The right - hand representation in reference 101 , Figure 1, without calcium
ion and with thapsigargin to stabilize the calcium binding site, is labeled
E2(TG). Its X - ray crystallographic structure is deposited as PDB: 1IWO.^98
Figure 1 of reference 98 shows the movement of the N, P, and A domains as
the Ca 2+ - ATPase molecule moves from the E1Ca 2+ to the E2(TG) state. The
E1 label refers to the high Ca 2+ affi nity state in the Ca 2+ - ATPase cycle (Figure
6.28 ), whereas the E2 label refers to the low Ca 2+ affi nity state of the enzyme.
In E2(TG), TG refers to thapsigargin, a Ca 2+ - ATPase cycle inhibitor believed
to fi x the enzyme in an E2 form. Ca 2+ - ATPase denatures in its calcium - free
form without the presence of such an inhibitor. Signifi cant shifts in position
and orientation of the transmembrane helices take place between the two
states, along with as large movements of the enzyme domains that protrude
into the cytoplasm. First, we will discuss the M1 – M10 helical domains of PDB:
1SU4 and 1IWO, followed by discussion of the cytoplasmic domains.
Figure 1 of reference 98 represents a detailed diagram of the E2(TG) (E 2
in Figure 6.28 ) and E1Ca 2+ (Ca 2 E 1 in Figure 6.28 ) structures that will be helpful
in understanding the differences described below. Figure 6.29 represents the
Ca 2 E 1 state. The Ca 2+ - ATPase enzyme portion residing in the membrane con-
sists of 10α - helices, usually referred to in the literature as helices M1 – M10.
The M1 – M2 helical domain is comprised of enzyme aa residues 44 – 123. The
M1α - helix contains an amphipathic (containing both polar and nonpolar
domains) sector (M1 ′ ) thought to interface between the cytoplasm and mem-
brane. The orientation of M1 ′ changes substantially between the E2(TG)
structure and that of E1Ca 2+ (E 1 in Figure 6.28 ). In addition, M1 contains a
glu58 (E58) and an asp59 (D59), either or both of which are thought to help
in guiding calcium ions to their binding location within the membrane portion
of Ca 2+ - ATPase. Glu309 (E309) of M4 is another acidic residue that may have
access to the cytoplasm and assist calcium ions entering the enzyme. The
calcium ions enter the enzyme sequentially and cooperatively; also, the fi rst
calcium ion occupies Site I, followed by the second in Site II. M1 and M2 move
upwards toward the cytoplasm in moving from E1Ca 2+ (E 1 in Figure 6.28 ) to
other states in the cycle. The M3 – M6 helical region, residues 239 – 329 and
740 – 821, contains many of the calcium ions ligands. In passing from the E1Ca 2+
state to the E2(TG) state, M4 moves toward the luminal (extracellular) side
by one turn of anα - helix, the upper part of M5 moves toward M4, and the
unwound part of M6 rotates by nearly 90 °.^102 All of these movements have the
effect of decreasing the availability of coordinating oxygen ligands for Ca 2+.^103
Helices M4 – M6 (moving during the E1Ca 2+ to E2(TG) states) and M8 (unmov-
ing) contain the ligands binding Ca 2+ to the enzyme. Membrane helices M7 –
M10, residues 822 – 994 are essentially unchanged during the E1Ca 2+ to E2(TG)
transition. Site I calcium ions ligands are side - chain oxygens of asn768 (N768)
and glu771 (E771) of M5, thr799 (T799) and asp800 (D800) of M6, glu908
(E908) of M8, and two water molecules. Site II ligands include a second side -
chain carboxyl oxygen of asp800 plus side - chain oxygens donated by asn796
(N796) of M6 and glu309 (E309) of M4 as well as main - chain carbonyl oxygen
PHOSPHORYL TRANSFER: P-TYPE ATPASES 331