Identification of the two zinc-bound cysteines in the ferric uptake regulation protein from Escherichia coli: chemical modification and mass spectrometry analysis.
Selective chemical modification of thiol groups combined with mass spectrometry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein. Fur is a metalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake in Gram-negative bacteria such as Escherichia coli. In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines. Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein. The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylation of the thiol groups monitored by quenching the reaction at different times and measuring the extent of alkylation by mass spectrometry. Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkylation by the zinc atom. Enzymatic digestion of the modified protein and analysis of the peptide mixture by mass spectrometry enabled fast identification of reactive and protected thiol groups. Two cysteines, Cys92 and Cys95, were thus assigned as zinc ligands. Examination of the sequence comprising the zinc site indicates that it may belong to a new type of structural zinc site. Furthermore, Cys132 was shown to be the fastest reacting cysteine, implying it is a surface-exposed residue.
Gonzalez de Peredo
A
A
Laboratoire de Spectrométrie de Masse des Protéines, Institut de Biologie Structurale, 38027 Grenoble Cedex 1, France.
Saint-Pierre
C
C
Adrait
A
A
Jacquamet
L
L
Latour
J M
JM
Michaud-Soret
I
I
Forest
E
E
D000478:Alkylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry*; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D007460:Iodoacetamide / Q000737:chemistry; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D005290:Ferric Compounds; D008667:Metalloproteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D004492:Edetic Acid; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D015032:Zinc; D003545:Cysteine; D007460:Iodoacetamide