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Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation

Y B Lee¹, Y A Joe, E C Wolff

¹Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340, USA.

The Biochemical Journal

|May 7, 1999

Summary

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Deoxyhypusine synthase forms a highly stable complex with its substrate, eukaryotic initiation factor 5A precursor (eIF5A). This interaction is crucial for hypusine synthesis, a vital post-translational modification.

Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Deoxyhypusine synthase (DHPS) is key in hypusine biosynthesis.
Hypusine modification is essential for the function of eukaryotic initiation factor 5A (eIF5A).
DHPS acts on a specific protein substrate, eIF5A precursor.

Purpose of the Study:

To investigate the stable complex formation between human DHPS and human recombinant eIF5A precursor (ec-eIF5A).
To characterize the stoichiometry and binding affinity of the DHPS-ec-eIF5A complex.
To understand the conditions influencing complex formation and product generation.

Main Methods:

Affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A.
Gel mobility-shift assays.

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Analytical ultracentrifugation and N-terminal amino acid sequencing.

Main Results:

DHPS selectively bound to immobilized His.Tag-ec-eIF5A.
A stable 1:1 complex of DHPS tetramer to ec-eIF5A monomer was identified.
A very strong binding affinity (Kd ≤ 0.5 nM) was determined for the DHPS-ec-eIF5A interaction.
Complex formation occurred across a pH range of 7.0-9.2 and was independent of NAD+ or spermidine.
An enzyme-product complex and the modified eIF5A were detected under complete reaction conditions.

Conclusions:

DHPS and ec-eIF5A form a highly stable complex with a precise 1:1 stoichiometry.
The strong interaction facilitates the essential hypusine modification of eIF5A.
Understanding this complex is vital for deciphering eIF5A's role in translation regulation.