Chen Baowei, Squier Thomas C, Bigelow Diana J
Cell Biology Group, Biological Sciences Division, Fundamental Science Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
Biochemistry. 2004 Apr 13;43(14):4366-74. doi: 10.1021/bi0356350.
High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 degrees involving the nucleotide-binding (N) domain is required to permit the transfer of the gamma-phosphoryl group of ATP to Asp(351) in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientations of the N domain relative to the P domain in the different crystal structures of the Ca-ATPase following calcium activation and the structures of other P-type ATPases suggests the presence of conformational heterogeneity in solution, which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer to measure the spatial separation and conformational heterogeneity between donor (i.e., 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid) and acceptor (i.e., fluorescein 5-isothiocyanate) chromophores covalently bound to the P and N domains, respectively, within the Ca-ATPase stabilized in different enzymatic states associated with the transport cycle. In comparison to the unliganded enzyme, the spatial separation and conformational heterogeneity between these domains are unaffected by enzyme phosphorylation. However, calcium activation results in a 3.4 A increase in the average spatial separation, from 29.4 to 32.8 A, in good agreement with the 4.3 A increase in the distance estimated from high-resolution structures where these sites are respectively separated by 31.6 A (1IWO.pdb) and 35.9 A (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. These results suggest that the approximation of cytoplasmic domains accompanying calcium transport, as observed from crystal structures, occurs in solution within the context of large amplitude domain motions important for catalysis. We suggest that these domain motions enhance the rates of substrate (ATP) access and product (ADP) egress and the probability of a productive juxtaposition of the gamma-phosphoryl moiety of ATP with Asp(351) on the phosphorylation domain to facilitate enzyme phosphorylation and calcium transport.
在两种构象下获得的钙 - ATP酶的高分辨率晶体结构表明,在偶联钙转运过程中,需要核苷酸结合(N)结构域进行大约50度的大规模刚体结构域重排,以使ATP的γ - 磷酰基转移至磷酸化(P)结构域中的天冬氨酸(Asp351)。然而,在钙激活后钙 - ATP酶的不同晶体结构以及其他P型ATP酶中,N结构域相对于P结构域的取向存在变异性,这表明溶液中存在构象异质性,这种异质性可能受到晶体内接触相互作用的调节。因此,为了研究溶液中这些结构域之间构象异质性的程度,我们利用荧光共振能量转移来测量分别共价连接到处于与转运循环相关的不同酶状态的钙 - ATP酶的P和N结构域上的供体(即5 - [[2 - [(碘乙酰基)氨基]乙基]氨基]萘 - 1 - 磺酸)和受体(即异硫氰酸荧光素)发色团之间的空间距离和构象异质性。与未结合配体的酶相比,这些结构域之间的空间距离和构象异质性不受酶磷酸化的影响。然而,钙激活导致平均空间距离增加3.4 Å,从29.4 Å增加到32.8 Å,这与从高分辨率结构估计的距离增加4.3 Å非常吻合,在这些高分辨率结构中,这些位点分别相距31.6 Å(1IWO.pdb)和35.9 Å(1EUL.pdb)。因此,晶体结构准确地反映了钙 - ATP酶的平均溶液结构。这些结果表明,从晶体结构观察到的伴随钙转运的细胞质结构域的近似情况,发生在溶液中对于催化至关重要的大幅度结构域运动的背景下。我们认为这些结构域运动提高了底物(ATP)进入和产物(ADP)排出的速率,以及ATP的γ - 磷酰基部分与磷酸化结构域上的天冬氨酸(Asp351)有效并列的概率,以促进酶的磷酸化和钙转运。