Metal binding specificities of a P1B4-ATPase from M. smegmatis.

RAIMUNDA, DANIEL; ARGÜELLO, JOSÉ M

Conferencia; The 13th International ATPase Conference: Na,K-ATPase and Related P-type Transport ATPases; 2011

P1B-type ATPases are ubiquitous metal efflux systems that function either in metal detoxification or in the maturation of extracellular metalloproteins.  An unusual number of alkali and heavy metal P-type ATPases genes are found in Mycobacterium tuberculosis.  Whole genome genetic screens suggest that ctpD is likely required for bacterial growth or survival during infection. Characterization of CtpD bacterial and eukaryote homologs suggests disparate metal specificities (Co2+, Cu+, Ca2+, Zn2+, etc.). In order to characterize the biological function of CtpD, a M. smegmatis deletion of the homologous SMEG_5403 gene was generated. This strain did not grow in the presence of high Co2+ (IC50= 7.5 µM) or Ni2+ (IC50= 120µM) in the culture media.  Cellular Co2+ accumulation was detected with no changes in the levels of other metals.  Up-regulation of gene transcription in wild type strain was induced by sub-lethal Co2+ concentrations but not by Ni2+.  Stoichiometric binding to Co2+, Ni2+ and Zn2+ to heterologously expressed, affinity purified M. smegmatis CtpD was observed. Interestingly, the enzyme binds Zn2+ with higher affinity (Zn2+ K1/2= 53 nM, Co2+ K1/2= 2.6 µM, Ni2+ K1/2= 2.6 µM). These results could explain the specificities observed in vitro for P1B4-ATPases and point to a primary role in Co2+ homeostasis in vivo.