Two high-resolution structures of the human E3 ubiquitin ligase Siah1

Abstract

Siah1 is an E3 ubiquitin ligase that contributes to proteasome-mediated degradation of multiple targets in key cellular processes and which shows promise as a therapeutic target in oncology. Structures of a truncated Siah1 bound to peptide-based inhibitors have been reported. Here, new crystallization conditions have allowed the determination of a construct encompassing dual zinc-finger subdomains and substrate-binding domains at significantly higher resolution. Although the crystals appear isomorphous, two structures present distinct states in which the spatial orientation of one zinc-finger subdomain differs with respect to the rest of the dimeric protein. Such a difference, which is indicative of conformational freedom, infers potential biological relevance related to recognition of binding partners. The crystallization conditions and improved models of Siah1 may aid future studies investigating Siah1-ligand complexes.

The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear

“…Whereas ZnF2 packs on the SBD and has a buried surface area of 626 Å 2 , the ZnF1 has limited contacts with the SBD. The overall structure of the SIAH2 is similar to that of SIAH1 (PDB ID: 4CA1) [25,26] (Fig. S1A).…”

“…Such movement is probably limited by a conserved proline, P165, in the linker region, which is known to cause structural rigidity of domain linkers [42]. Similar to the structure of SIAH1 [25][26][27], SIAH2 also forms a dimer in the asymmetric unit. This is consistent with the elution profile of the protein on size exclusion chromatography (data not shown).…”

“…The crystal structure of the substrate binding domain of mouse and human SIAH1 in apo form [25,26] and in complex with peptides from adaptors Siah-interaction protein (SIP) [27], phyllopod (PHYL) [28] and a co-valent peptidomimetic inhibitors [10] have been reported previously. We report here the crystal structures of SIAH2 in the apo form, and SIAH1 in complex with a peptide from deubiquitinase USP19.…”

The SIAH E3 ubiquitin ligases promote Wnt/β-catenin signaling through mediating Wnt-induced Axin degradation

“…The crystal structure of the SIAH1-SBD/AXIN1 (375-394) complex was solved by the molecular replacement method using PHASER in the PHENIX suite (Adams et al 2010) with one monomer of SIAH1 (PDB 4CA1) (Rimsa et al 2013) as the search model. Iterative cycles of refinement and manual model building were carried out with PHENIX refinement programs and COOT (Emsley and Cowtan 2004), respectively, at 2.1 Å resolution.…”

The phylogenetic functional conservation ofDrosophilaSeven-In-Absentia (SINA) E3 ligase and its two human paralogs, SIAH1 and SIAH2, inDrosophilaeye development

“…Secondly, X-ray structural and biochemical analysis of mouse SIAH1a and human SIAH1 utilized the N-terminal truncated forms of SIAH1, missing ~80-90 amino acids to delete the entire catalytic RING-domain (missing nearly one third of SIAH1). Such a large N-terminal-truncated mutant SIAH1 might have unknown and unpredictable structural consequences on correct protein folding and thus may yield a possibly incorrect SIAH1 3D structure inappropriate and thus premature for conducting large-scale small molecule inhibitor screens [55][56][57][58][59]. Thirdly, several published anti-SIAH inhibition strategies were designed using such truncated SIAH proteins, and several newly identified SIAH small molecule inhibitors were chemically screened and identified by targeting these RING-domaindeleted SIAH mutant proteins [60][61][62][63][64].…”