Structural basis for GTP-dependent dimerization of hydrogenase maturation factor HypB.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Maturation of [NiFe]-hydrogenase involves sequential insertion of metals like nickel, iron, cyanide, and carbon monoxide.
• HypB (HypB) is a crucial metal-binding GTPase protein essential for nickel delivery during hydrogenase maturation.

Purpose of the Study:

• To elucidate the structural and functional mechanisms of HypB in nickel delivery.
• To investigate the role of GTP binding and dimerization in HypB activity.

Main Methods:

• Crystal structure determination of Archeoglobus fulgidus HypB (AfHypB) in its apo-form.
• Biochemical assays to assess guanine nucleotide binding and GTPase activity.
• Site-directed mutagenesis (K148A substitution) and in vivo complementation studies in Escherichia coli.

Main Results:

• AfHypB recognizes guanine nucleotides via Asp-194, despite a non-canonical G4 motif.
• GTP binding induces conformational changes in the switch I region, leading to the formation of an intermolecular salt-bridge between Asp-72 and Lys-148.
• Substitution of Lys-148 abolished GTP-dependent dimerization but not nucleotide binding or GTPase activity.
• The invariant Lys-148 is essential for in vivo hydrogenase maturation.

Conclusions:

• GTP-dependent dimerization of HypB is a critical step for hydrogenase maturation.
• Nickel ions are likely loaded at the dimeric interface of GTP-bound HypB and transferred to the hydrogenase post-GTP hydrolysis.