Crucial steps in the structure determination of the Na+/H+ antiporter NhaA in its native conformation.
Sodium proton antiporters are ubiquitous membrane proteins. Their importance for cell viability is the result of their role in homeostasis of intracellular pH, cellular Na+ content and cell volume. Recently, the first structure of this family of secondary transporters, namely of NhaA from Escherichia coli, revealed a novel fold and elucidated the molecular basis for the mechanism of transport and its regulation by pH. Here, we describe the key steps for the structure determination of NhaA, an iterative process of improving protein quality as well as crystallization conditions. Protein quality was optimized by shortening the purification to a single step and by changing the expression host. The major steps for crystal improvement were the exchange of the detergent during protein purification from the beta- to the alpha-anomer of DDM, the addition of OG to the crystallization set ups, and the growth of the crystals under conditions suitable for cryo-temperatures. Unexpectedly, the dimeric association of the transporter in the 3D crystal lattice is non-physiological. A comparison of the X-ray structure with the electron density map from cryo-electron microscopy of 2D crystals demonstrates that the NhaA helix packing in the 3D crystal is identical with the one in the lipid environment. Thus, the antiporter is in a native conformation in the 3D crystals.
Screpanti
Emanuela
E
Department of Molecular Membrane Biology, Max-Planck-Institute of Biophysics, Max-von-Laue-Str. 3, D-60438 Frankfurt, Germany.
Padan
Etana
E
Rimon
Abraham
A
Michel
Hartmut
H
Hunte
Carola
C
D000595:Amino Acid Sequence; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers