A new structural paradigm in copper resistance in Streptococcus pneumoniae

Abstract

Copper resistance has emerged as an important virulence determinant of microbial pathogens. In Streptococcus pneumoniae, copper resistance is mediated by the copper-responsive repressor CopY, CupA, and CopA, a copper effluxing P1B-type ATPase. We show here that CupA is a novel cell membrane-anchored Cu(I) chaperone, and that a Cu(I)-binding competent, membrane-localized CupA is obligatory for copper resistance. The crystal structures of the soluble domain of CupA (sCupA) and the N-terminal metal binding domain (MBD) of CopA (CopAMBD) reveal isostructural cupredoxin-like folds each harboring a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site on sCupA to the high-affinity site of CopAMBD. However, copper binding by CopAMBD is not essential for cellular copper resistance, consistent with a primary role of CupA in cytoplasmic Cu(I) sequestration and/or direct delivery to the transmembrane site of CopA for cellular efflux.

Cu(I)-mediated Allosteric Switching in a Copper-sensing Operon Repressor (CsoR)

“…Apo-wild-type CsoR and N-terminally truncated CsoR11 contained the full complement of reduced thiols (1.9 free thiols per monomer; 2 expected). Copper-loaded CsoRs were prepared by saturating apo-CsoRs anaerobically with the addition of 1.0 protomer molar eq of freshly prepared CuCl stock solution in fully degassed Buffer B (25 mM HEPES, pH 7.0, 200 mM NaCl) in an anaerobic glove box essentially as described previously (34). Apo-and Cu(I)-Gt CsoR R65A and K101A for the fluorescence anisotropy DNA binding assay were expressed and purified as wild-type protein.…”

Stress Physiology of Lactic Acid Bacteria

“…lactis CopR, the CopY homolog, controls a regulon of 14 mostly uncharacterized genes (70). Recently, a different copper resistance mechanism was described for S. pneumoniae (71). This organism lacks a CopZ homolog.…”

“…The primary roles of CupA are proposed to be the sequestering of Cu ϩ and its transfer to CopA, activities that are essential for copper resistance. If the concentration of Cu ϩ exceeds the capacity of CupA, free cytoplasmic Cu ϩ may be bound by the repressor CopY, thus relieving repression of copY, cupA, and copA (71).…”

“…This organism lacks a CopZ homolog. Instead, the resistance system consists of the membrane-bound Cu chaperone CupA, a copper exporter (CopA), and a CopY-type regulator (71). The copY, cupA, and copA genes are arranged in an operon whose expression was shown to be induced by Cu and repressed by CopY (72).…”

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

“…The preference of Cu(I) for soft bases (His, Cys, and Met) is manifest in the coordination environments of cytoplasmic metallochaperones (35)(36)(37) and the Met-rich sites found in periplasmic copperbinding proteins (38)(39)(40)(41). However, in the context of Cu(I) transport through membrane channels, kinetic lability may be more important than thermodynamic stability, and indeed high binding affinity is likely to inhibit transport, unless accompanied by energy input that can toggle high-and low-affinity states via conformational change, as has been proposed as a mechanism for intramembrane transport in P1B-type ATPases (42).…”