The L2A and β4-5 loops of Sad1's ZnF-UBP domain are contracted relative to other ZnF-UBPs. (A) Structural alignment of Sad1's ZnF-UBP domain (pale orange) to the ZnF-UBP domains from H. sapiens HDAC6 (violet; PDB ID 3C5K) and H. sapiens USP5 (cyan; PDB ID 2G43). The zinc atoms of HDAC6 and USP5 are depicted as gray spheres, and the zinc atom from Sad1 is depicted as an orange sphere. Structural alignments were performed in Pymol. (B) Structural alignment of Sad1's ZnF-UBP main-chain (pale orange) to all other ZnF-UBP domain main-chains available in the PDB (gray; 2G45, 2G43, 3IHP, 3C5K, 3PHD, 3GV4, 2UZG, 2I50, 2L80, 2IDA, 3M99, 3MHH, and 3MHS). Sad1 is an outlier with respect to the distance between its L2A and β4-5 loops. (C) Structural alignment of Sad1's ZnF-UBP domain to the apo ZnF-UBP domain from USP5 (PDB ID 2G43). The ubiquitin interacting residue R221 of USP5 is pointed toward solvent. In contrast, the corresponding residue in Sad1, R70, is held close to the surface through two hydrogen bonds to Y58 and N102. Hydrogen bonds were determined by Pymol with a 3.1 Å cut-off. (D) Structural alignment of Sad1's ZnF-UBP to the ZnF-UBP domain from USP5 bound to ubiquitin (raspberry; PDB ID 2G45), shows that the ubiquitin tail as it binds to USP5 would clash with the unique placement of Sad1 R70. (E) Surface representations of the ZnF-UBP domains from USP5 and Sad1, either with or without ubiquitin. The structural alignment from 4D was used to model Sad1 with ubiquitin, and USP5 was removed for clarity. The deep depression present on the surface of USP5's ZnF-UBP domain is entirely filled by the unique placement of Sad1 R70. (F) Structural alignment of the A site zinc finger of HDAC6 to the corresponding regions of USP5 and Sad1. The residues of USP5 that are engaged in compensatory hydrogen bond interactions are labeled. (G) Primary sequence alignment of USP5 homologs aligned with the T-coffee server. The H. sapiens USP5 residues that engage in compensatory A-site zinc finger hydrogen bond interactions are highlighted. Shown below the alignment for reference are the equivalent residues in Sad1.
