Structure and electrostatic property of cytoplasmic domain of ZntB transporter.

ZntB is the distant homolog of CorA Mg(2+) transporter within the metal ion transporter superfamily. It was early reported that the ZntB from Salmonella typhimurium facilitated efflux of Zn(2+) and Cd(2+), but not Mg(2+). Here, we report the 1.90 A crystal structure of the intracellular domain of ZntB from Vibrio parahemolyticus. The domain forms a funnel-shaped homopentamer that is similar to the full-length CorA from Thermatoga maritima, but differs from two previously reported dimeric structures of truncated CorA intracellular domains. However, no Zn(2+) or Cd(2+) binding sites were identified in the high-resolution structure. Instead, 25 well-defined Cl(-) ions were observed and some of these binding sites are highly conserved within the ZntB family. Continuum electrostatics calculations suggest that the central pore of the funnel is highly attractive for cations, especially divalents. The presence of the bound Cl(-) ions increases the stability of cations along the pore suggesting they could be important in enhancing cation transport.

Chapter 3. High-throughput protein purification for x-ray crystallography and NMR.

In structural biology, the most critical issue is the availability of high-quality samples. "Structural-biology-grade" proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. Where the ultimate goal of structural biology is the same-to understand the structural basis of proteins in cellular processes, the structural genomics approach is different in that the functional aspects of individual protein or family are not ignored, however, emphasis here is on the number of unique structures, covering most of the protein folding space and developing new technologies with high efficiency. At the Midwest Center Structural Genomics (MCSG), we have developed semiautomated protocols for high-throughput parallel protein purification. In brief, a protein, expressed as a fusion with a cleavable affinity tag, is purified in two immobilized metal affinity chromatography (IMAC) steps: (i) first IMAC coupled with buffer-exchange step, and after tag cleavage using TEV protease, (ii) second IMAC and buffer exchange to clean up cleaved tags and tagged TEV protease. Size exclusion chromatography is also applied as needed. These protocols have been implemented on multidimensional chromatography workstations AKTAexplorer and AKTAxpress (GE Healthcare). All methods and protocols used for purification, some developed in MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Disease (CSGID) purification pipeline, are discussed in this chapter.