The Prp19 U-box crystal structure suggests a common dimeric architecture for a class of oligomeric E3 ubiquitin ligases - PubMed (original) (raw)

Comparative Study

. 2006 Jan 10;45(1):121-30.

doi: 10.1021/bi051787e.

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Comparative Study

The Prp19 U-box crystal structure suggests a common dimeric architecture for a class of oligomeric E3 ubiquitin ligases

Craig W Vander Kooi et al. Biochemistry. 2006.

Abstract

Prp19 is an essential splicing factor and a member of the U-box family of E3 ubiquitin ligases. Prp19 forms a tetramer via a central coiled-coil domain. Here, we show the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. A high-resolution structure of the homodimeric state of the Prp19 U-box was determined by X-ray crystallography. Mutation of the U-box dimer interface abrogates U-box dimer formation and is lethal in vivo. The structure of the U-box dimer enables construction of a complete model of Prp19 providing insights into how the tetrameric protein functions as an E3 ligase. Finally, comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/BARD1 RING-finger domains uncovers a common architecture for a family of oligomeric U-box and RING-finger E3 ubiquitin ligases, which has mechanistic implications for E3 ligase-mediated polyubiquitination and E4 polyubiquitin ligases.

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Figures

Figure 1

Figure 1

The U-box domain of Prp19 exists as a functional dimer. A) Prp19 contains three domains: an N-terminal U-box, a central tetrameric coiled-coil, and a C-terminal WD-40 repeat domain. B) Analytical ultracetrifugation reveals that the coiled-coil domain drives the formation of a single functional tetramer in the context of a construct containing both U-box and coiled-coil domains. C) The construct containing U-box and coiled-coil domains is well structured with primarily helical content. D) NMR reveals that the U-box domain is integrally connected to Prp19 and not flexibly linked, as is the case for the C-terminal WD40 repeats. E) The U-box of Prp19 exists in a monomer/dimer equilibrium. F) A model of the U-box and coiled-coil regions of Prp19 showing the symmetric U-box dimers formed around the coiled-coil tetramer.

Figure 2

Figure 2

Basis for Prp19 dimerization A) Ribbon diagram of the Prp19 U-box dimer. Molecular graphics were generated using MOLMOL (48). B) Prp19 contains a central hydrophobic patch at the dimer interface composed of four long-chain hydrophobic residues, shown with a zoom showing the electron density at the dimer interface. C) Representative 2FO-FC electron density map of the dimer interface contoured at 2σ (generated with PYMOL;

http://www.pymol.org

). D) Mutation of the central hydrophobic L15 to glutamate disrupts the ability of the U-box to dimerize. AU profile of Prp19(1-73) L15E shows only one peak with a sedimentation coeffiecient consistent with the monomeric species. E) 15N-1H HSQC of the same construct shows excellent line width and chemical shift dispersion, characteristics of a well folded domain. Additionally, the chemical shifts are virtually superimposable with the wild-type protein (19) demonstrating that the L15E mutation causes minimal structural perturbation and adopts a native-like structure. F) Mutations to the residues in the dimer interface abrogate Prp19 function in vivo either in rescuing a temperature sensitive strain (Upper panel) or rescuing growth for the null allele (Lower panel).

Figure 3

Figure 3

Stabilization centers of the U-box domain. A) U-box domain is stabilized by two networks of hydrogen bonds and salt bridges in Prp19. B) In contrast the RING-finger domain of Rag1 (1RMD) is stabilized by chelation of two zinc atoms. C, D) The C-terminal stabilization center of Prp19 is much more compact and largely recapitulates the organization of the RING-finger center (D), but with Asp38 extended to occupy the position of the central zinc atom. E) The N-terminal stabilization center of the U-box contains an extended network of inter-residue interactions including many between side chain and main chain amides. Distances between potential hydrogen bond donors and acceptors are color coded: d < 3.0Å as green, 3.0< d <3.25 as pink, 3.25< d <3.50 as tan. F) The zinc mediated RING-finger contains the four zinc-sulfur bonds.

Figure 4

Figure 4

Comparison of related E3 ligase domain stabilization A) Multiple sequence alignment of Prp19, Rag1, and three human Miz-finger domains. Residues contributing to the N-terminal center of the U-box are highlighted in blue. Residues contributing to the C-terminal center of the RING-finger domain are highlighted in red. B) Schematic of the cross-braced stabilization of U-box and RING-finger proteins with hydrogen-bonding and zinc centers in cyan and red, respectively. Both domains contain a core helix (large rectangle) and three short β-strands (small rectangles). The cross-brace is mediated through the central strand, represented by the dotted line connecting the two stabilization centers. The Miz-finger domain found in related SUMO E3 ligases appears to represent a hybrid of these domains.

Figure 5

Figure 5

Model of the quaternary structure of Prp19. A) Prp19 exists as a functional tetramer with dimeric U-box domains and flexibly attached WD40 domains. WD40 repeat domain modeled from Groucho/Tle1 (49). B) The basic domain structure of an E3 ligase requires E2 and substrate recruitment domains. C) The quaternary structure of Prp19 suggests a common architecture for oligomeric E3 ubiquitin ligases that brings the E2 recruitment domain into close proximity with a substrate recognition domain.

Figure 6

Figure 6

The Prp19 dimer as a general model for oligomeric E3 ligases. Comparison of the dimerization interfaces of A) Prp19 B) BRCA1/BARD1 (1JM7) and C) Rag1 (1RMD), respectively, show different characterized dimerization interfaces. The U-box or RING-finger dimerization interface appears structurally conserved between Prp19 and BRCA1 D) Multiple sequence alignment of dimeric RING-finger domains of Mdm2 and MdmX with Prp19. The four hydrophobic residues at the Prp19 dimer interface are highlighted.

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