X-ray absorption fine structure (XAFS) spectra have been measured for a series of structurally characterized zinc model complexes that mimic the zinc sites found in metalloproteins. These include both inorganic zinc coordination complexes and small zinc binding peptides. These data have been analyzed to determine the extent to which Zn XAFS can be used to determine reliably the ligation environment of the zinc. Because Zn−N and Zn−S XAFS oscillations are nearly out of phase over the accessible energy range, it is difficult to determine the relative number of sulfur and nitrogen scatterers, and in some cases, it is even difficult to determine whether a low-Z (N or O) ligand is bound in the presence of high-Z (S) ligands. We describe a protocol that, by controlling the number of variable parameters, can be used to obtain an accurate quantitation of the number of low-Z ligands. We also show that two of the variables that are often treated as freely adjustable parameters, the scale factor and shift in the threshold energy, can lead to erroneous results if not carefully controlled.

We have determined the solution structure of a complex containing the K50 class homeodomain Pituitary homeobox protein 2 (PITX2) bound to its consensus DNA site (TAATCC). Previous studies have suggested that residue 50 is an important determinant of differential DNA-binding specificity among homeodomains. Although structures of several homeodomain-DNA complexes have been determined, this is the first structure of a native K50 class homeodomain. The only K50 homeodomain structure determined previously is an X-ray crystal structure of an altered specificity mutant, Engrailed Q50K (EnQ50K). Analysis of the NMR structure of the PITX2 homeodomain indicates that the lysine at position 50 makes contacts with two guanines on the antisense strand of the DNA, adjacent to the TAAT core DNA sequence, consistent with the structure of EnQ50K. Our evidence suggests that this side chain may make fluctuating interactions with the DNA, which is complementary to the crystal data for EnQ50K. There are differences in the tertiary structure between the native K50 structure and that of EnQ50K, which may explain differences in affinity and specificity between these proteins. Mutations in the human PITX2 gene are responsible for Rieger syndrome, an autosomal dominant disorder. Analysis of the residues mutated in Rieger syndrome indicates that many of these residues are involved in DNA binding, while others are involved in formation of the hydrophobic core of the protein. Overall, the role of K50 in homeodomain recognition is further clarified, and the results indicate that native K50 homeodomains may exhibit differences from altered specificity mutants.