Structural insights into the U-box, a domain associated with multi-ubiquitination

Melanie D. Ohi; Craig W. Vander Kooi; Joshua A. Rosenberg; Walter J. Chazin; Kathleen L. Gould

doi:10.1038/nsb906

Structural insights into the U-box, a domain associated with multi-ubiquitination

Melanie D. Ohi, Craig W. Vander Kooi, Joshua A. Rosenberg, Walter J. Chazin, Kathleen L. Gould

Research output: Contribution to journal › Article › peer-review

244 Scopus citations

Abstract

The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.

Original language	English
Pages (from-to)	250-255
Number of pages	6
Journal	Nature Structural Biology
Volume	10
Issue number	4
DOIs	https://doi.org/10.1038/nsb906
State	Published - Apr 1 2003

Bibliographical note

Funding Information:
We thank S. Hatakeyama and K.I. Nakayama from the Department of Molecular and Cellular Biology at Kyushu University for the kind gift of GST-Ubc3. We also gratefully acknowledge J.A. Smith for valuable technical assistance with structure calculations and molecular graphics. Work in our laboratories was supported by the National Institutes of Health in the form of operating grants to K.L.G. and W.J.C., training grant positions to M.D.O., C.W.V.K. and J.A.R., and core facility grants to the Vanderbilt Ingram Cancer Center and the Vanderbilt Center in Molecular Toxicology. K.L.G. is an Associate Investigator of the Howard Hughes Medical Institute.

Funding

We thank S. Hatakeyama and K.I. Nakayama from the Department of Molecular and Cellular Biology at Kyushu University for the kind gift of GST-Ubc3. We also gratefully acknowledge J.A. Smith for valuable technical assistance with structure calculations and molecular graphics. Work in our laboratories was supported by the National Institutes of Health in the form of operating grants to K.L.G. and W.J.C., training grant positions to M.D.O., C.W.V.K. and J.A.R., and core facility grants to the Vanderbilt Ingram Cancer Center and the Vanderbilt Center in Molecular Toxicology. K.L.G. is an Associate Investigator of the Howard Hughes Medical Institute.

Funders	Funder number
Vanderbilt Center in Molecular Toxicology
Vanderbilt Ingram Cancer Center
National Institutes of Health (NIH)
National Institute of General Medical Sciences	R01GM047728

ASJC Scopus subject areas

Structural Biology
Biochemistry
Genetics

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title = "Structural insights into the U-box, a domain associated with multi-ubiquitination",

abstract = "The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.",

author = "Ohi, {Melanie D.} and {Vander Kooi}, {Craig W.} and Rosenberg, {Joshua A.} and Chazin, {Walter J.} and Gould, {Kathleen L.}",

note = "Funding Information: We thank S. Hatakeyama and K.I. Nakayama from the Department of Molecular and Cellular Biology at Kyushu University for the kind gift of GST-Ubc3. We also gratefully acknowledge J.A. Smith for valuable technical assistance with structure calculations and molecular graphics. Work in our laboratories was supported by the National Institutes of Health in the form of operating grants to K.L.G. and W.J.C., training grant positions to M.D.O., C.W.V.K. and J.A.R., and core facility grants to the Vanderbilt Ingram Cancer Center and the Vanderbilt Center in Molecular Toxicology. K.L.G. is an Associate Investigator of the Howard Hughes Medical Institute.",

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N2 - The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.

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Structural insights into the U-box, a domain associated with multi-ubiquitination

Abstract

Bibliographical note

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