Stability and interactions of the amino-terminal domain of ClpB from Escherichia coli.

Abstract

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates aggregated proteins. The sequence of ClpB contains two ATP-binding regions that are enclosed between the N- and C-terminal extensions. Whereas it has been found that the N-terminal region of ClpB is essential for the chaperone activity, the structure of this region is not known, and its biochemical properties have not been studied. We expressed and purified the N-terminal fragment of ClpB (residues 1-147). Circular dichroism of the isolated N-terminal region showed a high content of alpha-helical structure. Differential scanning calorimetry showed that the N-terminal region of ClpB is thermodynamically stable and contains a single folding domain. The N-terminal domain is monomeric, as determined by gel-filtration chromatography, and the elution profile of the N-terminal domain does not change in the presence of the N-terminally truncated ClpB (ClpBDeltaN). This indicates that the N-terminal domain does not form strong contacts with ClpBDeltaN. Consistently, addition of the separated N-terminal domain does not reverse an inhibition of ATPase activity of ClpBDeltaN in the presence of casein. As shown by ELISA measurements, full-length ClpB and ClpBDeltaN bind protein substrates (casein, inactivated luciferase) with similar affinity. We also found that the isolated N-terminal domain of ClpB interacts with heat-inactivated luciferase. Taken together, our results indicate that the N-terminal fragment of ClpB forms a distinct domain that is not strongly associated with the ClpB core and is not required for ClpB interactions with other proteins, but may be involved in recognition of protein substrates. Study holds ProTherm entries: 14875 Extra Details: 10% glycerol and 1mM DTT was added in the experiment,amino-terminal domain ClpB; molecular chaperone; folding domain; protein stability; protein-protein,interactions; differential scanning calorimetry

Submission Details

ID: TeYRV28t

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Tek V;Zolkiewski M,Protein Sci. (2002) Stability and interactions of the amino-terminal domain of ClpB from Escherichia coli. PMID:11967375
Additional Information

Study Summary

Number of data points 2
Proteins Chaperone protein ClpB ; Chaperone protein ClpB
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dHcal ; Experimental Assay: Tm
Libraries Mutations for sequence HMQALKKYTIDLTERAEQGKLDPVIGRDEEIRRTIQVLQRRTKNNPVLIGEPGVGKTAIVEGLAQRIINGEVPEGLKGRRVLALDMGALVAGAKYRGEFEERLKGVLNDLAKQEGNVILFIDELHTMVGAGKADGAMDAGNMLKPALARGELHCVGATTLDEYRQYIEKDAALERRFQKVFVAEPSVEDTIAILR

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Chaperone protein ClpB P63285 CLPB_ECO57
100.0 Chaperone protein ClpB P63286 CLPB_ECOL6
100.0 Chaperone protein ClpB P63284 CLPB_ECOLI
99.5 Chaperone protein ClpB Q7AMH5 CLPB_SALTI
99.5 Chaperone protein ClpB Q7CQ01 CLPB_SALTY
99.5 Chaperone protein ClpB Q7UBW5 CLPB_SHIFL
97.4 Chaperone protein ClpB Q7N788 CLPB_PHOLL
93.8 Chaperone protein ClpB Q74X11 CLPB_YERPE
90.7 Chaperone protein ClpB Q9PGC1 CLPB_XYLFA
90.7 Chaperone protein ClpB Q8EBE6 CLPB_SHEON
90.7 Chaperone protein ClpB Q87S63 CLPB_VIBPA
90.7 Chaperone protein ClpB Q8PHQ4 CLPB_XANAC
90.7 Chaperone protein ClpB Q8P6A0 CLPB_XANCP
90.2 Chaperone protein ClpB Q87AX8 CLPB_XYLFT
90.7 Chaperone protein ClpB Q9KU18 CLPB_VIBCH
90.2 Chaperone protein ClpB Q7VNH1 CLPB_HAEDU
90.2 Chaperone protein ClpB Q8DEV2 CLPB_VIBVU
90.2 Chaperone protein ClpB Q7MNK1 CLPB_VIBVY
90.2 Chaperone protein ClpB P44403 CLPB_HAEIN