Motivations for CadA topology

Enterococcus hirae CopA and the special Cu²⁺ binding affinity to the N-terminal peptide of this ATPase (Bayle et al., 1998). As determined experimentally by in vitro transcription/translation method, H. pylori CopA also contains four pairs of TMs, and shares the same patterns of motifs with the predicted Helicobacter CadA.

Alignment of all the P-type ATPases suggests that the Helicobacter pumps and other homologous heavy metal ATPases have an additional pair of membrane sequences preceding the first pair of membrane segments that are present in the non-heavy metal ATPases. The

Helicobacter pumps and other similar P-type ATPases, on the other hand, do not have sequences following the TM8 (Melchers et al., 1996). The heavy metal transporting CPx-type ATPases set themselves apart from the rest of the P-type ATPases not only at the sequence aspect, but also at the membrane topology concerns (Solioz and Vulpe, 1996).

1.4 Motivations for CadA topology

Although the topological study of CPx-type ATPases has been done in Helicobacter models, some uncertainties were remained. Firstly, the experimental data presented to this topology model for this transition metal ATPase was from in vitro study and only 9 in vivo phoA fusion data in a subsequent study (Melchers et al., 1996; Melchers et al., 1999; Fig.

4). However, there are still 3 large cytoplasmic domains of this protein has not been explored, which are the N-terminal first 70 residues, and the regions ranging from amino acid positions 177 to 309 and 371 to 493.

Additionally, the third and forth TMs (TM3 and TM4) suggested in these

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studies were originally predicted to be as low hydrophobic domains, according to hydropathy programs (Fig. 2c). It seems that they were not able to traverse the lipid bilayer. In the in vitro study, the suggested TM4 of the CadA homologous ATPase was not present as completed

transmembrane domain (Melchers et al., 1996). The in vivo alkaline phosphatase fusions were too few to cover the full length of the protein including this region (TM3 to TM4) (Melchers et al., 1999). Therefore, this information would not be possible to serve as the perfect topological model for the CPx-type ATPases.

In addition to the reasons we have argued above, some differences exist between these two ATPases. From the sequence comparison data shown in Fig. 4, staphylococcal CadA has a longer N-terminal portion of amino acid sequences and more charged C-terminal portion of amino acid residues than those of the Helicobacter one. Moreover, some CPx-type ATPases including staphylococcal CadA have another putative

transmembrane domain, just behind the GDGXNDXP conserved motif, which were not predicted as a transmembrane domain in the ATPases of Helicobacter spp (Fig. 2). The similar domain was also shown in other CPx-type ATPases like CadA ATPases from S. aureus Tn554, Bacillus firmus, Listeria monocytogenes as well as PacS and CtaA from

Synechococcus spp (Chikramane and Dubin, unpublished; Ivey et al., 1992; Lebrun et al., 1994; Kanamaru et al., 1993; Phung et al., 1994).

Therefore, another membrane topology data would be necessary to clarify these suspicions.

1.4.1 Gene fusion strategy for CadA topology

Since the topography acquired from Helicobacter studies were not completed to truly represent the CPx-type ATPases, therefore, it will be necessary to re-examine the protein structure of the family. In this thesis study, we decide to use CadA as a model to further disclose the structural aspect of the CPx-type ATPase. There are many experimental techniques would be able to study the topological structure of membrane proteins, including side chain covalent modification, proteolysis, and epitope recognition (Jennings, 1989). However, these techniques are often unable to provide a full topological description of a membrane protein because of their reliance on the natural occurrence of residues that are susceptible to proteolytic cleavage, reactive side chains or accessible to antibody binding (Lee and Manoil, 1996). Another method, the in vitro

transcription/translation technique, seems amazing (Holland and

Drickamer, 1986), but difficult to reflect the intact protein folding in cell membrane because of using only those putative transmembrane fragments for detection. These limitations could be circumvented by the supplement with data from some other molecular biological methods, especially those of gene fusion strategies. The best-established gene fusion strategy for the study of this kind is the phoA- and lacZ-fusion combination method

(Manoil et al., 1990; Manoil, 1991; Fig. 3). This method has been used in many membrane topological studies, for example, the E. coli ArsB done by Wu et al. (1992), E. coli K-12 Mtr permease done by Sarsero and Pittard (1995), B. subtilis BofA done by Varcamonti et al. (1997), Lactococcal LcnC done by Franke et al. (1999), Helicobacter NixA done by Fulkerson and Mobley (2000). In this study, we take the similar strategy of using the phoA- and lacZ-fusion to determine the membrane topography of CadA for the first time.

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1.4.2 Properties of reporter enzymes

Alkaline phosphatase is a nonspecific phosphomonoesterase, which has a total of 471 amino acids including a signal sequence of 21 amino acids (Chang, 1986). It is ordinarily found in the periplasm of E. coli under the condition of phosphate starvation (Wanner 1996). If retained in the cytoplasm, alkaline phosphatase is enzymatically inactive (Michaelis 1983, Hoffman 1985), due to the inability of forming disulfide bonds (Derman and Beckwith, 1991). This property of alkaline phosphatase forms the basis for a convenient in vivo strategy for monitoring protein translocation across the cytoplasmic membrane (Manoil et al. 1990).

On the other hand, β-galactosidase is a tetramer, and each identical monomer is 116,353 Dalton in molecular weigth and 1,023 amino acid residues in length (Fowler and Zabin, 1978). It is active normally in the cytoplasm (Manoil et al., 1988; Slauch and Silhavy, 1991), but inactive or even toxic when secreted to the periplasm of E. coli (Snyder and Silhavy, 1995).

Using these two differently orientated reporter genes to fuse with membrane protein at different locations, completed membrane protein topography will then be possible (Froshauer et al., 1988).