Q000737:chemistry; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D007477:Ions; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011522:Protons; D013917:Thrombin / Q000737:chemistry; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology
D002147:Calmodulin; D007477:Ions; D008024:Ligands; D010455:Peptides; D011522:Protons; D002104:Cadmium; D014508:Urea; D013917:Thrombin; D002122:Calcium Chloride; D002118:Calcium