Structural Insights into Homo- and Heterotropic Allosteric Coupling in the Zinc Sensor S.aureus CzrA from Covalently Fused Dimers

摘要

The Zn(II)/Co(II)-sensing transcriptional represser,Staphylococcus aureus CzrA,is a homodimer containing a symmetry-related pair of subunit-bridging tetrahedral N_3O metal sensor coordination sites.A metal-induced quaternary structural change within the homodimer is thought to govern the biological activity of this and other metal sensor proteins.Here,we exploit covalent (Gly_4Ser)_n linkers of variable length in "fused" CzrAs,where n = 1 (designated 5L-fCzrA),2 (10L-fCzrA),or 3 (15L-fCzrA),as molecular rulers designed to restrict any quaternary structural changes that are associated with metal binding and metal-mediated allosteric regulation of DNA binding to varying degrees.While 15L-fCzrA exhibits properties most like homodimeric CzrA,shortening the linker in 10L-fCzrA abolishes negative homotropic cooperativity of Zn(II) binding and reduces DNA binding affinity of the apoprotein significantly.Decreasing the linker length further in 5L-fCzrA effectively destroys one metal site altogether and further reduces DNA binding affinity.However,Zn(II) negatively regulates DNA binding of all fCzrAs,with allosteric coupling free energies (DELTA G~1_C) of 4.6,3.1,and 2.7 kcal mol~(-1) for 15L-,10L-,and 5L-fCzrAs,respectively.Introduction of a single nonliganding H97N substitution into either the N-terminal or C-terminal protomer domain in 10L-fCzrA results in DELTA G~1_C = 2.6 kcal mol~(-1) or approx= 83% that of 10L-fCzrA;in contrast,homodimeric H97N CzrA gives DELTAG~1_C = 0.~1H-~(15)N HSQC spectra acquired for wt-,10L-fCzrA and H97N 10L-fCzrA in various Zn(II) ligation states reveal that the allosteric change of the protomer domains within the fused dimer is independent and not concerted.Thus,occupancy of a single metal site by Zn(II) introduces asymmetry into the CzrA homodimer that leads to significant allosteric regulation of DNA binding.