anchor) domain, (2) the N (nucleotide - binding) domain, and (3) the P (phos-
phorylation) domain. See Figure 6.29. Domain A is the smallest of these. It
contains approximately 110 residues, 1 – 43, the N - terminal end of the enzyme,
and 124 – 235 falling in sequence between M2 and M3. Domain P is comprised
of residues 330 – 359 and 601 – 739. The M4 and M5 helices are integrated into
the P domain as part of a Rossmann fold, a well - known protein motif contain-
ing (usually) a seven - stranded central parallel β - sheet associated with seven
surroundingα - helices. The P domain rotates toward the A domain in passing
from the E1Ca 2+ to E2(TG) states. The N domain, residues 360 – 600, also
changes position during the E1Ca 2+ to E2(TG) transition. Domain A rotates
and changes position in each of the X - ray and NMR structures known for the
Ca2+ - ATPase cycle and is thought to help control access to the opposite cyto-
solic and lumenal sides of the membrane. In the E1Ca 2+ enzyme (PDB: 1SU4)
the domains A, N, and P reside in an “ open ” position. On going to the E2(TG)
structure, the A domain rotates 110 ° , closing the cytoplasmic gate of the
enzyme.
The next discussion will center on changes that take place on moving from
the Ca 2 E 1 state to the Ca 2 E 1 · ATP bound state. In the Ca 2 E 1 state, there does
not appear to be a relationship between the N domain (nucleotide - binding)
and the P domain that contains the phosphorylation site at asp351. Upon addi-
tion of nucleotide, in this case the inactive ATP mimic called AMPPCP [aden-
soine ( β – γ - methylene) - triphosphate (PDB: 1T5S)^95 ], N loop region residues
576 – 581 form a nucleotide - binding crevice with P loop region residues 720 –
- Domain A rotates by approximately 45 ° to form a lid on one side of this
nucleotide - binding pocket. This is illustrated in Figure 2 of reference 95. As
the M2 helix continues as aβ - strand into the A domain, the rotation after
nucleotide binding pulls the M1 – M2 helix pair toward the cytoplasm. Helices
M1 – M2 are lifted by 8 – 9 Å relative to helices M3 – M10. A supplemental movie
to reference 95 illustrates the changes ( http://www.sciencemag.org/content/
vol304/issue5677/images/data/1672/DC1/1099366s1.mov ). In the movie, domain
A appears in yellow, domain N in red, domain P in blue, and membrane helices
M1 and M2 in magenta. A well - conserved amino acid sequence in Ca 2+ -
ATPases, TGES, starts at thr181 in domain A. This motif helps anchor the A
domain and M1 – M3 to the P domain, and it is important for carrying out the
phosphorylation of asp351 in the P domain. The N domain, residues 360 – 600
of the enzyme, binds the ATP molecule that enters from the cytoplasm. This
ATP will be hydrolyzed to ADP, providing the energy for the release of
calcium ions to the lumen. In going from Ca 2 E 1 (PDB: 1SU4) to Ca 2 E 1 · ATP
(PDB: 1T5S) the N domain rotates by 90 ° and closes with the P domain. The
ATP analog binds in a highly conserved nucleotide - binding pocket between
the N and P domains. Three arginine residues, arg489 and arg560 of the N
domain and arg687 of the P domain, stabilize the nucleotide in a bent confor-
mation.^95 The magnesium ion accompanying AMPPCP interacts with its γ -
phosphate as well as with side chains of thr353 and thr625 of the P domain
and with the main chain amide of gly626 (P domain).
PHOSPHORYL TRANSFER: P-TYPE ATPASES 333