Elucidation of DNA Recognition Mode for the Lon Protease α-domain from Brevibacillus thermoruber WR-249
Date Issued
2011
Date
2011
Author(s)
Chen, Yu-Da
Abstract
Lon protease is a multifunctional enzyme, and it’s functions include the degradation of damaged proteins, ATPase, and chaperone-like activities. It is highly conserved in prokaryotes and eukaryotic organelles. Our previous studies demonstrate the DNA binding ability as a novel function for a thermostable Lon protease from Brevibacillus thermoruber WR-249. However, the physiological role and structure of DNA binding by Lon was still poorly understood.
Bioinfomatic analysis reveals that the α-domain of Bt-Lon should be a new member of DNA binding domains with an uncharacterized recognition mode. With the aim of elucidating the DNA recognition mode of Bt-Lon, NMR technique and protein crystallography were employed to determine the structures of free and DNA-bound forms of α-domain. The α-domain was crystallized by the sitting drop, vapor diffusion method. X-ray diffraction data was collected at a synchrotron-radiation source and belonged to space group P23 , with unit cell parameters a = b = c = 93.8 Å , α = 90°, β = 90° , γ = 90° . The Bt-Lon α-domain contains four α helices and two parallel β strands and resembles similar domains found in a variety of ATPases. NMR chemical shift perturbation experiments suggest that there are three major sites responsible for DNA binding : (1) α4 ; (2) the loop between α1 and β1 ; (3) and the loop between α3 and β2. The feasible model of protein-DNA complex was docking with HADDOCK software based on the mutation and NMR perturbation data.
Subjects
Brevibacillus thermoruber WR-249
Lon
α-domain
X-ray crystallography
NMR
Type
thesis
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