Bodet-Lefevre, S. | Search bioRxiv

bioRxiv2026

A long-chain heparan sulfate capture mechanism directs paracrine GDNF-GFRα1 signalling through RET

The receptor tyrosine kinase RET is activated by GDNF and GFR1 together as a bipartite ligand, driving receptor activation and signalling in developmental and neuroprotective contexts. Evidence from developmental and cell models has suggested that heparan sulfate (HS) functions as a fourth component in RET signalling by binding to GDNF, but the molecular details remain unclear. Here, we present the cryo-EM structure of the heterohexameric RET:GDNF:GFR1 complex with a fully resolved heparin ligand, revealing an unexpected extended HS binding site spanning all three proteins. The architecture of the complex and binding mode of the HS chain in this complex enables the formation of a higher order 4:4:4 assembly bound to a single 30-saccharide HS chain which bridges two intimately bound complexes. This multi-protein interface selectively binds the highly sulfated domains of HS over other GAG classes, and is essential for RET activation in trans with soluble GFR1, but not in cis when GFR1 is membrane-bound. Our data suggest that HS shapes the dynamics of RET signalling at every stage, from ligand diffusion to signalling complex formation. Thus, GDNF-GFRa1 paracrine signalling reveals a surprising dependence for long-chain GAG function in which the glycan engages in both receptor complex assembly and clustering.

biochemistry↗

bioRxiv2026

Relaxase Asymmetry Drives Initiation of Bacterial Conjugation

Bacterial conjugation is the major route of horizontal gene transfer and a key driver of the dissemination of antibiotic resistance, exacerbating the global health crisis. Central to this process is TraI, a bifunctional relaxase-helicase encoded by the iconic F-plasmid family. TraI initiates single-stranded DNA (ssDNA) transfer by covalently attaching to plasmid DNA through its trans-esterase (TE) activity and subsequently unwinds the plasmid via its helicase activity. Although genetic and biochemical studies have postulated that two TraI molecules are required for efficient conjugative transfer, how these molecules coordinate their activities in space and time - and how strand nicking is coupled to helicase loading - has remained unknown. Here, we capture the elusive, functionally asymmetric TraI homodimer and report its cryo-EM structure, revealing that the interaction between the two TraI molecules is mediated predominantly by ssDNA. We further show that TE-driven duplex melting at the origin of transfer (oriT) by one TraI molecule enables helicase loading by a second TraI molecule on the transfer strand of the unnicked plasmid DNA. Although the TE domain is intrinsically competent for strand nicking, its activity is inhibited by the host factor IHF, and this inhibition is relieved upon helicase loading. Together, these findings define a regulatory loop that coordinates helicase loading with strand nicking, providing mechanistic insight into the earliest stages of conjugative DNA processing.

microbiology↗