Split NanoLuc technology allows quantitation of interactions between PII protein and its receptors with unprecedented sensitivity and reveals transient interactions

Rokhsareh Rozbeh,Karl Forchhammer

doi:10.1038/s41598-021-91856-2

Abstract

PII proteins constitute a widespread signal transduction superfamily in the prokaryotic world. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. Depending on bound effector molecule, PII proteins interact with and modulate the activity of multiple target proteins. To investigate the complexity of interactions of PII with target proteins, analytical methods that do not disrupt the native cellular context are required. To this purpose, split luciferase proteins have been used to develop a novel complementation reporter called NanoLuc Binary Technology (NanoBiT). The luciferase NanoLuc is divided in two subunits: a 18 kDa polypeptide termed “Large BiT” and a 1.3 kDa peptide termed “Small BiT”, which only weakly associate. When fused to proteins of interest, they reconstitute an active luciferase when the proteins of interest interact. Therefore, we set out to develop a new NanoBiT sensor based on the interaction of PII protein from Synechocystis sp. PCC6803 with PII-interacting protein X (PipX) and N-acetyl-L-glutamate kinase (NAGK). The novel NanoBiT sensor showed unprecedented sensitivity, which made it possible to detect even weak and transient interactions between PII variants and their interacting partners, thereby shedding new light in PII signalling processes.

Highlights

PII proteins constitute a widespread signal transduction superfamily in the prokaryotic world
Mainly through PII-co-purification approaches, novel interaction partners of PII proteins have been identified in cyanobacteria, such as the biotin carboxyl carrier protein (BCCP)-subunit of the AcetylCoA carboxylase[15], an ensemble of nitrogen uptake systems
We decided to separate the NanoLuc Binary Technology (NanoBiT) fragments in two distinct polypeptites: on PII and a PII-receptor protein, using the 16 aa linker to fuse the SmBiT fragment, which appeared most suitable from the initial experiments

Summary

Introduction

PII proteins constitute a widespread signal transduction superfamily in the prokaryotic world. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. PII signalling proteins are ubiquitous in nature, in particular in Prokaryotes and plastids of A rchaeplastida[1] Their general task is to sense the metabolic state of the cells by binding in an interactive manner the metabolite status reporter molecules ATP and ADP as well as 2-oxoglutarate[2]. System URT and ammonium transport protein AMT1)[16], the Phosphoenolpyruvate carboxylase (PepC)[17] and two regulatory peptides, termed PirC and PirA, the former regulating carbon flow through interaction with the Phosphoglycerate mutase (PGAM), and the second, the arginine synthesis p athway[18,19] For all the latter interaction partners, structural information is not yet available. This allows PipX to sense the energy status via PII interaction whereas at the same time, NAGK can read out the level of 2-oxoglutarate (a status reporter of the carbon/nitrogen balance), provided that PII is present in excess over the receptors, which appears to be the c ase[21]

Methods

Results

Conclusion