Troponin C/calmodulin chimeras as erythrocyte plasma membrane Ca2+-ATPase activators.
Calmodulin (CaM) and troponin C (TnC) are EF-hand proteins that play fundamentally different roles in animal physiology. TnC has a very low affinity for the plasma membrane Ca2+-ATPase and is a poor substitute for CaM in increasing the enzyme's affinity for Ca2+ and the rate of ATP hydrolysis. We use a series of recombinant TnC (rTnC)/CaM chimeras to clarify the importance of the CaM carboxyl-terminal domain in the activation of the plasma membrane Ca2+-ATPase. The rTnC/CaM chimera, in which the carboxyl-terminal domain of TnC is replaced by that of CaM, has the same ability as CaM to bind and transmit the signal to Ca2+ sites on the enzyme. There is no further functional gain when the amino-terminal domain is modified to make the rTnC/CaM chimera more CaM-like. To identify which regions of the carboxyl-terminal domain of CaM are responsible for these effects, we constructed the chimeras rTnC/3CaM and rTnC/4CaM, where only one-half of the C-terminal domain of CaM (residues 85-112 or residues 113-148) replaces the corresponding region in rTnC. Neither rTnC/3CaM nor rTnC/4CaM can mimic CaM in its affinity for the enzyme. Nevertheless, with respect to the signal transduction process, rTnC/4CaM, but not rTnC/3CaM, shows the same behaviour as CaM. We conclude that the whole C-terminal domain is required for binding to the enzyme while Ca2+-binding site 4 of CaM bears all the requirements to increase Ca2+ binding at PMCA sites. Such mechanism of binding and activation is distinct from that proposed for most other CaM targets. Furthermore, we suggest that Ala128 and Met124 from CaM site 4 may play a crucial role in discriminating CaM from TnC.
Fidalgo da Silva
Elizabeth
E
Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21941-590 Rio de Janeiro, RJ, Brazil.
Freire
Mônica M
MM
Barrabin
Hector
H
Sorenson
Martha M
MM
Tikunova
Svetlana
S
Johnson
J David
JD
Chandra
Murali
M
Pearlstone
Joyce R
JR
Scofano
Helena M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D013552:Swine; D019209:Troponin C / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D019209:Troponin C; D000252:Calcium-Transporting ATPases; D002118:Calcium