Mycobacterial DnaB helicase intein as oxidative stress sensor

Danielle S Kelley,Marlene Belfort,Christopher W Lennon,Zhong Li,Michael R Miller,Hongmin Li,Nilesh K Banavali

doi:10.1038/s41467-018-06554-x

Abstract

Inteins are widespread self-splicing protein elements emerging as potential post-translational environmental sensors. Here, we describe two inteins within one protein, the Mycobacterium smegmatis replicative helicase DnaB. These inteins, DnaBi1 and DnaBi2, have homology to inteins in pathogens, splice with vastly varied rates, and are differentially responsive to environmental stressors. Whereas DnaBi1 splicing is reversibly inhibited by oxidative and nitrosative insults, DnaBi2 is not. Using a reporter that measures splicing in a native intein-containing organism and western blotting, we show that H2O2 inhibits DnaBi1 splicing in M. smegmatis. Intriguingly, upon oxidation, the catalytic cysteine of DnaBi1 forms an intramolecular disulfide bond. We report a crystal structure of the class 3 DnaBi1 intein at 1.95 Å, supporting our findings and providing insight into this splicing mechanism. We propose that this cysteine toggle allows DnaBi1 to sense stress, pausing replication to maintain genome integrity, and then allowing splicing immediately when permissive conditions return.

Highlights

Inteins are widespread self-splicing protein elements emerging as potential post-translational environmental sensors
The first M. smegmatis (Msm) intein, DnaBi1, lacks a homing endonuclease domain (HEN), necessary for invasion of novel sites, and is considered a miniintein (Fig. 1a). This intein localizes to the P-loop of the DnaB ATPase domain at insertion site b, where the P-loop serine that participates in Mg2+ coordination in the mature protein serves as a catalytic residue for intein splicing (Fig. 1b, c)
Inteins are emerging as pervasive post-translational regulatory elements in microbes, where splicing is often coupled to environmental conditions critical to the survival of the host organism or function of the invaded protein[2,3,4,5]

Summary

Introduction

Inteins are widespread self-splicing protein elements emerging as potential post-translational environmental sensors. Inteins have been found to be responsive to a range of stressors and environmental conditions, including temperature[4], DNA damage[2], salt[5,22], redox[6,23], and reactive oxygen and nitrogen species (ROS/RNS)[1,3] These conditions are often highly relevant to the environmental niche of the organism, such as salt with a halophile[5,22], or they relate to the function of the intein-containing protein, like a RadA recombinase intein and its enhanced splicing in the presence of the RadA substrate ssDNA2. A recent study showed that the intein in iron–sulfur scaffold protein SufB of M. tuberculosis is highly sensitive to splicing inhibition by oxidation and modifications caused by ROS and RNS stressors[3]

Methods

Results

Conclusion