within an approximate 2 - Å bonding distance of the asp351. The nearly linear
arrangement of the AlF 4 −’s aluminum ion and asp351 carboxyl moieties suggest
an associative nucleophilic reaction mechanism (S N 2) for the phosphoryl
transfer involving a pentavalent phosphorus intermediate. Side chains of two
aa residues, thr353 and thr625, form hydrogen bonds to the β - phosphate and
the AlF 4 −, respectively, and the main chain amides of gly626 and thr353 form
hydrogen bonds to the AlF 4 −, further stabilizing the structure. In the PDB:
1T5T structure, two calcium ions are occluded. The reference 95 authors
believe that changes in the A domain have aligned hydrophobic residues in
membrane helices M1 (phe57, val62, leu65) and M2 (ile94, leu97, leu98) to
close off Ca 2+ ion access to the cytoplasm — the occluded state.
The Toyoshima group has determined the structure of a Ca 2+ - ATPase that
they believe mimics the Ca 2 E 1 P · ADP state as well.^100 This structure, PDB:
1WPE, contains an AlF 3 · ADP molecule, two calcium ions, and two magnesium
ions. In agreement with the reference 95 work, the structure is very similar to
the Ca 2 E 1 · ATP model (PDB: 1T5S). The P domain is hardly altered from the
Ca 2 E 1 · ATP structure, and the N and P domains are strongly associated to hold
the ADP molecule in place. The important TGES motif in the A domain,
beginning at thr181 and continuing through gly182, glu183, and serine 184,
participates in hydrogen bonding near the phosphorylation site. Glu183 is an
especially important residue, hydrogen bonding through a water molecule to
the phosphorylation residue asp 351. The TGES motif may also participate in
holding (or releasing) ADP. A movie included as supplemental information
with reference 100 illustrates many of the points being discussed here. The
movie can be obtained as supplemental information in the reference 100 citing
available at http://www.nature.com/nature.
The next structures describe the transition from Ca 2 E 1 P · ADP to the E 2 P
and the E 2 P i states. During this transition, calcium ions have been released to
the lumen and ADP has been released as well. Two models have been described.
The structure described in reference 101 has been deposited in the PDB as
1XP5 and most closely models the E 2 P state, whereas the Toyoshima group
structure,^100 PDB: 1WPG, models E 2 P i. The E 2 P state is called the ADP - insensi-
tive state. This state is incapable of regenerating ATP, because the enzyme has
reconfi gured itself once again to allow the release of ADP. In the dephosphory-
lation transition state depicted by the PDB: 1XP5 structure, calcium ions are
no longer occluded and proton counterions have found their way into the
enzyme from the lumen. The PDB: 1XP5 structure contains a tetrafl uoroalu-
minate ion, AlF 4 −, a potassium ion, a magnesium ion, and a thapsigargin mol-
ecule, necessary for stabilizing the now empty calcium ion sites. Release of
ADP upon phosphoryl transfer initiates an approximate 50 ° rotation of the N
domain relative to the P domain. This, in turn, enables the A domain to rotate
108 ° and shift 8 Å around the P domain bringing the TGES motif in apposition
to the phosphorylation site. Connections of domain A to the membrane helices
M1 – M3 effect large changes in their orientation and position. The M1 – M3
movements affect the orientation of M4 through M6 relative to M7 – M10 that
PHOSPHORYL TRANSFER: P-TYPE ATPASES 335