Biology subjects

Botte, M.

Publications and source records attributed to Botte, M..

3 recordsLinked to original sources

bioRxiv2021

Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 reveal a novel binding site for positive modulators

Kv3 ion-channels constitute a class of functionally distinct voltage gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6 [A] resolution using full length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well resolved Zinc site and displaying a rotation by 35{degrees}. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator Lu AG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.

biochemistry↗

bioRxiv2021

Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies

Lipopolysaccharides (LPS) are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria 1. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex 2. Despite detailed insights from X-ray crystallography into the architecture of LptDE transporter complexes 3-5, no structure of a laterally open LptD transporter has been described, a transient state that occurs during LPS release 6. To facilitate the acquisition of hitherto unknown conformations we subjected LptDE of N. gonorrhoeae to cryo-EM analyses. In complex with newly designed rigid chaperones derived from nanobodies (Pro-Macrobodies, PMbs) we obtained a map of a partially opened LptDE transporter at 3.4 [A] resolution and in addition we captured a laterally fully opened LptDE complex from a subset of particles. Our work offers new insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a novel, highly rigid and widely applicable chaperone scaffold to enable structural biology of challenging protein targets.

biochemistry↗

bioRxiv2020

Cryo-EM structural studies of the agonist complexed human TRPV4 ion-channel reveals novel structural rearrangements resulting in an open-conformation

The human transient receptor potential vanilloid 4 (hTRPV4) ion channel plays a critical role in a variety of biological processes. Whilst the activation of hTRPV4 gating properties has been reported for a broad spectrum of stimuli, including synthetic 4-phorbols, the molecular basis of the activation is poorly understood. Here we report the novel cryo-EM structure of the hTRPV4 determined in the presence of the archetypical phorbol acid agonist, 4-PDD. Complementary mutagenesis experiments support the EM-identified binding site as well as allowing rationalization of disruptive mutants located outside of the 4-PDD binding site. This work represents the first structural information of hTRPV4 in a ligand-induced open conformation. Together, our data reveal the underlying molecular mechanisms resulting in the opening of the central pore and ion-channel activation and provide a structural template for designing inhibitors targeting the open-state conformation of hTRPV4.

molecular biology↗