Ratiometric pulsed alkylation/mass spectrometry of the cysteine pairs in individual zinc fingers of MRE-binding transcription factor-1 (MTF-1) as a probe of zinc chelate stability.
Metal-response element (MRE)-binding transcription factor-1 (MTF-1) is a zinc-regulated transcriptional activator of metallothionein (MT) genes in mammalian cells. The MRE-binding domain of MTF-1 (MTF-zf) has six canonical Cys(2)-His(2) zinc finger domains that are distinguished on the basis of their apparent affinities for zinc and their specific roles in MRE-binding. In this paper, pulsed alkylation of the zinc-liganding cysteine thiolate pairs with the sulfhydryl-specific alkylating reagent d(5)-N-ethylmaleimide (d(5)-NEM) is used as a residue-specific probe of the relative stabilities of the individual zinc finger coordination complexes in Zn(6) MTF-zf. A chase with excess H(5)-N-ethylmaleimide (H(5)-NEM) to fully derivatize MTF-zf concomitant with complete proteolysis, followed by MALDI-TOF mass spectrometry allows quantitation of the mole fraction of d(5),d(5)-, d(5),H(5)-, and H(5),H(5)-NEM derivatized peptides corresponding to each individual zinc finger domain as a function of d(5)-NEM pulse time. This experiment establishes the hierarchy of cysteine thiolate reactivity in MTF-zf as F5 > F6 >> F1 > F2 approximately F3 approximately F4. The apparent second-order rate of reaction of F1 thiolates is comparable to that determined for the DNA binding domain of Sp1, Zn(3) Sp1-zf, under identical solution conditions. The reactivities of all Cys residues in MTF-zf are significantly reduced when bound to an MREd-containing oligonucleotide. An identical experiment carried out with Zn(5) MTF-zf26, an MTF-zf domain lacking the N-terminal F1 zinc finger, reveals that MTF-zf26 binds to the MREd very weakly, and is characterized by strongly increased reactivity of nonadjacent F4 thiolates. These findings are discussed in the context of existing models for metalloregulation by MTF-1.
Apuy
J L
JL
Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-2128, USA.
Chen
X
X
Russell
D H
DH
Baldwin
T O
TO
Giedroc
D P
DP
D000478:Alkylation; D000595:Amino Acid Sequence; D002614:Chelating Agents; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins; D004355:Drug Stability; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011874:Radiometry; D017384:Sequence Deletion; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D015032:Zinc; D016335:Zinc Fingers; D000097809:Transcription Factor MTF-1
D002614:Chelating Agents; D003545:Cysteine; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1