P1B-ATPases exhibit eight transmembrane domains, two cytosolic loops (one containing the actuator site and the other one the ATP binding and hydrolysing sites), and cytoplasmic N- and C- terminus domains (Fig. 1).
The transmembrane (TM) domains 6-8 contain several metal binding sites and the TM 6 has a CPX motif (mainly CPC, but also CPS, CPA, CPT and CPD). The cysteine in the CPX domain is essential for the activity of the protein, and its mutation fully impairs the transport activity of the protein (Yoshimizu et al. 1998; Fan and Rosen 2002; Mandal and Arguello 2003; Lowe et al. 2004). The presence of SPC motif constitutes an exception that can be found in the Cu+-ATPases such as AtHMA1 which is widespread in plants (Arguello et al. 2007). Since this specific domain is found in most P-ATPases, it is not sufficient to determine the enzyme specificity. Also, it has been proposed that the specificity would result from the combination of the CPX sequence and the presence of specific residues located on TM 7 and 8 (Mills et al. 2005; Dutta et al. 2007). Finally, a new classification of the P1B-ATPases has been established using the nature of these residues describing
several sub-classes matching with different substrate specificity (Arguello et al. 2007). The amino acid sequence of the cytosolic loops is well conserved among the different members of the P1B-ATPases. The cytosolic loop between the TM 4 and 5 constitutes the actuator domain. Structure analyses of CopA and Serca (Sazinsky et al. 2006a) have shown that a highly conserved domain exhibiting a GE sequence can interact with the ATP-binding domain at the level of the second cytosolic loop responsible for conformation changes of the protein essential for the cation transport. The second cytosolic loop, between TM 6 and 7, is divided into two domains: the P domain is the nearest to the membrane and contains the DKTGT sequence, which is the ATP-hydrolysis site, the Pg being transferred to the D residue; the other part is the one called N (for nucleotide-binding), and contains the ATP-binding sequence (Sazinsky et al. 2006b). Most P1B-ATPase N-termini share a pappa folding exhibiting metal binding sites. Specific to plant Zn2+-ATPases is that the sequence of the metal binding domain (MBD) contains CC in contrast to CXXC in the other P-ATPase members. This N-terminus domain is involved in the regulation of enzyme activity. Interestingly, the deletion of the entire domain of AtHMA2 results in a loss of the activity of this protein in plants (Wong et al. 2009b). In addition, it has been shown that the CCXXE is crucial for the ATPase activity and that a mutation in this site in AtHMA2 and AtHMA4 impairs the transport activity (Verret et al. 2005). Finally, a truncated AtHMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites (Eren et al. 2007).
Plant Zn2+-ATPases have an unusual long C-terminus with numerous His and Cys mainly localized in MBDs (CC, poly-His) as for AtHMA2 and AtHMA4. Surprisingly, the function of the C-terminus does not seem to be conserved from a protein to another. For example, the deletion of the whole C-terminus of AtHMA2 does not fully impair the activity of the protein in plants (studied by complementation of hma2, hma4 mutant plants) but seems to cause a mislocation of the protein and this shortened form of the protein did not complement the female sterility of the double mutant (Wong et al. 2009b). Interestingly, the C-terminal domain of AtHMA4 contains 13 Cys doublets followed by a stretch of 11His which constitutes a high affinity Zn2+ and Cd2+ chelator domain that would be able to bind 10 Zn2+ ions (Baekgaard et al. 2010). The expression of this C-terminus domain alone from 7cHMA4 or AtHMA4 resulted in an increase in zinc and cadmium content and tolerance in yeast but also in planta (Papoyan and Kochian 2004; Bernard et al. 2004; Siemianowski et al. 2011). However, there is still a debate concerning the regulatory role of the C-terminus of AtHMA4. Indeed, it has been reported that the deletion of the terminal His stretch was sufficient to suppress the ability of AtHMA4 to complement Cd2+ and Zn2+ sensitive yeast strains (Verret et al. 2005). In contrast, other work has reported an increased activity of AtHMA4 when the C-terminus was deleted (Mills et al. 2010; Baekgaard et al. 2010). In any case, expression of a delta C-terminus in hma2, hma4 double mutants did not rescue the sterile phenotype of these mutants demonstrating that the C-terminus of the protein plays an essential role in plants (Mills et al. 2010).
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