Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1).

Metal response element (MRE) binding transcription factor-1 (MTF1) is a six Cys(2)His(2) zinc finger-containing transcription factor required for basal and zinc-induced transcription of metallothionein genes. The cobalt(II) and zinc(II) affinities of a protein fragment comprising the six zinc finger domains have been examined to reveal apparent dissociation constants (for the six domains collectively) of 0.5 +/- 0.2 microM for cobalt(II) and 31 +/- 14 pM for zinc(II). Two approaches have been used to determine the metal ion affinities of the individual domains. First, the six domains have been examined as single domain peptides revealing dissociation constants ranging from 0.3 to 1.7 microM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (K(d)(Co(II)) 0.3-0.5 microM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (K(d)(Co(II)) 1.6-1.7 microM). Second, we examined the affinity of each domain in the context of the six zinc finger domain protein by individually mutating one metal-binding His residue to Cys to allow independent monitoring of the cobalt(II) occupancy of each site. The affinity of each domain was higher in this context than as a single domain peptide with affinities (corrected for the effect of the mutation) ranging from 0.02 to 0.5 microM. The increase in affinity for the individual domains ranged from factors of 1.1 to 20. The order of affinities (from higher to lowest) was observed to be 4 > 2 approximately 5 > 6 approximately 3 approximately 1. These results reveal that none of the Cys(2)His(2) zinc finger domains of MTF1 have dramatically low metal ion affinities, certainly none low enough to respond to changes in free zinc ion concentrations in the micromolar range. Nonetheless, the metal ion affinities of some domains do differ by a factor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains.