The
comparison of the loop structures of membrane binding sites between human and bovine
annexins IV
Michiko Konno, Yae Kanzaki, Kayoko
Mochizuki, Nahomi Fushinobu, Ayano Sato, Kyoko Aikawa and Isamu Matsumoto
Department of Chemistry, Ochanomizu
University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610 Japan (konno@cc.ocha.ac.jp)
Annexins are a ubiquitous family of structurally related eukaryotic
proteins capable of binding phospholipid membranes in calcium-dependent manner [1]. Membrane binding properties of these
proteins have suggested that they are involved in numerous processes such as
exocytosis, endocytosis, vesicular trafficking, membrane fusion, and
ion-channel formation. It has been reported that
annexin I, II and IV proteins aggregate synthetic phospholipid vesicles and
chromaffin granules. The mRNA of human annexin IV is highly expressed in normal
pancreas, lung and kidney and also in various adenocarcinoma cell lines. On the
basis of idea that a little structural difference between the loops of
annexins, which interact with membranes, should reflect a little difference of
circumstances within vesicles, in order to compare in detail with loop structures
of membrane binding sites of reported bovine annexins IV, we have determined
the crystal structure of recombinant human annexin IV.
Recombinant human annexin IV was crystallized at 20 oC by
a hanging drop vapor diffusion method. Crystals were grown from a drop of
6mg/ml protein, 6 % PEG 6000, 1.5mM CaCl2, 3 % dioxane, 50mM
tris-HCl (pH 7.5) equilibrated against a reservoir of 20 % PEG 6000, 20 mM CaCl2,
6 % dioxane, and 100 mM tris-HCl (pH7.5). X-ray data were collected at 100 K at
Photon Factory (Tsukuba). Data were processed and scaled using DENZO and
SCALEPACK to give Rmerge = 5.1 % and completeness = 92.1 % at 2.0 resolution.
Crystals adopt the space group P21, with two molecules per
asymmetric unit. Lattice constants are a = 44.223 , b = 55.132 , c = 127.147
, b
= 91.45o. The structure was solved by molecular replacement using as
a search model the structure of bovine annexin IV (Protein DataBank accession
no. 1ANN) and was refined to Rfactor = 23.3 %, Rfree =
31.1 % at 2.0 using XPLOR.
In recombinant human annexin a
C-terminal core has four domains each which fold into five a-helices and an
N-terminal tail region of fifteen residues (MAMATKGGTVKAASG) lies across the concave protein surface. One calcium ion is bound in domain II, two in domain III and one
in domain IV. Three Ca2+ ions are bound in type II-binding sites of
AB loop and one in type III-binding site of DE loop. The calcium binding groups
of AB loop of domain I coordinate e-amino group of Lys99 of adjacent molecule. On the other hand, in bovine annexin IV two Ca2+ ions are bound in the AB loops of domains I and IV
[2]. The conformation of the AB loop of 184GEKKWGTDEV193 of domain III
bound Ca2+ ion of human annexin IV is different largely from that of
the corresponding loop of the same residues without Ca2+ ion in
bovine annexin IV. In the former the side chain of Trp188 is intruded out,
while in the latter that of Trp185 adopts a buried conformation. In human
annexin IV the AB loop of domain II is ordered and binds Ca2+ ion, while
in bovine annexin IV that is totally disordered and does not any binds Ca2+
ion.
References
1
Gerke, V. and
Moss, S. E. (2001) Physiol Rev. 82, 331-371.
2
Sutton, R.B., and
Sprang, S.R. (1996) Annexin: molecular structure to cellular function (Seaton, B., Ed) pp 31-42, R.G.Landes Company,
Austin, TX.