The roles of conserved amino acids on substrate binding and conformational integrity of ClpB N-terminal domain.
Escherichia coli heat shock protein ClpB disaggregates denatured protein in cooperation with the DnaK chaperone system. Several studies showed that the N-terminal domain is essential for the chaperone activity, but its role is still largely unknown. The N-terminal domain contains two structurally similar subdomains, and conserved amino acids Thr7 and Ser84 share the same position in two apparent sequence repeats. T7A and S84A substitutions affected chaperone activity of ClpB without significantly changing the native conformation [Liu, Z. et al. (2002) J. Mol. Biol. 321, 111-120]. In this study, we aimed to better understand the roles of several conserved amino acid residues, including Thr7 and Ser84, in the N-terminal domain. We investigated the effects of mutagenesis on substrate binding and conformational states of ClpB N-terminal domain fragment (ClpBN). Fluorescence polarization analysis showed that the T7A and S84A substitutions enhanced the interaction between ClpBN and protein aggregates. Interestingly, further analyses suggested that the mechanisms by which they do so are quite different. For T7A substitution, the increased substrate affinity could be due to a conformational change in the hydrophobic core as revealed by NMR spectroscopy. In contrast, for S84A, increased substrate binding would be explained by a unique conformational state of this mutant as revealed by pressure perturbation analysis. The thermal transition temperature of the S84A mutant, monitored by DSC, was 6.1 degrees C lower than that of wild-type. Our results revealed that conserved amino acids Thr7 and Ser84 both participated in maintaining the conformational integrity of the ClpB N-terminal domain.
Tanaka
Naoki
N
Department of Bio-molecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan. tanaka@kit.ac.jp
Tani
Yasushi
Y
Tada
Tomoko
T
Lee
Yin-Fai
YF
Kanaori
Kenji
K
Kunugi
Shigeru
S
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D013912:Threonine; D012694:Serine; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli