The Release Pathway of Copper Transporting Ptype ATPases

Abstract

Cellular levels of heavy metals are carefully regulated by the PIB class of P-type ATPases in all kingdoms of life and mutations of the human members ATP7A and ATP7B are the cause of the severe Menkes' and Wilson's diseases. Recently, a crystal structure of a homologous Cu+ ATPase from Legionella pneumophila (LpCopA), trapped in a transition state of dephosphorylation (E2Pi), suggested that copper extrusion employs an intramembranous exit site, but the release pathway remained elusive and the transmembrane (TM) domain was inferred to be occluded. However, by molecular dynamics (MD) simulations, we find that extracellular bulk water solvates the proposed exit and high-affinity ion-binding sites deep within the membrane. This view found further support by a 2.8 resolution LpCopA crystal structure trapped in the E2P state (associated with extracellular exchange in well-known PII-type ATPases such the sarcoplasmic reticulum Ca2+-ATPase, SERCA) showing a similar structure of the TM-domain, and delineating the same pathway by crystal water positions. We conclude that the E2P and E2Pi states are equally open, indicating that Cu+ ATPases couple the conformational changes associated with ion extrusion differently to dephosphorylation as compared to SERCA; in accordance with structural differences. The observed copper extrusion conduit was further validated by mutational studies and shown to involve the PIB-specific MA segment, which is absent in e.g. Co2+ ATPases and thus different unloading schemes may apply within PIB-ATPases. The pathway further explains why Menkes' and Wilson's mutations at the extracellular side impair protein function and constitutes a favorable site for novel inhibitors targeting pathogens from the extracellular environment.