Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA.

Eva I Hyde,Scott A White,Rohanah Hussain,Peter Timmins,Christopher M Thomas,Giuliano Siligardi,Trushar R Patel,Karthik V Rajasekar,David J Scott,Philip Callow

doi:10.1042/bcj20170281

Abstract

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.

Highlights

A large proportion of proteins have stretches of 50 residues or more that do not fold but exist as dynamic ensembles with little or no rigid secondary structure
The circular dichroism (CD) spectra show that the secondary structure of KorB is minimally affected on complex formation
In a small-angle X-ray scattering (SAXS) study of a comparable ParB protein from Mycobacterium tuberculosis, a reduction in overall radius of gyration of electron scattering was observed on DNA binding [42] and, it was proposed that the protein became more compact upon complex formation

Summary

Introduction

A large proportion of proteins have stretches of 50 residues or more that do not fold but exist as dynamic ensembles with little or no rigid secondary structure (reviewed in ref. [1]). These intrinsically disordered regions (IDRs) act as sites for interactions with multiple targets and are over-represented in classes of proteins that form network hubs, such as transcription factors and signalling proteins Proteins with such regions, extremely important for cellular processes, are difficult to characterise structurally, as they cannot be crystallised and are often too large for NMR studies and too small for CryoElectron Microscopy. Two further regions of KorB are predicted from the amino acid sequence to be intrinsically disordered: one between the DNA-binding domain and the C-terminal dimerisation domain (residues 253–293), and the other at the N-terminus (residues 1–54) These structural predictions are consistent with our previous studies of the free protein and deletion mutants, using NMR spectroscopy, circular dichroism (CD) spectroscopy and small-angle X-ray scattering (SAXS) [21]. Solvent matching has been used previously to examine macromolecules selectively in binary complexes; we have extended this technique to examine KorB directly in a ternary complex with KorA and DNA, by partial deuteration of KorA to give the same match point as the DNA

Experimental procedures

Results

Discussion