Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase upon Calcium Binding

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• 作者：Baowei Chen ; James E. Mahaney ; M. Uljana Mayer ; Diana J. Bigelow ; Thomas C. Squier
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：November 25, 2008
• 年：2008
• 卷：47
• 期：47
• 页码：12448-12456
• 全文大小：331K
• 年卷期：v.47,no.47(November 25, 2008)
• ISSN：1520-4995

文摘

Calcium-dependent domain movements of the actuator (A) and nucleotide (N) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH-EDT₂). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT₂. Distance measurements using the nucleotide analog 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP), which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 Å increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound, respectively, to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both A- and N-domains display virtually identical calcium dependencies (i.e., K_d = 4.8 ± 0.4 μM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates phosphoenzyme formation from ATP upon occupancy of the second high-affinity calcium site.