Interchange of autoinhibitory domain conformations in plasma-membrane Ca2+-ATPase-calmodulin complexes.
The Ca2+ signaling protein calmodulin (CaM) stimulates Ca2+ pumping in the plasma-membrane Ca2+-ATPase (PMCA) by binding to an autoinhibitory domain, which then dissociates from the catalytic domain of PMCA to allow full activation of the enzyme. We measured single-molecule fluorescence trajectories with polarization modulation to track the conformation of the autoinhibitory domain of PMCA pump bound to fluorescently labeled CaM. Interchange of the autoinhibitory domain between associated and dissociated conformations was detected at a physiological Ca2+ concentration of 0.15 microM, where the enzyme is only partially active, but not at 25 microM, where the enzyme is fully activated. In previous work we showed that the conformation of the autoinhibitory domain in PMCA-CaM complexes could be monitored by the extent of modulation of single-molecule fluorescence generated with rotating excitation polarization. In the present work, we determined the timescale of association and dissociation of the autoinhibitory domain with the catalytic regions of the PMCA. Association of the autoinhibitory domain was rare at a high Ca2+ concentration (25 microM). At a lower Ca2+ concentration (0.15 microM), conformations of the autoinhibitory domain interchanged with a dissociation rate of 0.042 +/- 0.011 sec(-1) and an association rate of 0.023 +/- 0.006 sec-1. The results indicate that the response time of PMCA upon a reduction in Ca2+ is limited to tens of seconds by autoinhibitory dynamics. This property may reduce the sensitivity of PMCA to transient reductions in intracellular Ca2+. We suggest that the dynamics of the autoinhibitory domain may play a novel role in regulating PMCA activity.
Mandal
Abhijit
A
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Liyanage
Mangala Roshan
MR
Zaidi
Asma
A
Johnson
Carey K
CK
D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012235:Rhodamines / Q000737:chemistry
D002147:Calmodulin; D005456:Fluorescent Dyes; D012235:Rhodamines; C005358:tetramethylrhodamine; D053499:Plasma Membrane Calcium-Transporting ATPases