PDBsum entry 2zbe

Hydrolase

PDB id: 2zbe

Contents

Protein chains

995 a.a.

Ligands

BEF ×2

Metals

_MG ×2

References listed in PDB file

Key reference

Title

How processing of aspartylphosphate is coupled to lumenal gating of the ion pathway in the calcium pump.

Authors

C.Toyoshima, Y.Norimatsu, S.Iwasawa, T.Tsuda, H.Ogawa.

Ref.

Proc Natl Acad Sci U S A, 2007, 104, 19831-19836. [DOI no: 10.1073/pnas.0709978104]

PubMed id

18077416

Abstract

Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is the best-studied member of the P-type or E1/E2 type ion transporting ATPases. It has been crystallized in seven different states that cover nearly the entire reaction cycle. Here we describe the structure of this ATPase complexed with phosphate analogs BeF(3)(-) and AlF(4)(-) in the absence of Ca(2+), which correspond to the E2P ground state and E2 approximately P transition state, respectively. The luminal gate is open with BeF(3)(-) and closed with AlF(4)(-). These and the E1 approximately P.ADP analog crystal structures show that a two-step rotation of the cytoplasmic A-domain opens and closes the luminal gate through the movements of the M1-M4 transmembrane helices. There are several conformational switches coupled to the rotation, and the one in the cytoplasmic part of M2 has critical importance. In the second step of rotation, positioning of one water molecule couples the hydrolysis of aspartylphosphate to closing of the gate.

Figure 3.
Fig. 3. Cross-sections of the transmembrane region of Ca^2+-ATPase. van der Waals surfaces of E1·AlF ·ADP (a) and E2·BeF (–TG) (b). Red colors represent acidic residues. Dotted circles in b indicate the positions of Ca^2+ observed in E1·AlF ·ADP (a). The images shown were prepared with PyMol (34).

Figure 7.
Fig. 7. A cartoon illustrating two-step rotation in the processing of aspartylphosphate and gating of the ion pathway. Small arrows indicate the movements of the TM helices. The M1–M4 (green) and A1–A3 (yellow) helices are numbered. P-D351 refers to phosphorylated Asp-351.

Secondary reference #1

Title

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 a resolution.

Authors

C.Toyoshima, M.Nakasako, H.Nomura, H.Ogawa.

Ref.

Nature, 2000, 405, 647-655. [DOI no: 10.1038/35015017]

PubMed id

10864315

Figure 2.
Figure 2: Architecture of the sarcoplasmic reticulum Ca^2+-ATPase. alpha--Helices are represented by cylinders and beta--strands by arrows, as recognized by DSSP46. Cylinders are not used for one-turn helices. Colour changes gradually from the N terminus (blue) to the C terminus (red). Three cytoplasmic domains are labelled (A, N and P). Transmembrane helices (M1-M10) and those in domains A and P are numbered. The model is orientated so that transmembrane helix M5 is parallel to the plane of the paper. The model in the right panel is rotated by 50° around M5. The M5 helix is 60 Å long and serves as a scale. Several key residues are shown in ball-and-stick, and TNP-AMP by CPK. D351 is the residue of phosphorylation. Two purple spheres represent Ca^2+ in the transmembrane binding sites. The binding sites for phospholamban (PLN)16 and thapsigargin (TG)17 are marked, as are major digestion sites for trypsin5 (T1 and T2) and proteinase K28 (PrtK). The arrow specifies the direction of view in Fig. 6b. Figure prepared with MOLSCRIPT47.

Figure 4.
Figure 4: Details of the transmembrane Ca^2+-binding sites. The refined model is superimposed with a 2|F[o]| - |F[c]| composite-omit map prepared with CNS (blue meshes, contoured at 1.5 ). The meshes in pink show omit |F[ o]| - |F[c]| map for Ca^2+ and a bound water (cut-off at 3 ). Blue spheres represent Ca^ 2+, and red spheres water molecules. Viewed roughly normal to the membrane from the cytoplasmic side (a) and parallel to the membrane (b) in stereo. The coordinations of oxygen atoms to Ca^2+ are indicated by white dotted lines, and possible hydrogen bonds stabilizing the coordination geometry by green dashed lines. The viewing direction in b is also specified in Fig. 3a. Water molecule nearest to the Ca^ 2+-binding site is marked (asterisk). Note the unwinding of helices M4 and M6 and rows of exposed carbonyl oxygen atoms along these helices in b.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #2

Title

Structural changes in the calcium pump accompanying the dissociation of calcium.

Authors

C.Toyoshima, H.Nomura.

Ref.

Nature, 2002, 418, 605-611. [DOI no: 10.1038/nature00944]

PubMed id

12167852

Figure 3.
Figure 3: Interface between the transmembrane helices (M3 -M5) and the P domain of Ca^2+-ATPase. Superimposition of the Ca^2+-bound (E1Ca^2+, violet) and thapsigargin-bound (E2(TG), light green) forms fitted with the P domain. The residues (in atom colour) represent those in E2(TG). Links between the P1 and M3 helices involve hydrogen bonds between E340 and NH of L249 (not seen) as well as OH of T247 near the top of M3. Also see Supplementary Information Animation 3.

