Effects of T1084d and T1084A Point Mutations in the NtTkr Tail of Nicotiana tabacum on Coiled-helix Structure and Interaction with Target Proteins

doi:10.13560/j.cnki.biotech.bull.1985.2018-0957

Abstract

Abstract: As a novel member of kinesin family,NtTkr is predominantly expressed in the vigorous division tissues of tobacco,and in it there are 3 coiled-coils（CC1,CC2 and CC3）. It is known that the point mutation and T deletion of T1084 in the conventional CC3 motif of the tail may affect the distribution of NtTkr,and which is inferred to be affected by the protein substrate binding with it. In order to determine the effects of deletion and point mutations of T1084 on the interaction with candidate proteins,T1084 deletion（T1084d）and T1084A point mutations were introduced into the tobacco NtTkr tail by overlap extension PCR,cloned into pGBKT7 to construct bait expression vector,and transferred to AH109 and mated to Y187 with pGADT7-H2A/Sulfer/NT3 for two-hybrid analysis. The results showed that T1084A was similar to T,but bluing earlier and the color was darker than T;while T1084d did not perform as T did. The primary structure and Coiled-coil software analysis showed that T1084 was located in the ‘a’ position of CC3 and was a polar hydrophilic amino acid. Substituting it with non-polar hydrophobic alanine enhanced the hydrophobicity of heptapeptide repeat domain（HR domain）,and resulting in the prolongation of repeated sequence of heptapeptide,while the deletion of T1084 resulted in its shortening. The results indicate that increasing the hydrophilicity of HR domain and the length of heptapeptide repeat sequence may enhance the interaction of NtKrp with the candidate protein,while shortening of coiled coil may result in a decrease of their interaction.

Key words: NtTkr, coiled-coil, site-directed mutagenesis, protein interaction

XU Lin-na, HU Meng-ke, TONG Wen-yan, LI Fen. Effects of T1084d and T1084A Point Mutations in the NtTkr Tail of Nicotiana tabacum on Coiled-helix Structure and Interaction with Target Proteins[J]. Biotechnology Bulletin, 2019, 35(5): 64-69.

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