Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10&ndash;5-10&ndash;6 M for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.<br /

GerN, an Antiporter Homologue Important in Germination of Bacillus cereus Endospores

A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to l-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 μM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 μM l-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of l-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations

Dynamics and Stability of the Metal Binding Domains of the Menkes ATPase and Their Interaction with Metallochaperone HAH1

International audience: Human copper-ATPases ATP7A and ATP7Bare essential for intracellular copper homeostasis. The mainroles of the Menkes protein, ATP7A, are the delivery of copperto the secretory pathway and the export of excess copper fromthe enterocytes. The N-terminal domain of membrane proteinATP7A consists of six repetitive sequences of 60−70 aminoacids (Mnk1−Mnk6) that fold into individual metal bindingdomains (MBDs) and bind a single copper ion in the reducedCuI form via two cysteine residues. The structure of each individual MBD is known from nuclear magnetic resonanceexperiments. Here, we were interested in the stability and dynamics of each isolated MBD in their apo and holo forms and theirinteractions with the soluble metallochaperone HAH1 that delivers copper to ATP7A. Using molecular dynamics simulations ofthe MBDs under different conditions, we show that some MBDs (Mnk1 and Mnk5) present large root-mean-square deviationsfrom initial structures or large root-mean-square fluctuations, and great care has to be taken in setting up the simulations. Wepropose that the first MBD, Mnk1, probably important in the transfer of copper between the metallochaperone and ATPase,could be stabilized by interactions with other MBDs, including a domain located in the loop between Mnk1 and Mnk2. Animportant result of this work is the apparent direct correlation between the difference in the fluctuations of the metal binding siteloop in its apo and holo forms and the measured affinity of the MBD for copper. This difference decreases from Mnk1 to Mnk6,Mnk4, and Mnk2 in this order. The study of the exposure to the solvent of the metal and the residues of the metal binding loopof the MBDs also shows different behavior for each MBD. In particular, copper in serine-rich domain Mnk3 and largelyfluctuating domain Mnk5 appears to be more solvent-exposed than in the other MBDs. In the second part of this work, weinvestigated the importance of electrostatics in the MBD−chaperone interactions using different docking programs. Mnk1 andMnk4 present a large electrostatic dipole moment and large stabilizing interaction energies with HAH1. Finally, we propose amodel structure of ATP7A from Mnk6 (E561) to P1413 based on the crystal structure of LpCopA and docking simulations