Allosteric modulation of the GTPase activity of a bacterial LRRK2 homolog by conformation-specific Nanobodies

Abstract

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer–monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer–monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations.

Exploring cellular biochemistry with nanobodies

“…Nanobodies can inhibit intracellular signaling proteins, such as the transcription factor STAT3 (151), the uridyltransferase TUT4 (152), apicomplexan calcium-dependent kinase (153), the actin-capping protein CapG (154), the AMPylation enzyme HypeE (155) and the Salmonella ADP-ribosylation enzyme SpvB (156). A nanobody that targets an allosteric site on a bacterial LRRK2 GTPase homologue modulates oligomerization and enzymatic activity (157). The ER-localized E2 enzyme UBC6e is targeted by a nanobody that augments enzymatic activity without obvious biological consequences (158).…”

Transforming nanobodies into high-precision tools for protein function analysis

“…This does not necessarily qualify them as therapeutic agents but in the first place as tools to study protein dynamics and conformational changes in protein structure causing alterations in protein function. In Parkinson's disease, the LRRK2 leucine-rich repeat kinase 2 and α-synuclein both play a very important role (92). Apart from phosphorylating substrates, LRRK2 also displays GTPase activity.…”

Nanobodies as allosteric modulators of Parkinson’s disease-associated LRRK2

“…To determine the binding affinity (dissociation constant K D ) of the ten Nbs, two methods were used in parallel: microscale thermophoresis (MST) and bio-layer interferometry (BLI). For the MST experiment, we site-specifically labeled the ten Nbs with an m-TAMRA fluorophore at its Cterminus, using sortase-mediated coupling (24), and then titrated increasing amounts of full-length LRRK2 to these Nbs (Fig. 3b, Fig.…”

“…One way to modulate the dynamics, regulation and activity of proteins is via the use of Nanobodies (Nbs), the small and stable singledomain fragments derived from camelid heavy chain-only antibodies (23). Accordingly, we recently reported the identification of Nbs that allosterically activate the GTPase activity of a bacterial LRRK2 homologue by interfering with the protein's monomer-dimer cycle (24).…”