title: Dynamics differentiate between active and inactive inteins.
abstract: The balance between stability and dynamics for active enzymes can be somewhat quantified by studies of intein splicing and cleaving reactions. Inteins catalyze the ligation of flanking host exteins while excising themselves. The potential for applications led to engineering of a mini-intein splicing domain, where the homing endonuclease domain of the Mycobacterium tuberculosis RecA (Mtu recA) intein was removed. The remaining domains were linked by several short peptides, but splicing activity in all was substantially lower than the full-length intein. Native splicing activity was restored in some cases by a V67L mutation. Using computations and experiments, we examine the impact of this mutation on the stability and conformational dynamics of the mini-intein splicing domain. Molecular dynamics simulations were used to delineate the factors that determine the active state, including the V67L mini-intein mutant, and peptide linker. We found that (1) the V67L mutation lowers the global fluctuations in all modeled mini-inteins, stabilizing the mini-intein constructs; (2) the connecting linker length affects intein dynamics; and (3) the flexibilities of the linker and intein core are higher in the active structure. We have observed that the interaction of the linker region and a turn region around residues 35-41 provides the pathway for the allostery interaction. Our experiments reveal that intein catalysis is characterized by non-linear Arrhenius plot, confirming the significant contribution of protein conformational dynamics to intein function. We conclude that while the V67L mutation stabilizes the global structure, cooperative dynamics of all intein regions appear more important for intein function than high stability. Our studies suggest that effectively quenching the conformational dynamics of an intein through engineered allosteric interactions could deactivate intein splicing or cleaving.
authors.affiliation: Basic Science Program, Leidos Biomedical Research, Inc., Cancer and Inflammation Program, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Cronin
authors.affiliation: Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH 43210, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Coolbaugh
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: David
authors.initials: D
authors.lastname: Nellis
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, 800 DongChuan Road, Shanghai 200240, China.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH 43210, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Wood
authors.affiliation: Basic Science Program, Leidos Biomedical Research, Inc., Cancer and Inflammation Program, National Cancer Institute, Frederick, MD 21702, USA; Sackler Inst. of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Ruth
authors.initials: R
authors.lastname: Nussinov
authors.affiliation: Basic Science Program, Leidos Biomedical Research, Inc., Cancer and Inflammation Program, National Cancer Institute, Frederick, MD 21702, USA. Electronic address: mabuyong@mail.nih.gov.
authors.forename: Buyong
authors.initials: B
authors.lastname: Ma
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics; D047668:Inteins / Q000235:genetics*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D019154:Protein Splicing; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D011938:Rec A Recombinases; D004720:Endonucleases
keywords: Allostery; Conformational dynamics; Enzyme dynamics; Mini-intein; Non-linear Arrhenius plot; Protein splicing
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