HIGH-RESOLUTION STRUCTURE OF THE DIPHTHERIA-TOXIN REPRESSOR COMPLEXEDWITH COBALT AND MANGANESE REVEALS AN SH3-LIKE 3RD DOMAIN AND SUGGESTSA POSSIBLE ROLE OF PHOSPHATE AS CO-COREPRESSOR

The crystal structure of diphtheria toxin repressor (DtxR) in complex with the corepressor Co2+ has been determined at 2.0 Angstrom resoluti on and in complex with Mn2+ at 2.2 Angstrom resolution. The structure of the flexible third domain could be determined at this high resoluti on. It appears to contain five antiparallel strands exhibiting a fold very similar to the SH3 domain. A superposition of 46 equivalent C alp ha atoms of DtxR and alpha-spectrin SH3 resulted in an rms deviation o f 3.0 Angstrom. The sequence identity is only 7%. This third domain of DtxR appears to have no interactions with the DNA binding domain nor with the metal binding domain of the repressor. Yet, flexibility in th e region between the second and the third domain allows in principle s ignificant conformational changes such as might occur upon DNA binding . The two metal binding sites in the second domain have been unraveled in considerable detail. Metal binding site 1 was well occupied in bot h the cobalt and manganese structures and showed a surprising sulfate ion as ligand. The sulfate was proven beyond doubt by the high peak at its position in a selenate versus sulfate difference Fourier. The pre sence of the intriguing sulfate ion at such a crucial position near th e metal corepressor suggests the possibility that under physiological conditions phosphate may act as a ''co-corepressor'' for this class of metal-regulated DNA binding proteins in Corynebacteria, Mycobacteria, and related organisms. The second metal binding sire is significantly different in these two DtxR structures. In the 2.0 Angstrom cobalt st ructure, the site is not occupied by a metal ion. In the 2.2 Angstrom manganese structure the site is well occupied, at approximately the sa me position as observed previously in cadmium DtxR. The ligands are Gl u105, His106, the carbonyl oxygen of Cys102, and a water molecule. The reasons for differential occupancy of this site in different structur es are intriguing and require further investigations.