Crystallization and structure determination of gramicidin D and the cytosolic domain of CzrB

Crystallization of membrane proteins and peptides represents a challenge in the field of structural biology. Lipidic cubic phase (LCP) has become an important medium for crystallogenesis of membrane proteins of different molecular weight. Here, the small membrane peptide gramicidin is used as an example peptide to test if LCP can produce diffraction quality crystals for membrane proteins and peptides in the lower molecular weight range. This approach was initially tested with the standard LCP lipid monoolein and later on extended to a variety of different monoacylglycerols varying in their acyl chain length. Data sets for three different crystal forms were obtained. In each case gramicidin was found in the double stranded double helical (DSDH) conformation. One crystal form shows stabilizing hydrogen bonds between adjacent tryptophan residues indicating how DSDH can be stabilized as an aggregate in the membrane. The cytoplasmic domain of the putative zinc transporter CzrB was solved in the apo and zinc-bound forms. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP.