Biology subjects

Steyaert, J.

Publications and source records attributed to Steyaert, J..

3 recordsLinked to original sources

bioRxiv2018

Characterisation and structure determination of a llama-derived nanobody targeting the J-base binding protein 1

The J-base Binding Protein 1 (JBP1) contributes to biosynthesis and maintenance of base J ({beta}-D-glucosyl-hydroxymethyluracil), a modification of thymidine confined to some protozoa. Camelid (llama) single domain antibody fragments (nanobodies) targeting JBP1 were produced for use as crystallization chaperones. Surface plasmon resonance (SPR) screening identified Nb6 as a strong binder, recognising JBP1 with a 1:1 stoichiometry and high affinity (kD=30nM). Crystallisation trials of JBP1 in complex with Nb6, yielded crystals diffracting to 1.47[A] resolution.\n\nHowever, the asymmetric unit dimensions and molecular replacement with a nanobody structure, clearly showed that the crystals of the expected complex with JBP1 were of the nanobody alone. Nb6 crystallizes in spacegroup P31 with two molecules in the asymmetric unit; its crystal structure was refined to a final resolution of 1.64[A]. Ensemble refinement suggests that on the ligand-free state one of the complementarity determining regions (CDRs) is flexible while the other two adopt well-defined conformations.\n\nSynopsisA camelid single domain antibody fragment (nanobody) is shown to have high affinity towards its recognition target, the J-base binding protein 1 (JBP1). The serendipitous crystallisation of this nanobody alone, and its crystal structure solution and refinement to 1.64[A] resolution are described. Ensemble refinement suggests that on the ligand-free state one of the complementarity determining regions (CDRs) is flexible while the other two adopt well-defined conformations.

biochemistry↗

bioRxiv2018

Heteromeric GABAA receptor structures in positively-modulated active states

Type-A {gamma}-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels (pLGICs), typically consisting of /{beta}/{gamma} subunit combinations. They are the principal mediators of inhibitory neurotransmission throughout the central nervous system and targets of major clinical drugs, such as benzodiazepines (BZDs) used to treat epilepsy, insomnia, anxiety, panic disorder and muscle spasm. However, the structures of heteromeric receptors and the molecular basis of BZD operation remain unknown. Here we report the cryo-EM structure of a human 1{beta}3{gamma}2 GABAAR in complex with GABA and a nanobody that acts as a novel positive allosteric modulator (PAM). The receptor subunits assume a unified quaternary activated conformation around an open pore. We also present crystal structures of engineered 5 and 5{gamma}2 GABAAR constructs, revealing the interfacial site for allosteric modulation by BZDs, including the binding modes and the conformational impact of the potent anxiolytic and partial PAM, bretazenil, and the BZD antagonist, flumazenil. These findings provide the foundation for understanding the mechanistic basis of GABAAR activation.

biophysics↗

bioRxiv2018

A genetically encoded biosensor reveals location bias of opioid drug action

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. One way that drugs can differ is by imposing selective effects on the conformational equilibrium of individual ORs. We wondered if drugs can also impose selective effects on the location of OR activation in individual OR-expressing neurons. Here we develop a genetically encoded biosensor that directly localizes ligand-induced activation and deactivation of ORs in living cells, and use it to generate the first real-time map of the spatiotemporal organization of - and {delta}-OR activation in neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by uniquely driving OR activation in the somatic Golgi apparatus and extending throughout the dendritic arbor in Golgi outposts. These results demonstrate a new approach to probe the cellular basis of neuromodulation and reveal that drugs profoundly distort the spatiotemporal landscape of neuronal OR activation.

neuroscience↗