Florent Guillain

Nickel binding and immunological properties of the C‐terminal domain of the Helicobacter pylori GroES homologue (HspA)

Helicobacter pylori synthesizes a heat‐shock protein of the GroES class. The gene encoding this protein (heat‐shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N‐terminal domain (domain A) homologous with other GroES proteins, and a C‐terminal domain (domain B) corresponding to 27 additional residues resembling a metal‐binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose‐binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP‐fused proteins allowed us to conclude that HspA binds nickel ions by means of its C‐terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP–HspA binds nickel ions with an apparent dissociation constant (Kd) of 1.8 μM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99–100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co‐expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant‐encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.

The cadmium transport sites of CadA, the Cd2+-ATPase from Listeria monocytogenes.

CadA, the Cd2+-ATPase from Listeria monocytogenes, belongs to the Zn2+/Cd2+/Pb2+-ATPase bacterial subfamily of P1B-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd2+, CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd2+ (Wu, C. C., Bal, N., Pérard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) Biochem. Biophys. Res. Commun. 324, 1034–1040). This phenotype, which reflects in vivo Cd2+-transport activity, was used to select from 33 point mutations, shared out among the eight transmembrane (TM) segments of CadA, those that affect the activity of the protein. Six mutations affecting CadA were found: M149A in TM3; E164A in TM4; C354A, P355A, and C356A in TM6; and D692A in TM8. Functional studies of the six mutants produced in Sf9 cells revealed that Cys354 and Cys356 in TM6 as well as Asp692 in TM8 and Met149 in TM3 could participate at the Cd2+-binding site(s). In the canonical Cys-Pro-Cys motif of P1B-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys354 being directly involved in Cd2+ binding, while Cys356 seems to be required for Cd2+ occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu+-ATPase, also act at different steps. In TM4, Glu164, which is conserved among P1B-ATPases, may be required for Cd2+ release. Finally, analysis of the role of Cd2+ in the phosphorylation from ATP and from Pi of the mutants suggests that two Cd2+ ions are involved in the reaction cycle of CadA.

Ca2+ translocation across sarcoplasmic reticulum ATPase randomizes the two transported ions.

Cytoplasmic Ca2+ dissociation is sequential, and the Ca2+ ions bound to the nonphosphorylated ATPase are commonly represented as superimposed on each other, so that the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is not. Under conditions where ADP-sensitive phosphoenzyme accumulates (leaky vesicles, 5°C, pH 8, 300 mM K+), luminal Ca2+ dissociation is sequential as well, so that the representation of two superimposed Ca2+ ions still holds on the phosphoenzyme, with the superficial Ca2+ facing the lumen freely exchangeable and the deeper Ca2+ blocked by the superficial Ca2+. Under the same conditions, we have investigated whether a prebuilt Ca2+ order is maintained during membrane translocation. Starting from a prebuilt order on the cytoplasmic side, we showed that the Ca2+ ions cannot be identified after translocation to the luminal side. The same result was obtained starting from a prebuilt order on the luminal side and following the luminal to cytoplasmic translocation. We conclude that the two Ca2+ ions are mixed during ATP-induced phosphorylation as well as during ADP-induced dephosphorylation.

HOW DO CA2+ IONS PASS THROUGH THE SARCOPLASMIC RETICULUM MEMBRANE

We propose an overview of the mechanism of Ca2+ transport through the sarcoplasmic reticulum membrane via the Ca2+-ATPase. We describe cytoplasmic calcium binding, calcium occlusion in the membrane and lumenal calcium dissociation. A channel-like structure is discussed and related to structural data on the membranous domain of the Ca2+-ATPase.

Dimethyl sulfoxide favours the covalent phosphorylation and not the binding of Pi to sarcoplasmic reticulum ATPase

Competition between Ca2+ binding and Pi phosphorylation showed that the affinity of Ca(2+)-ATPase for Pi was not changed by 20% DMSO. Thus, the enhancement of Pi phosphorylation in the presence of DMSO should not be attributed to a solvent effect on the affinity for Pi, but rather on the phosphorylation reaction itself.