Figure 5.
Figure 5: Conformation changes around the Ca^2+-binding sites. a, C trace and the side chains of the coordinating residues in E1Ca^2+. b, Composite omit map37 (at 1.2 ) and the model for the corresponding area in E2(TG). c, Stereo view of the composite of the models for E1Ca^2+ (violet) and E2(TG) (atom colour). The viewing direction is approximately down the M5 helix in E1Ca^2+. Two bound Ca^2+ appear as cyan spheres (a) or circles (c). Dashed lines in b show potential hydrogen bonds. Orange arrows in c show the movements of the corresponding residues during the change from E1Ca^2+ to E2(TG).

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #3

Title

Crystal structure of the calcium pump with a bound ATP analogue.

Authors

C.Toyoshima, T.Mizutani.

Ref.

Nature, 2004, 430, 529-535. [DOI no: 10.1038/nature02680]

PubMed id

15229613

Figure 3.
Figure 3: Transmembrane Ca^2+-binding sites (I and II) and the movement of the M1 helix.
a, E1 2Ca^2+; b, E1 AMPPCP; c, superimposition of E1 2Ca^2+ (violet) and E1 AMPPCP (atom colour) in stereo view. Cyan (a–c) and violet spheres (c) represent bound Ca^2+; 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 Ca^2+ and hydrogen bonds involving E309 and D800 side chains.

Figure 4.
Figure 4: Omit-annealed F[o] - F[c] map around AMPPCP at 5 sigma-(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 Mg^2+ (green) and two water molecules (red), which coordinate to the Mg^2+, 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).

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #4

Title

Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues.

Authors

C.Toyoshima, H.Nomura, T.Tsuda.

Ref.

Nature, 2004, 432, 361-368. [DOI no: 10.1038/nature02981]

PubMed id

15448704

Figure 5.
Figure 5: Details of the phosphorylation site in E2 [glyph.gif]MgF[4]^2-. In the enlarged view (b), the atomic model of aspartylphosphate taken unchanged from a related protein CheY (PDB accession code 1QMP)29 is incorporated. The blue net in a shows an omit annealed Fo -Fc map (at 5 ; temperature factor also refined) at 2.3 Å resolution. Small spheres represent water molecules (red) and Mg2+ (green). MgF[4]^2- is shown in ball-and-stick representation. Large yellow arrow indicates the expected water attack to the aspartylphosphate (P-D351). Conserved sequence motifs are shown in a. Broken lines in pink indicate likely hydrogen bonds, and those in light green coordinations of Mg2+.

Figure 6.
Figure 6: A cartoon depicting the structural changes of the Ca^2+-ATPase during the reaction cycle, based on the crystal structures in five different states.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #5

Title

Structural role of countertransport revealed in ca(2+) pump crystal structure in the absence of ca(2+).

Authors

K.Obara, N.Miyashita, C.Xu, I.Toyoshima, Y.Sugita, G.Inesi, C.Toyoshima.

Ref.

Proc Natl Acad Sci U S A, 2005, 102, 14489-14496. [DOI no: 10.1073/pnas.0506222102]

PubMed id

16150713

Figure 2.
Fig. 2. Superimposition of two E2 structures viewed in stereo. Yellow, E2(BHQ); light green, E2(TG) (PDB ID code 1IWO [PDB] ). Atomic models of TG and BHQ are shown in ball-and-stick. Transmembrane helices and three cytoplasmic domains are labeled.

Figure 7.
Fig. 7. Superimposition of the C^ -traces of E2(TG+BHQ) (green) and E1·2Ca^2+ (violet) around the Ca^2+-binding sites, viewed in stereo. Water molecules are represented by small spheres in red (E2(TG+BHQ); 1-6) and violet (E1·2Ca^2+; a-e). Ca^2+ in E1·2Ca^2+ appear as cyan spheres (marked I and II). Side chains [atom color for E2(TG+BHQ) and violet for E1·2Ca^2+] are shown for some of the residues that may be involved in moving water or binding Ca^2+. Arrows shows the movements of the transmembrane helices in the transition E1·2Ca^2+ E2(TG+BHQ). Orange broken lines represent hydrogen bonds, and cyan lines show Ca^2+-coordination.

Secondary reference #6

Title

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors.

Authors

M.Takahashi, Y.Kondou, C.Toyoshima.

Ref.

Proc Natl Acad Sci U S A, 2007, 104, 5800-5805. [DOI no: 10.1073/pnas.0700979104]

PubMed id

17389383

Figure 3.
Fig. 3. The CPA binding site in E2(CPA+CC) superimposed with that of E2(TG+BHQ) viewed in stereo approximately perpendicular to the membrane. Yellow, E2(CPA+CC); light green, E2(TG+BHQ). Cylinders represent transmembrane helices (M1–M5). The pink net represents an |F[obs]| – |F[calc]| electron density map at 2.8-Šresolution contoured at 4 before CPA (shown in ball-and-stick) was introduced into the atomic model. Dotted lines show likely hydrogen bonds. The atomic model is shown in stick representation for E2(CPA+CC). Side chains of Asn-101 and Gln-56 also are shown for E2(TG+BHQ).

Figure 6.
Fig. 6. Differences in orientation of the transmembrane helices in three crystal structures. E2(CPA) (yellow), E2(TG+BHQ) (light green), and E2·MgF (cyan) are superimposed with the M7–M10 helices and are shown in stereo in the same direction as in Fig. 1. M2, M3, M5, and M7 are represented with two to three cylinders, although they are continuous helices. Some of the residues used in helix–helix contacts are shown.