A ubiquitin-interacting motif from Hrs binds to and occludes the ubiquitin surface necessary for polyubiquitination in monoubiquitinated proteins

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Abstract

The ubiquitin-interacting motif (UIM) is a short, approximately 20 residue, structural element, which is present in, but not limited to, the proteins involved in endocytotic and proteasomal degradation. UIMs facilitate endocytotic vesicular sorting of the monoubiquitinated proteins and may be important for the targeting of the polyubiquitinated proteins to the proteasome. Using heteronuclear NMR backbone and side-chain chemical shift mapping of the ubiquitin interaction surface, the UIM from the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, specifically binds to the ubiquitin hydrophobic surface using UIM's well-conserved central helical LALAL motif. Molecular modeling of the ubiquitin:UIM_Hrs complex suggests that binding occurs through a specific interaction of Leu263 and Leu267 of the UIM_Hrs with two ubiquitin hydrophobic patches located in close proximity to the ubiquitin major polyubiquitination site, Lys48. Intramolecular binding of ubiquitin to a UIM in monoubiquitinated proteins would render Lys48 unavailable for further ubiquitination, thus, explaining the absolute requirement of UIMs for monoubiquitination. Two leucines, Leu265 and Leu269, located on the opposite face of UIM_Hrs can also interact, albeit less favorably than Leu263 and Leu267, with the ubiquitin hydrophobic patches, suggesting a possible mode for polyubiquitin:UIM binding and apparent preference of UIMs for polyubiquitins.

Section snippets

Materials and methods

Sample preparation. [U-13C, 15N]ubiquitin was produced using a pET3a (Novagen) plasmid, which was a gift of Dr. C.M. Pickart. The plasmid containing a gene for human ubiquitin was transformed into BL21(DE3) cell line and grown at 37 °C on M9 media supplemented with [13C]glucose and 15NH4Cl as sole sources of carbon and nitrogen. Protein expression was induced by adding 1 mM IPTG once A600 reached 0.6. The cells were then grown at 30 °C overnight, harvested by centrifugation (5000 g for 20 min), and

A well-defined helical structure encompasses the hydrophobic central part of the UIM_Hrs peptide

Secondary structure prediction programs from the PredictProtein server [27] indicate that amino acids from the hydrophobic part of the peptide have a predisposition to form α-helix. A CD spectrum of the UIM_Hrs dissolved in water indicated that UIM_Hrs has 45% α-helix (Fig. 1A). To characterize the secondary structure of the UIM_Hrs at the residue specific level, we performed two-dimensional homonuclear experiments, [1H–1H] TOCSY and [1H–1H] NOESY (data not shown). Sequential assignments of

Acknowledgements

This work was supported by NIH Grant GM 47021 and NCI Fellowship F037244.

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