Biology subjects

Zhang, W.-P.

Publications and source records attributed to Zhang, W.-P..

3 recordsLinked to original sources

bioRxiv2019

A key interfacial residue identified with in-cell structure characterization of a Class-A GPCR dimer

G protein coupled receptors (GPCRs) have been shown homo-dimeric. Despite extensive studies, no single residue has been found essential for dimerization. Lacking an efficient method to shift the monomer-dimer equilibrium also makes functional relevance of GPCR dimer elusive. Here, using fluorescence lifetime-based imaging for distance measurements, we characterize the dimeric structure of GPR17, a class A GPCR, in cells. The structure reveals transmembrane helices 5 and 6 the dimer interface, and pinpoints F229 a key residue, mutations of which can render GPR17 monomeric or dimeric. Using the resulting mutants, we show that GPR17 dimer is coupled to both Gi and Gq signaling and is internalized, whereas GPR17 monomer is coupled to Gi signaling only and is not internalized. We further show that residues equivalent to F229 of GPR17 in several other class A GPCRs are also important for dimerization. Our findings thus provide fresh insights into GPCR structure and function.

biochemistry↗

bioRxiv2019

Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13

The interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13NTD) and Ub monomer has been characterized, and it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among three distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have solved the complex structure between Rpn13NTD and K48-diUb. In the structure, Rpn13NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13NTD similarly as the Ub monomer in the known Rpn13NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13NTD. Thus, a charge reversal mutation in Rpn13NTD can weaken the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, blockage of the access of the distal Ub to Rpn13NTD with a proximity attached Ub monomer can also disrupt the interaction between Rpn13 and K48-diUb. Together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.

biophysics↗

bioRxiv2019

Macrocyclic colibactin induces DNA double-strand breaks via copper-mediated oxidative cleavage

Colibactin is an as-yet-uncharacterized human gut bacterial genotoxin, whose biosynthesis is linked to clb genomic island that distributes widespread in pathogenic and commensal human enterobacteria. Colibactin-producing gut microbes promote colon tumor formation and enhance progression of colorectal cancer (CRC) via DNA double-strand breaks (DSBs)-induced cellular senescence and death; however, the chemical basis contributing to the pathogenesis at the molecular level remains elusive. Here we report the discovery and the mechanism of action of colibactin-645 as the highly sought final colibactin metabolite with a novel molecular scaffold. Colibactin-645 recapitulates its previously assumed genotoxicity and cytotoxicity, exhibiting a strong DNA DSBs activity in vitro and in human cell cultures via a unique copper-mediated oxidative mechanism. We also present a complete model for colibactin biosynthesis, revealing an unprecedented dual function of the aminomalonate-utilizing polyketide synthases. This work thus provides the first molecular basis for colibactins genotoxic activity and facilitates further mechanistic study of colibactin-related CRC incidence and prevention.

microbiology↗