Abstract:
A high-resolution structure has been obtained by three-dimensional NMR spectroscopy for the recombinant target recognition domain (rTRD) of zoocin A. The rTRD is a 128-residue protein responsible for targeting the cell wall of sensitive bacteria. It has a globular domain consisting of an unstructured N-terminal region, two pairs of short anti-parallel beta sheets, two sets of three-strand β-sheet and an α-helix at the c-terminal. A search for the similar fold with DALI server and SWISS-MODEL server yields no significant match, suggesting the novel folding of the rTRD. A hypothesis for the location of the binding site was proposed after observing that EDTA bound specifically to the rTRD. Chemical shifts of backbone amides of T64, G67, T70, G79, Y80 and V82 are affected by EDTA binding, and these residues are close to the poorly-structured region (residues 71-74) of the rTRD, which could provide flexibility for the rTRD to bind to its natural substrate. Additionally, the metal binding site of zoocin A has been studied by 113Cd-NMR and 15N-HSQC experiments. The two 113Cd resonances at 113.6 ppm and 107.2 ppm suggested that two nitrogen and two oxygen atoms ligate to metal center according to the correlation between the chemical shifts of Cd resonances and coordination environment of 113Cd complexes. As the metal binding site is only located at the catalytic domain (CAT) of zoocin A, two-dimensional 15N HSQC experiments for the recombinant catalytic domain (rCAT), metal-free rCAT and reconstituted Zn-rCAT provide evidence for Zn2+ as the metal cofactor. Therefore, zoocin A is also a Zn metalloprotein like its homologous proteins lysostaphy and LytM.
