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Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

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The basis for selective E1-E2 interactions in the ISG15 conjugation system.

Larissa A Durfee¹, Melissa L Kelley, Jon M Huibregtse

¹Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, 2500 Speedway, Austin, TX 78712, USA.

The Journal of Biological Chemistry

|June 28, 2008

Summary

This summary is machine-generated.

The E1 enzyme Ube1L preferentially activates the E2 enzyme UbcH8 over UbcH7 for ISG15 conjugation. Specific interactions between Ube1L and UbcH8

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Area of Science:

Biochemistry
Molecular Biology
Cellular Signaling

Background:

Ubiquitin (Ub) and ubiquitin-like proteins (Ubls) conjugation involves E1 and E2 enzymes.
ISG15 is an interferon-induced Ubl, with Ube1L and UbcH8 as its specific E1 and E2 enzymes.
UbcH7, closely related to UbcH8, does not conjugate ISG15 in vivo.

Purpose of the Study:

To investigate the molecular basis for Ube1L's preference for UbcH8 over UbcH7.
To determine if UbcH8 activation by Ube1L is comparable to its activation by the general ubiquitin E1 enzyme (E1(Ub)).

Main Methods:

Kinetic analyses (K(m) determinations) of wild-type and chimeric E2 enzymes.
Thioester assays using chimeric E2 enzymes.
Site-directed mutagenesis and domain swapping experiments.

Main Results:

Ube1L showed a 29-fold higher affinity (lower K(m)) for UbcH8 compared to UbcH7.
Two regions in UbcH8's N-terminus (alpha1-helix and beta1-beta2) are critical for Ube1L specificity.
The C-terminal ubiquitin fold domain (UFD) of Ube1L is essential for ISG15 transfer and binding to UbcH8.
Replacing Ube1L's UFD with E1(Ub)'s UFD resulted in ISG15 transfer to UbcH7.

Conclusions:

Ube1L discriminates between UbcH8 and UbcH7 through specific interactions involving the Ube1L UFD and UbcH8's N-terminal determinants.
The N-terminal regions of E2 enzymes play a crucial role in E1-E2 specificity during Ubl conjugation.