Volume 11, Issue 1, Pages 249-259 (January 2003) Conformational Flexibility Underlies Ubiquitin Ligation Mediated by the WWP1 HECT Domain E3 Ligase  Mark A Verdecia, Claudio A.P Joazeiro, Nicholas J Wells, Jean-Luc Ferrer, Marianne E Bowman, Tony Hunter, Joseph P Noel  Molecular Cell  Volume 11, Issue 1, Pages 249-259 (January 2003) DOI: 10.1016/S1097-2765(02)00774-8

Figure 1 Comparison of the HECT Domains of WWP1/AIP5 and E6AP (A) The structure of the HECT domain of WWP1/AIP5 consists of two lobes connected by a hinge loop (residues 803–806) colored gold. The N lobe consists of residues 546–802 and is colored blue. The C lobe, comprising residues 807–917, is colored rose. (B) The structure of the E6AP HECT domain (Huang et al., 1999; accession code 1C4Z). The color scheme is the same as for (A). The figure was prepared with MOLSCRIPT (Kraulis, 1991) and PovRay (Amundsen et al., 1997). (C) Secondary structure of the HECT domains of WWP1/AIP5 (gold) and E6AP (blue) are shown above and below their respective sequences. Residues involved in C lobe/N lobe interactions are highlighted red for E6AP and blue for WWP1/AIP5. E2 interacting residues are colored orange, and hinge loop residues are colored gold. Residues in the catalytic region are highlighted green. Highlighted in aqua are the residues missing from the C terminus of WWP1/AIP5. Molecular Cell 2003 11, 249-259DOI: (10.1016/S1097-2765(02)00774-8)

Figure 2 Analysis of the H1′ Helix from WWP1/AIP5 (A) Orthogonal views of the structure of WWP1/AIP5 highlighting the location of the H1′ helix. The H1′ helix is located behind the C lobe at the opposite end from the E2 binding region. (B) Surface view of the N lobe region with which the H1′ helix interacts. Conserved residues from the H1′ helix that are buried in a hydrophobic patch on the N lobe surface are shown in red. The hydrophobic patch on the WWP1/AIP5 N lobe, covered by the H1′ helix, is shown as a white surface with the surrounding features colored blue. Solvent-exposed residues on the H1′ helix are colored yellow, blue, green, and purple to highlight apparent differences in conservation among HECT domain sequences. (C) Sequence alignment of the N-terminal region corresponding to the H1′ helix in WWP1/AIP5 with 20 HECT domains. Positions of residues on the H1′ helix that are buried against the N lobe are displayed in red. The N lobe binding residues on the H1′ helix align on one side of the helix and are highly conserved among different HECT domains from various species (highlighted red). Solvent-exposed residues are not as conserved and appear to segregate into groups and are displayed in yellow, blue, green, and purple backgrounds to highlight these differences. Molecular Cell 2003 11, 249-259DOI: (10.1016/S1097-2765(02)00774-8)

Figure 3 Structure Alignment of the HECT Domain of WWP1/AIP5 with the HECT Domain of E6AP (A) WWP1/AIP5 has an inverted T shape (⊥) and E6AP has an L shape (L). The C lobe rotates approximately 100° between the L to ⊥ shapes. Bond rotations around phi (φ) and psi (ψ) angles within the hinge loop are given that allow for rotation from the ⊥ shape to adopt the L shape. (B) WWP1/AIP5 (residues 546–922) was used to determine wild-type activity. Deletion mutants are completely inactive in autoubiquitylation assays as compared to wild-type protein. Insertion mutants appear to be more active. (C) The Met804Pro/Gln805Pro double mutation has significant loss of rotational freedom; however, it can compensate by increasing rotation around the Q805P phi (φ) bond. The Gln805Pro/Glu806Pro double mutant also has a loss of conformational freedom but can adopt the L shape by switching the direction of phi (φ) and psi (ψ) rotations around Met804Pro and the psi (ψ) bond in Glu806Pro as well as increasing the amount of rotation around the Glu806Pro psi (ψ) bond. Molecular Cell 2003 11, 249-259DOI: (10.1016/S1097-2765(02)00774-8)

Figure 4 Conformations of the HECT Domain Ubiquitin was docked onto the E2 using Ubc1-ubiquitin thioester model coordinates (Hamilton et al., 2001; accession code 1FXT), using the programs O (Jones et al., 1991) and MIDAS (Ferrin et al., 1988). HECT domain C lobes are colored rose, N lobes are colored blue, and hinge loops are colored gold. Dashed red lines indicate point-to-point distances between the HECT domain catalytic cysteine and the modeled thioester bond. (A) E6AP-UbcH7 complex with UbcH7 colored green and ubiquitin modeled in red. The E6AP-UbcH7 protein complex depicted is an experimentally determined structure while the ubiquitin chain shown is modeled. (B) WWP1/AIP5 in the ⊥ shape conformation. The UbcH5-ubiquitin complex was positioned based on the E6AP-UbcH7 complex structure. UbcH5 is colored green and ubiquitin is colored red. (C) WWP1/AIP5 modeled in a putative catalytic conformation. Molecular Cell 2003 11, 249-259DOI: (10.1016/S1097-2765(02)00774-8)

Figure 5 Stabilizing Interactions at the Base of the Hinge Loop (A) Residue contacts within E6AP in the L shape conformation. Lys549 interacts with Asn822, Gln553 hydrogen bonds with Thr819, and Asp607 appears to interact with His609. (B) Residue contacts within WWP1/AIP5 in the ⊥ shape conformation. Arg613 interacts with Gly610, and His621 hydrogen bonds with Asp675. Both pairs of interactions bridge the H3 helix to loop regions (S2/H3 and H4/H5 loops). (C) WWP1/AIP5 (residues 546–922) was used as a reference for wild-type autoubiquitylation activity. Molecular Cell 2003 11, 249-259DOI: (10.1016/S1097-2765(02)00774-8)

Figure 6 C Lobe and N Lobe Interactions in WWP1/AIP5 (A) Residue contacts between the N and C lobes within WWP1/AIP5 in the ⊥ shape conformation. Asp793 on the N lobe interacts with Arg845 on the C lobe. Gln848 and Arg855 on the C lobe form hydrogen bonds with Glu798 and the carbonyl oxygen of Thr676. Asp793 and Arg845 are highly conserved, and Glu798, Gln848, and Arg855 are absolutely conserved among 22 HECT domain sequences analyzed (data not shown). (B) Ubiquitin transfer assays. Molecular Cell 2003 11, 249-259DOI: (10.1016/S1097-2765(02)00774-8)

Figure 7 Schematic Representation of the Indexation Model of Ubiquitin Transfer and Ubiquitin Chain Elongation by HECT Domain Ubiquitin Ligases (A–F) The C lobe is shown in rose, the N lobe is shown in blue, the E2 is shown in green, and ubiquitin molecules are displayed in pink, orange, yellow, and red. The catalytic cysteine on the C lobe, as well as the thioester bond between the C lobe and the first ubiquitin molecule, is shown as a yellow bond in the schematic diagram. Lys48 of each acceptor ubiquitin and isopeptide bonds between ubiquitins are colored light blue in the schematic. The series of models depicted have been drawn approximately to scale to reflect, at least as well as modeling can, the dimensions of the growing polyubiquitin chain relative to the E2 and E3. Molecular Cell 2003 11, 249-259DOI: (10.1016/S1097-2765(02)00774-8)