Structural Model of the hUbA1-UbcH10 Quaternary Complex: In Silico and Experimental Analysis of the Protein-Protein Interactions between E1, E2 and Ubiquitin

Abstract

UbcH10 is a component of the Ubiquitin Conjugation Enzymes (Ubc; E2) involved in the ubiquitination cascade controlling the cell cycle progression, whereby ubiquitin, activated by E1, is transferred through E2 to the target protein with the involvement of E3 enzymes. In this work we propose the first three dimensional model of the tetrameric complex formed by the human UbA1 (E1), two ubiquitin molecules and UbcH10 (E2), leading to the transthiolation reaction. The 3D model was built up by using an experimentally guided incremental docking strategy that combined homology modeling, protein-protein docking and refinement by means of molecular dynamics simulations. The structural features of the in silico model allowed us to identify the regions that mediate the recognition between the interacting proteins, revealing the active role of the ubiquitin crosslinked to E1 in the complex formation. Finally, the role of these regions involved in the E1–E2 binding was validated by designing short peptides that specifically interfere with the binding of UbcH10, thus supporting the reliability of the proposed model and representing valuable scaffolds for the design of peptidomimetic compounds that can bind selectively to Ubcs and inhibit the ubiquitylation process in pathological disorders.

Dissecting the Specificity of Adenosyl Sulfamate Inhibitors Targeting the Ubiquitin-Activating Enzyme

“…MLN4924 is clearly inferior to the other two ligands, mainly because of its missing hydroxyl group and the resulting inability to form electrostatic interactions in the bound state on a comparable level. Since these results are in good qualitative agreement with the experimentally measured inhibitory values, MLN7243 was also investigated as the best ligand on the basis of the human UBA1 homology model published earlier (Correale et al, 2014). In this complex the electrostatic contribution was found to be slightly less favorable.…”

“…Alternate locations were selected after visual inspection and the system was protonated using Protonate3D (Labute, 2009) with default settings at pH 7.4 and locally minimized with the Amber12:EHT force field implemented in MOE using an rms gradient of 0.1 kcal/(mol•Å ). Furthermore, a complex of human UBA1 and MLN7243 was generated on the basis of the published homology model of human UBA1 without ligand (Correale et al, 2014). After preparation of the protein according to the presented workflow and modeling the side chains of M505 and R551 (corresponding to N471 and K519 in yeast Uba1) in conformations not blocking the binding pocket, MLN7243 was placed therein, in analogy to the binding mode observed in the corresponding yeast Uba1 complex structure.…”

Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism

“… 96 , 104 , 109 In each case, the thioester-linked ubiquitin is bordered by the E2, the FCCH domain, and ubiquitin in the adenylation site, although the models differ somewhat. In two cases the thioester-linked ubiquitin contacts the FCCH domain, 104 , 109 while in another case the thioester-linked ubiquitin contacts the E2. 96 In the later case, ubiquitin was positioned in a similar but not identical conformation to the closed E2∼ubiquitin conformation (see below).…”

Ubiquitination Enzymes

“…In human, ten UBA orthologues have been identified that can activate ubiquitin or UBLs. Ubiquitin-like modifier-activating enzyme 1 (UBA1) is mainly responsible for ubiquitin-activation and can also activate the NEDD8 UBL peptide [26][27][28]. UBA2 (or UBLE1B) is also known as SUMO-activating enzyme subunit 2 (SAE2), and activates the SUMO UBL peptide as heterodimer with SAE1 [29].…”