Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1.

The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.

Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways.

Despite the similar enzyme cascade in the Ubiquitin and Ubiquitin-like peptide(Ubl) conjugation, the involvement of single or heterodimer E1 activating enzyme has been a mystery. Here, by using a quantitative Förster Resonance Energy Transfer (FRET) technology, aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we elucidate in detail the functional properties of each subunit of the E1 heterodimer activating-enzyme for NEDD8, UBA3 and APPBP1. In contrast to SUMO activation, which requires both subunits of its E1 heterodimer AOS1-Uba2 for its activation, NEDD8 activation requires only one of two E1 subunits, UBA3. The other subunit, APPBP1, only contributes by accelerating the activation reaction rate. This discovery implies that APPBP1 functions mainly as a scaffold protein to enhance molecular interactions and facilitate catalytic reaction. These findings for the first time reveal critical new mechanisms and a potential evolutionary pathway for Ubl activations. Furthermore, this quantitative FRET approach can be used for other general biochemical pathway analysis in a dynamic mode.

Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay.

Ubiquitin and ubiquitin-like proteins (Ubls), such as SUMO, are covalently conjugated to their targets by related, but distinct enzymatic conjugation reactions that involve the dynamic E1-E2-E3 enzyme cascade. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, with the help of ATP, to form a covalent thioester bond. Subsequently, Ubls are transferred to E2 to generate a thioester-linked product. In previous studies, we showed the dynamic processes and thioester intermediates of SUMO with its E1 and E2 conjugating enzymes. Studies of the enzyme specificity of the Ubl conjugation cascade are normally carried out by tedious biochemical processes, and the reaction intermediates are often difficult to capture because they are unstable and have short half-lives. Here, using our recently developed robust quantitative FRET-based technology, we describe systematic investigations of enzymatic specificity and thioester intermediate determination of ubiquitin with its E1-E2 ligases in conjugation with SUMO and its ligases. Our technology easily determined the strong specificity of enzyme-substrate interactions and thioester intermediates in ubiquitination and SUMOylation cascades. The traditional FRET pair ECFP/EYFP lacked adequate signals for these assays. However, in contrast, the highly sensitive FRET pair CyPet/YPet was easily harnessed to define the reaction specificities and intermediates. In addition, the thioester intermediates can be readily monitored by a newly defined FRET index parameter. These results provide an example of a systems biology approach to determine Ubl conjugation specificity and demonstrate that a robust FRET technology can be used to identify enzymes and substrates in other Ubl pathways.