ABSTRACT
The ZntB Zn(2+) efflux system is important for maintenance of Zn(2+) homeostasis in Enterobacteria. We report crystal structures of ZntB cytoplasmic domains from Salmonella enterica serovar Typhimurium (StZntB) in dimeric and physiologically relevant homopentameric forms at 2.3 Å and 3.1 Å resolutions, respectively. The funnel-like structure is similar to that of the homologous Thermotoga maritima CorA Mg(2+) channel and a Vibrio parahaemolyticus ZntB (VpZntB) soluble domain structure. However, the central α7 helix forming the inner wall of the StZntB funnel is oriented perpendicular to the membrane instead of the marked angle seen in CorA or VpZntB. Consequently, the StZntB funnel pore is cylindrical, not tapered, which may represent an "open" form of the ZntB soluble domain. Our crystal structures and isothermal titration calorimetry data indicate that there are three Zn(2+) binding sites in the full-length ZntB, two of which could be involved in Zn(2+) transport.
Subject(s)
Bacterial Proteins/chemistry , Carrier Proteins/chemistry , Cation Transport Proteins/chemistry , Recombinant Proteins/chemistry , Salmonella typhimurium/chemistry , Zinc/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Calorimetry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli , Ion Transport , Models, Molecular , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Salmonella Infections/virology , Salmonella typhimurium/genetics , Salmonella typhimurium/metabolism , Thermotoga maritima/chemistry , Thermotoga maritima/metabolism , Vibrio parahaemolyticus/chemistry , Vibrio parahaemolyticus/metabolismABSTRACT
Amphipathic polymers ("amphipols") were introduced several years ago (Tribet, C.; Audebert, R.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15047-15050) as an alternative method for solubilizing integral membrane proteins in stable, nativelike conformations. However, direct maintenance of full membrane protein functionality in amphipol solutions has not previously been demonstrated in the absence of added lipid or detergent. In this contribution, the first zwitterionic amphipol "PMAL-B-100" is introduced. PMAL-B-100 not only maintains membrane protein structure and solubility, but also supports the full catalytic activity of an integral membrane enzyme, diacylglycerol kinase, in the complete absence of additional lipid or detergent. All of the roles which a lipid bilayer normally plays in maintaining diacylglycerol kinase's structure and in facilitating catalysis are satisfied by the environment and interactions supplied by PMAL-B-100.
