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.