DNA recognition by Escherichia coli CbpA protein requires a conserved arginine-minor-groove interaction.

Kiran Chintakayala,Shivani S Singh,Ilja Westerlaken,Rajesh Shahapure,Laura E Sellars,Anne S Meyer,David C Grainger,Remus T Dame

doi:10.1093/nar/gkv012

Abstract

Curved DNA binding protein A (CbpA) is a co-chaperone and nucleoid associated DNA binding protein conserved in most γ-proteobacteria. Best studied in Escherichia coli, CbpA accumulates to >2500 copies per cell during periods of starvation and forms aggregates with DNA. However, the molecular basis for DNA binding is unknown; CbpA lacks motifs found in other bacterial DNA binding proteins. Here, we have used a combination of genetics and biochemistry to elucidate the mechanism of DNA recognition by CbpA. We show that CbpA interacts with the DNA minor groove. This interaction requires a highly conserved arginine side chain. Substitution of this residue, R116, with alanine, specifically disrupts DNA binding by CbpA, and its homologues from other bacteria, whilst not affecting other CbpA activities. The intracellular distribution of CbpA alters dramatically when DNA binding is negated. Hence, we provide a direct link between DNA binding and the behaviour of CbpA in cells.

Highlights

Escherichia coli curved DNA binding protein A (CbpA) was first isolated on the basis of its propensity to bind intrinsically curved, AT-rich, DNA molecules [1,2]
Conserved in many ␥ -proteobacteria, CbpA consists of three domains; the Nterminal J-domain is separated from two C-terminal domains (CTDI and CTDII) by a flexible linker (Figure 1A)
CbpA was initially isolated alongside the histone-like nucleoid structuring (H-NS) protein in a screen of E. coli cell extracts for proteins binding AT-rich DNA fragments [1,2]

Summary

Introduction

Escherichia coli curved DNA binding protein A (CbpA) was first isolated on the basis of its propensity to bind intrinsically curved, AT-rich, DNA molecules [1,2]. It has subsequently been shown that CbpA is multifunctional, having both co-chaperone and DNA binding activities [3,4,5,6]. Whilst the relationship between these activities is unknown it is clear that CbpA is a stress response protein; CbpA is produced during periods of starvation and DNA binding protects nucleic acids from damage [7]. Conserved in many ␥ -proteobacteria, CbpA consists of three domains; the Nterminal J-domain is separated from two C-terminal domains (CTDI and CTDII) by a flexible linker (Figure 1A). The J-domain, a highly conserved feature of DnaJ-like co-chaperones, was shown to interact with DnaK (a chaperone) and CbpM (a CbpA inhibitor) but was dispensable for DNA binding, an activity that locates to the linker-CTDI region. CbpM forms a dimer that can bind two copies of the CbpA J-domain (Figure 1Bi). The CTDI–CTDII dimer forms a 30 Acleft (Figure 1Bii)

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