Nickel binding and [NiFe]-hydrogenase maturation by the metallochaperone SlyD with a single metal-binding site in Escherichia coli.
SlyD (sensitive to lysis D) is a nickel metallochaperone involved in the maturation of [NiFe]-hydrogenases in Escherichia coli (E. coli) and specifically contributes to the nickel delivery step during enzyme biosynthesis. This protein contains a C-terminal metal-binding domain that is rich in potential metal-binding residues that enable SlyD to bind multiple nickel ions with high affinity. The SlyD homolog from Thermus thermophilus does not contain the extended cysteine- and histidine-rich C-terminal tail of the E. coli protein, yet it binds a single Ni(II) ion tightly. To investigate whether a single metal-binding motif can functionally replace the full-length domain, we generated a truncation of E. coli SlyD, SlyD155. Ni(II) binding to SlyD155 was investigated by using isothermal titration calorimetry, NMR and electrospray ionization mass spectrometry measurements. This in vitro characterization revealed that SlyD155 contains a single metal-binding motif with high affinity for nickel. Structural characterization by X-ray absorption spectroscopy and NMR indicated that nickel was coordinated in an octahedral geometry with at least two histidines as ligands. Heterodimerization between SlyD and another hydrogenase accessory protein, HypB, is essential for optimal hydrogenase maturation and was confirmed for SlyD155 via cross-linking experiments and NMR titrations, as were conserved chaperone and peptidyl-prolyl isomerase activities. Although these properties of SlyD are preserved in the truncated version, it does not modulate nickel binding to HypB in vitro or contribute to the maturation of [NiFe]-hydrogenases in vivo, unlike the full-length protein. This study highlights the importance of the unusual metal-binding domain of E. coli SlyD in hydrogenase biogenesis.
Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, ON, Canada M5S 3H6.
Harini
H
Kaluarachchi
Matthias
M
Altenstein
Sonia R
SR
Sugumar
Jochen
J
Balbach
Deborah B
DB
Zamble
Caroline
C
Haupt
D020816:Amino Acid Motifs; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D056511:Metallochaperones / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D029968:Escherichia coli Proteins; D008024:Ligands; D056511:Metallochaperones; D008670:Metals; C085606:SlyD protein, E coli; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D019696:Peptidylprolyl Isomerase