Abstract
The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2b isoform possesses an extended C terminus (SERCA2b tail) forming an 11th transmembrane (TM) helix, which slows conformational changes of the Ca2+-pump reaction cycle. Here, we report that a Darier disease (DD) mutation of SERCA2b that changes a glutamate to a lysine in the cytoplasmic loop between TM8 and TM9 (E917K) relieves these kinetic constraints. We analyzed the effects of this mutation on the overall reaction and the individual partial reactions of the Ca2+ pump compared with the corresponding mutations of the SERCA2a and SERCA1a isoforms, lacking the SERCA2b tail. In addition to a reduced affinity for Ca2+, caused by the mutation in all three isoforms examined, we observed a unique enhancing effect on the turnover rates of ATPase activity and Ca2+ transport for the SERCA2b E917K mutation. This relief of kinetic constraints contrasted with inhibitory effects observed for the corresponding SERCA2a and SERCA1a (E918K) mutations. These observations indicated that the E917K/E918K mutations affect the rate-limiting conformational change in isoform-specific ways and that the SERCA2b mutation perturbs the interactions of TM11 with other SERCA2b regions. Mutational analysis of an arginine in TM7 that interacts with the glutamate in SERCA1a crystal structures suggested that in wildtype SERCA2b, the corresponding arginine (Arg-835) may be involved in mediating the conformational restriction by TM11. Moreover, the E917K mutation may disturb TM11 through the cytoplasmic loop between TM10 and TM11. In conclusion, our findings have identified structural elements of importance for the kinetic constraints imposed by TM11.
Highlights
The sarco(endo)plasmic reticulum Ca2؉-ATPase (SERCA) 2b isoform possesses an extended C terminus (SERCA2b tail) forming an 11th transmembrane (TM) helix, which slows conformational changes of the Ca2؉-pump reaction cycle
The expression levels of these mutants in the COS-1 cells were significantly lower than the expression levels of the respective wildtypes, and more so for the SERCA2 mutants (ϳ5- and 10-fold reduced for SERCA2b and SERCA2a, respectively) than for the SERCA1a mutant (2-fold reduced)
In agreement with previous findings [11, 15, 20], the present data show that the maximal turnover rate of ATPase activity of wildtype SERCA2b is only about one-third that of the SERCA1a wildtype, and the same is the case for the maximal turnover rate of the ATP-dependent active Ca2ϩ uptake in the microsomal vesicles determined under conditions of saturation of the highaffinity Ca2ϩ-binding sites (5 M free Ca2ϩ, Fig. 1, and Table 1)
Summary
The sarco(endo)plasmic reticulum Ca2؉-ATPase (SERCA) 2b isoform possesses an extended C terminus (SERCA2b tail) forming an 11th transmembrane (TM) helix, which slows conformational changes of the Ca2؉-pump reaction cycle. In addition to a reduced affinity for Ca2؉, caused by the mutation in all three isoforms examined, we observed a unique enhancing effect on the turnover rates of ATPase activity and Ca2؉ transport for the SERCA2b E917K mutation This relief of kinetic constraints contrasted with inhibitory effects observed for the corresponding SERCA2a and SERCA1a (E918K) mutations. The X-ray diffraction analysis of SERCA1a in conjunction with site-directed mutagenesis studies has defined six residues with oxygen-containing side chains in the transmembrane segments TM4, TM5, TM6, and TM8 as Ca2ϩ ligands at two high-affinity Ca2ϩ transport sites [17, 21,22,23,24] These residues are conserved among the SERCA isoforms, and all are believed to possess Ca2ϩ-binding sites similar to those of SERCA1a. SERCA2b is characterized by significantly lower rates of the major rate-limiting conformational changes of the enzyme cycle compared with the other isoforms, which may be ascribed to the presence of the SERCA2b tail [15, 20]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
