Structural Insights into the GTP-Driven Monomerization and Activation of a Bacterial LRRK2 Homologue Using Allosteric Nanobodies

Core Concepts

GTP binding induces large-scale conformational changes in the bacterial Roco protein CtRoco, leading to monomerization and activation. Binding of conformation-specific nanobodies stabilizes the active GTP-bound state and reveals the underlying structural mechanisms.

Abstract

The content provides structural insights into the GTP-driven activation mechanism of the bacterial Roco protein CtRoco, a homologue of the Parkinson's disease-associated human protein LRRK2.
Key highlights:

The cryo-EM structure of GTPγS-bound CtRoco, stabilized by two conformation-specific nanobodies (NbRoco1 and NbRoco2), reveals an elongated monomeric conformation in contrast to the compact dimeric state observed in the nucleotide-free structure.
GTP binding induces large-scale conformational changes, including a 135° rotation of the LRR domain away from the Roc-COR domains, facilitated by the LRR-Roc linker region and the α0-helix.
Conformational changes in the Roc domain's P-loop and Switch 2 region upon GTP binding are incompatible with the dimeric arrangement, providing an initial trigger for monomerization.
The CORB domain undergoes a 30° rotational movement, using a conserved Roc-CORB interface as a hinge point, which would clash with the adjacent protomer in the dimer, driving monomerization.
NbRoco1 and NbRoco2 bind to the active GTP-bound conformation and stabilize it through allosteric mechanisms, with a synergistic effect observed when both nanobodies are present.
The structural changes observed in CtRoco upon activation are reminiscent of those reported for the activation of human LRRK2, suggesting conserved regulatory mechanisms within the Roco protein family.

Stats

The GTPγS-bound CtRoco monomer displays a 135° rotation of the LRR domain compared to the nucleotide-free dimeric state.
The CORB domain undergoes a 30° rotational movement upon GTP binding.

Quotes

"The cryo-EM structure shows the GTPγS-bound CtRoco as an elongated monomer, where the LRR domain has undergone an approximately 135° rotation away from the Roc and COR domains."
"Comparison of the nucleotide-free CtRoco dimer with the GTPγS-bound monomer suggests a direct and reciprocal link between monomerization and the conformational changes within the protomers."

Key Insights Distilled From

Structural insights in the GTP-driven monomerization and activation of a bacterial LRRK2 homologue using allosteric nanobodies

by Galicia,C., ... at www.biorxiv.org 11-13-2023

https://www.biorxiv.org/content/10.1101/2023.11.13.566844v2

Deeper Inquiries

How do the conformational changes observed in CtRoco relate to the regulation of the kinase activity in human LRRK2?

The conformational changes observed in CtRoco, particularly the movement of the LRR domain away from the Roc and COR domains upon GTP binding, have significant implications for understanding the regulation of the kinase activity in human LRRK2. In LRRK2, the LRR domain wraps around the kinase domain in the inactive state, blocking the entry of substrates into the ATP pocket. Upon activation, the N-terminal domains, including the LRR domain, become disordered, allowing access to the active site of the kinase domain. This conformational change in LRRK2 is similar to the movement observed in CtRoco, where the LRR domain rotates away from the Roc-COR domains upon GTP binding. Therefore, the activation mechanism of CtRoco provides insights into how the conformational changes in the LRR domain may regulate the kinase activity of LRRK2.

How do the potential implications of the allosteric activation mechanisms mediated by NbRoco1 and NbRoco2 for the development of small-molecule modulators targeting the Roc-COR domains of LRRK2?

The allosteric activation mechanisms mediated by NbRoco1 and NbRoco2 offer promising opportunities for the development of small-molecule modulators targeting the Roc-COR domains of LRRK2. By understanding how these nanobodies stabilize the active conformation of CtRoco and enhance its GTPase activity, researchers can design small molecules that mimic the allosteric effects of NbRoco1 and NbRoco2. These small molecules could potentially modulate the GTPase activity of LRRK2, which is dysregulated in Parkinson's disease. By targeting the Roc-COR domains, which play a crucial role in the GTPase activity of LRRK2, these small-molecule modulators could offer a novel approach for therapeutic intervention in Parkinson's disease.

Could the insights into the GTPase-driven activation of CtRoco provide clues about the physiological role and regulation of other Roco proteins across different organisms?

The insights into the GTPase-driven activation of CtRoco could indeed provide valuable clues about the physiological role and regulation of other Roco proteins across different organisms. Roco proteins are a family of GTPases with conserved Roc-COR domains, and the mechanisms of GTPase activation observed in CtRoco are likely to be relevant to other Roco proteins. By understanding how GTP binding induces conformational changes and regulates the activity of CtRoco, researchers can extrapolate these findings to other Roco proteins and investigate their roles in various cellular processes. The regulatory mechanisms identified in CtRoco could serve as a blueprint for studying the activation and function of Roco proteins in different organisms, shedding light on their physiological significance and potential implications in disease states.