Copper-transporting PIB-type ATPases (CopA-family) fulfill key roles in copper homeostasis by pumping copper across bacterial or intracellular and cytoplasmic eukaryotic membranes. Mutations of the copper-transporting ATPase gene cause Wilson and Menkes diseases. CopA consists of 8 transmembrane helices and three cytosolic domains; Nucleotide binding (N), Phosphorylation (P) and Actuator (A) domains. We have used synthetic peptides to identify minimal structural motifs and physical mechanisms of cytosolic domain interactions.Based on the crystal structure[1], we have designed decameric-peptides whose sequences are derived from the putative interaction site of the A-domain with the PN domain of CopA from Legionella pneumophila. The interaction of the synthetic A-domain peptide with the individually expressed PN domain was measured by Isothermal Titration Calorimetry(ITC), Circular Dichroism (CD), and stopped-flow fluorescence techniques. Preliminary ITC results show a favorable TΔS, indicating that binding of the peptide carrying the native sequence is driven by hydrophobic interactions with stoichiometry(N=1). Time-resolved fluorescence measurements with a tryptophan-carrying actuator peptide show association and dissociation rate constants with the PN domain of 22000M−2s−1 and 0.7M−1s−1, respectively. This corresponds to a Kd of the peptide for the PN-domain of 32μM. In the presence of the non-hydrolyzable nucleotide AMPPNP the best kinetic fit was obtained with rates of 20000M−2s−1 and 1.0M−1s−1, indicating an almost unaffected affinity of the peptide for the nucleotide-loaded PN-domain. In agreement with the kinetic and isothermal data, the deca-peptide also shifted the melting temperature of the PN domain from 330 to 332K as monitored by CD spectroscopy.In summary, we have good evidence that the actuator peptide binds in a predominantly hydrophobic mechanism to the PN domain in the 30-50μM Kd range with little dependence on the occupancy of the nucleotide binding site of the PN domain.1)Gourdon,P. et.al.2011 Nature475, 59.