具有一个结合的非水溶性ATP对应体的钙离子ATP酶的晶体结构（分辨率为2.9埃）的发表，标志着结构生物学中一个篇章的完成。现在，这一重要钙泵工作周期中三大中间体的结构也已知道，对其作用模式进行详细解析成为可能。由ATP提供动力的P-型泵确定穿越细胞和细胞器膜的离子浓度梯度，所以在很多生物学过程中起中心作用。
Crystal structure of the calcium pump with a bound ATP analogue
P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across cell and organelle membranes. Here, we describe the crystal structure of the Ca2+ pump of skeletal muscle sarcoplasmic reticulum, a representative member of the P-type ATPase superfamily, with an ATP analogue, a Mg2+ and two Ca2+ ions in the respective binding sites. In this state, the ATP analogue reorganizes the three cytoplasmic domains (A, N and P), which are widely separated without nucleotide, by directly bridging the N and P domains. The structure of the P-domain itself is altered by the binding of the ATP analogue and Mg2+. As a result, the A-domain is tilted so that one of the transmembrane helices moves to lock the cytoplasmic gate of the transmembrane Ca2+-binding sites. This appears to be the mechanism for occluding the bound Ca2+ ions, before releasing them into the lumen of the sarcoplasmic reticulum.
Figure 1 Front views (parallel to the membrane (x–y) plane) of Ca2+-ATPase with (E1AMPPCP) and without (E12Ca2+) AMPPCP (shown in space fill), an ATP analogue, in the presence of 10 mM Ca2+. Colours change gradually from the amino terminus (blue) to the carboxy terminus (red). Purple spheres (circled) represent bound Ca2+. Large arrows in E12Ca2+ indicate the direction of movements of the cytoplasmic domains (A, N and P) in the E12Ca2+ E1AMPPCP transition; the axis of tilting of the A-domain is also specified (thin red line). -Helices in the A-domain (A1 and A2) and those in the transmembrane domain (M1, M4, M5 and M10) are indicated. M1' is an amphipathic part lying on the bilayer surface. PtrK, a proteinase-K digestion site (around Glu 243 (ref. 16)); SR, sarcoplasmic reticulum; T2, a trypsin digestion site at Arg 198 (ref. 10); ATP, the binding pocket for the adenosine moiety of ATP. Several key residues—E183 (A), F487 (N, adenine binding), D351 (P, phosphorylation site), R560 (N, -phosphate binding) and those involved in interdomain hydrogen bonds (including T171 and E486)—are shown in ball-and-stick representation. Dotted circles indicate three contact spots between A- and N-domains (magenta) and between A- and P-domains (yellow). The figure was prepared with Molscript44. Inset is a simplified reaction scheme (showing only the forward direction); the two states compared here are shown with a yellow backgrou.
Figure 2 Superimposition of the E12Ca2+ and E1AMPPCP forms of Ca2+-ATPase fitted with the transmembrane domain. E12Ca2+, violet; E1AMPPCP, cyan (A-domain and M1–M3 helices), light green (N-domain) and orange (P-domain and M4–M10 helices). -Helices are represented by cylinders and -strands by arrows. Both (a and b) are viewed along the membrane plane, but at a difference of 45°. M2, M3 and M5 are represented by two or three cylinders, although they are continuous helices. Helices in the A-domain (A1 and A2), P-domain (4–7) and transmembrane helices are indicated. The position of a preserved hydrogen bond between the A- and P-domains (Gly 156–Ala 725) is indicated by a double circle for E12Ca2+. Large arrows indicate the direction of movements of the cytoplasmic domains (A, N and P) in the E12Ca2+ E1AMPPCP transition, and small ones those of transmembrane helices. Broken lines enclose the A-domain (a) and the N-domain (b). A thin blue line in b specifies the axis of tilting of the A-domain.
Figure 3 Transmembrane Ca2+-binding sites (I and II) and the movement of the M1 helix. a, E12Ca2+; b, E1AMPPCP; c, superimposition of E12Ca2+ (violet) and E1AMPPCP (atom colour) in stereo view. Cyan (a–c) and violet spheres (c) represent bound Ca2+; red spheres indicate water molecules in the crystals. Owing to the resolution limitation of the diffraction data, no water molecules are shown in b. Dotted lines in c show the coordination of Ca2+ and hydrogen bonds involving E309 and D800 side chains.
Figure 4 Omit-annealed Fo - Fc map around AMPPCP at 5 (a) and the hydrogen-bonding network around AMPPCP (b). AMPPCP is shown in ball-and-stick representation; the N- and P-domains are coloured light green and orange, respectively. Light-green broken lines in b show likely hydrogen bonds. A part of the N-domain is removed for clarity. Small spheres represent Mg2+ (green) and two water molecules (red), which coordinate to the Mg2+, together with -phosphate, carboxyl groups of Asp 351 and Asp703, and a carbonyl group of Thr 353 (orange broken lines in a and dark-green lines in b).
Figure 5 Superposition of the P-domain in E12Ca2+ and E1AMPPCP, fitted with the 15 residues at the N-terminal end of the M5 helix. a, b, Top (a) and side (b) views. Colours representing E12Ca2+ and different regions of the P-domain in E1AMPPCP, as recognized by Dyndom45, are indicated. A part of the P-domain (containing helices 4 and 4') is removed. Small spheres represent Mg2+ (green) and two water molecules (red). Two long lines specify the positions of the axis of the N-domain inclination for E2(TG) (green) and E1AMPPCP (purple) as determined by Dyndom45. The green arrow in a indicates the movement of -strands 1–4 in the E12Ca2+ E1AMPPCP transition, whereas those in red show the tilting of the -helices 5–7.
Figure 6 Water-accessible surface of the cytoplasmic domains in E1AMPPCP showing the A–N interface. Inset is an enlarged view of the boxed area, where the A- and N-domains appear to be mechanically coupled.
