Functional characterization of the active site residues in the HNH motif of colicin E7

• Main Authors: Shen, Yong Liang, 沈勇良
• Other Authors: Hanna S. Yuan
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/99473680926373321282

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 91 === Colicins are toxic proteins released by Escherichia coli when bacteria encounter environmental stresses. This type of toxin kills the related bacteria strain nearby to eliminate the opponents of host cells. Depending on the receptor required to enter target cells, colicins can be classified into several groups. Colicin E7 (ColE7) belongs to the family of E group colicins which invade their targets via BtuB, a receptor originally used to transport vitamin B12 in E. coli. The toxicity of ColE7 comes from its C-terminal DNase domain, where an HNH motif is located. ColE7 kills target cells by degrading their chromosomal DNA. The HNH motif has been identified in many endonucleases and the HNH stands for the most conserved His and Asn residues in this motif. In ColE7, the three residues correspond to His545, Asn560 and His569. To find out the roles of His545 and Asn560 in DNA hydrolysis in the DNase domain of ColE7 (nuclease-ColE7), several nuclease-ColE7 mutants were constructed using site-directed mutagenesis, including H545A, H545E, H545Q, N560A, N560D, and N560H. Moreover, two basic residues located at the C-terminus of HNH motif, Lys567 and Arg568, were mutated to alanine (K567A and R568A). The conformation integrity of these mutants was first checked by circular dichroism (CD) and tryptophan fluorescence emission. The DNase activity for the wild-type and the mutant proteins were then monitored by plasmid nicking experiments and the fluorescence resonance energy transfer (FRET) method. We find that the CD and tryptophan emission spectra of all mutants are almost identical to those of wild type, except for N560D which shows minor variation in tryptophan fluorescence emission spectra. This implies that mutations of these residues do not change the protein overall conformation. The plasmid nicking experiment shows that K567A and R568A remain ~100 % DNase activity; N560A, N560D and N560H contain ~10 % activity; and H545A, H545E and H545Q, contain only ~2 % activity as compared to that of the wild-type enzyme. Similarly in the FRET method, H545A, H545D and H545E show no detectable DNase activity, while N560A, N560D and N560H have reduced activity with kcat of 2.6 %, 7 % and 1.5 % and Km of 6, 6.5 and 4.1 times to those of the wild type enzyme, respectively. These results support the earlier suggestion that His545 is the general base in DNA hydrolysis, therefore the mutation at this position abolishes the DNase activity. Asn560 was suggested to play a simple structural role to stabilize the protein conformation, since this conserved residue is located far away from the active site. However, in this study, we find that the protein conformation for the Asn560 mutants are mostly retained, but their DNase activities decrease significantly. The main chain carbonyl oxygen of Val555 in the loop region in the HNH motif makes hydrogen bond with the imidazole ring of His545. This interaction may fix the side-chain conformation and increase the pKa of His545. It is likely that Asn560 stabilizes only the loop structure in the HNH motif but not the overall structure. Asn560 therefore likely affects enzyme activity by stabilizing the local loop structure to maintain the hydrogen bond between Val555 and His545.