Functional Analysis of CbpA, a DnaJ Homolog and Nucleoid-associated DNA-binding Protein

Jeremy G Bird,Suveena Sharma,Sara C Roshwalb,Joel R Hoskins,Sue Wickner

doi:10.1074/jbc.m603365200

Jeremy G Bird, Suveena Sharma + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m603365200

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Abstract

DnaK/Hsp70 proteins are universally conserved ATP-dependent molecular chaperones that help proteins adopt and maintain their native conformations. DnaJ/Hsp40 and GrpE are co-chaperones that assist DnaK. CbpA is an Escherichia coli DnaJ homolog. It acts as a multicopy suppressor for dnaJ mutations and functions in vitro in combination with DnaK and GrpE in protein remodeling reactions. CbpA binds nonspecifically to DNA with preference for curved DNA and is a nucleoid-associated protein. The DNA binding and co-chaperone activities of CbpA are modulated by CbpM, a small protein that binds specifically to CbpA. To identify the regions of CbpA involved in the interaction of CbpA with CbpM and those involved in DNA binding, we constructed and characterized deletion and substitution mutants of CbpA. We discovered that CbpA interacted with CbpM through its N-terminal J-domain. We found that the region C-terminal to the J-domain was required for DNA binding. Moreover, we found that the CbpM interaction, DNA binding, and co-chaperone activities were separable; some mutants were proficient in some functions and defective in others.

Highlights

A Data not shown. ϩϩϩ, wild type CbpA activity or property; ϩϩ, less than wild type; ϩ, much less than wild type; Ϫ, not detected. b Numbers in parentheses refer to the position and substitution in site-directed mutants. c NT, not tested
The apparent molecular masses of CbpA(J) and CbpA(J,L,CTDI) did not change following BS3 treatment, suggesting that those mutants existed as monomers (Fig. 7, lanes 4 –7)
The following is a summary of our findings. (i) The CbpM interacting region is within the J-domain. This is somewhat surprising in that the J-domain is highly conserved between E. coli DnaJ and CbpA, yet CbpM interacts with CbpA and not DnaJ. (ii) Residues essential for DNA binding are C-terminal to the J-domain, including the linker region and CTD I, with some contribution from CTD II. (iii) The co-chaperone, DNA binding, and CbpM interaction functions of CbpA are separable. (iv) Dimerization of CbpA is through CTD II

Summary

EXPERIMENTAL PROCEDURES

Plasmids—CbpA domain deletions were constructed by generating cbpA PCR fragments coding for CbpA(1–78), CbpA(1– 201), CbpA(79 –306), and CbpA(202–306) that contained 5Ј and 3Ј NdeI and HindIII sites, respectively, and lacked a terminal stop codon. DNA Binding Assay—Reaction mixtures (20 ␮l) contained Buffer B (20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol (v/v)), 100 mM KCl, 0.005% Triton X-100 (v/v), 50 ␮g/ml bovine serum albumin, 9 fmol [3H]oriP1 plasmid DNA (2700 cpm/fmol), and 720 nM CbpA unless otherwise indicated. Chaperone Assay—To measure activation of RepA, reaction mixtures (20 ␮l) contained Buffer B, 1 mM ATP, 100 mM KCl, 10 mM MgOAc, 50 ␮g/ml bovine serum albumin, 0.005% Triton X-100 (v/v), 4 nM RepA, CbpA or CbpA mutant protein as indicated, 228 nM GrpE, and 430 nM DnaK. CbpA-RepA Binding Assay—Reaction mixtures (100 ␮l) contained 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol (v/v), 0.005% Triton X-100 (v/v), 100 ␮g/ml bovine serum albumin, 125 nM [3H]RepA (960 cpm/pmol), and CbpA or CbpA mutant protein as indicated. Retentates containing CbpA1⁄7RepA complexes were recovered in 100 ␮l of 10% SDS, and the radioactivity measured

RESULTS

To identify the region of the

CbpM interaction

NTc NTc NTc NTc

DISCUSSION