The predicted secondary structures of the nucleotide‐binding sites of six cation‐transporting ATPases lead to a probable tertiary fold

Abstract

Six cation-dependent transporting ATPases have homologous sequences in the region assigned by chemical labelling to nucleotide binding. Comparison of the most highly conserved segments with other nucleotide-binding domains showed that the sequences were consistent with a mononucleotide-binding fold and enabled a number of likely folding topologies to be limited to two or three alternatives. One of these possible folds was topologically equivalent to adenylate kinase; this was taken as a model in which the significance of conserved amino acids was investigated. In this model conserved amino acids were grouped around a postulated ATP-binding cleft, satisfactorily accounting for their degree of conservation.The recent determination of the amino acid sequences of six cation-transporting ATPases has shown high similarity (30 -50% identity) in limited regions accounting for about one quarter of the molecule [I]. One of the best defined domains, which follows the central tryptic cleavage site of the Ca2 + -ATPase, is labelled by fluorescein isothiocyanate (FITC) in competition with ATP [2-41. Since erythrosin (tetraiodofluorescein) is known to occupy the adenine pocket of the coenzyme site of lactate dehydrogenase, we can regard FITC as a potential affinity label for the ATP-binding site.The nucleotide domain (98 -180 residues long) forms part of the largest extra-membranous region in all the ATPases (280-430 residues). It is situated between two other conserved domains within this region and separated from them by loops of varying length and sequence. In our preliminary analysis of the secondary structure of two Ca'+-transporting ATPases from sarcoplasmic reticulum [5, 61, we concluded that this region cosisted mainly of alternating p-strands and a-helices and that it probably formed a parallel-stranded psheet structure analogous to that of other nucleotide-binding domains [7]. The domain on the N-terminal side includes an aspartyl residue which is phosphorylated by ATP bound to the nucleotide domain. The segment on the C-terminal side is a short (64 residues) but highly conserved region which, by virtue of its direct connection to the membrane must be close to the N-terminal phosphorylation domain. By analogy with known structures of several kinases [8] we assigned a hinge-I like role to this C-terminal domain.Most nucleotide-binding domains contain parallel psheets which, in spite of marked structural differences, have Correspondence to N. M. Green,

Effect of Hailey-Hailey Disease Mutations on the Function of a New Variant of Human Secretory Pathway Ca2+/Mn2+-ATPase (hSPCA1)

“…It has been proposed that Thr-625 in SERCA1a, which is an integral part of the highly conserved TGD motif, might participate in the formation of the nucleotide-binding site (39). Conservative mutations in Thr-625 were shown to result in a Ca 2ϩ transport deficiency and impaired abilities to form a phosphorylated intermediate with ATP or P i (40).…”

Kinetic characterization of Na,K-ATPase inhibition by Eosin

“…This cytoplasmic segment is 430 amino acids in length of which, 180 residues comprise the ATP binding domain [9]. As hypothesized, the M4M5 cytoplasmic loop contains the nucleotide-binding site as evidenced by several studies utilizing chemical modification to inhibit ATP hydrolysis.…”

Prediction of the structural motifs of sandwich proteins

“…Another interesting approach to this problem is based on the analysis of the folding mechanism of ␤ proteins (5): it has been shown that the number of permissible topologies depends on the number of ␤-strands and on the folding pathway. Furthermore, a number of regularities for ␤-sheet motifs, such as the nonexistence of knots in the chain, has been discovered (15)(16)(17)(18)(19)(20). It has also been shown that loops do not cross (14,21).…”