Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4

Robert C. Benirschke; James R. Thompson; Yves Nominé; Emeric Wasielewski; Nenad Juranić; Slobodan Macura; Shigetsugu Hatakeyama; Keiichi I. Nakayama; Maria Victoria Botuyan; Georges Mer

doi:10.1016/j.str.2010.04.017

Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4

Robert C. Benirschke, James R. Thompson, Yves Nominé, Emeric Wasielewski, Nenad Juranić, Slobodan Macura, Shigetsugu Hatakeyama, Keiichi I. Nakayama, Maria Victoria Botuyan, Georges Mer

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Abstract

Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.

Original language	English (US)
Pages (from-to)	955-965
Number of pages	11
Journal	Structure
Volume	18
Issue number	8
DOIs	https://doi.org/10.1016/j.str.2010.04.017
State	Published - Aug 2010
Externally published	Yes

Bibliographical note

Funding Information:
We acknowledge the use of beamline 19-BM at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source (APS). Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. We thank Younchang Kim at APS for assistance with X-ray data collection. We thank Ema Stokasimov for her early contribution to this project. Partial support from National Institutes of Health grant CA109449 and a March of Dimes Basil O'Connor scholarship to G.M. is gratefully acknowledged. J.R.T. acknowledges support from the Minnesota Partnership for Biotechnology and Medical Genomics. Y.N. was a recipient of a Kendall-Mayo postdoctoral fellowship in biochemistry.

Keywords

Proteins

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title = "Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4",

abstract = "Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.",

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author = "Benirschke, {Robert C.} and Thompson, {James R.} and Yves Nomin{\'e} and Emeric Wasielewski and Nenad Jurani{\'c} and Slobodan Macura and Shigetsugu Hatakeyama and Nakayama, {Keiichi I.} and Botuyan, {Maria Victoria} and Georges Mer",

note = "Funding Information: We acknowledge the use of beamline 19-BM at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source (APS). Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. We thank Younchang Kim at APS for assistance with X-ray data collection. We thank Ema Stokasimov for her early contribution to this project. Partial support from National Institutes of Health grant CA109449 and a March of Dimes Basil O'Connor scholarship to G.M. is gratefully acknowledged. J.R.T. acknowledges support from the Minnesota Partnership for Biotechnology and Medical Genomics. Y.N. was a recipient of a Kendall-Mayo postdoctoral fellowship in biochemistry. ",

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AU - Thompson, James R.

AU - Nominé, Yves

AU - Wasielewski, Emeric

AU - Juranić, Nenad

AU - Macura, Slobodan

AU - Hatakeyama, Shigetsugu

AU - Nakayama, Keiichi I.

AU - Botuyan, Maria Victoria

AU - Mer, Georges

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N2 - Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.

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Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4

Abstract

Bibliographical note

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