title: Structural and mutational studies of a hyperthermophilic intein from DNA polymerase II of Pyrococcus abyssi.
abstract: Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 Å and a heavy atom root mean square deviation of 0.93 Å. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra β-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.
authors.affiliation: Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Du
authors.affiliation: Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Jiajing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Clayton D
authors.initials: CD
authors.lastname: Albracht
authors.affiliation: Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Alice
authors.initials: A
authors.lastname: Hsu
authors.affiliation: Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Wen
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Chemistry, College of the Holy Cross, Worcester, Massachusetts 01610.
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Marieni
authors.affiliation: Department of Chemistry, College of the Holy Cross, Worcester, Massachusetts 01610.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Colelli
authors.affiliation: Department of Chemistry, College of the Holy Cross, Worcester, Massachusetts 01610.
authors.forename: Jennie E
authors.initials: JE
authors.lastname: Williams
authors.affiliation: Department of Chemistry, College of the Holy Cross, Worcester, Massachusetts 01610.
authors.forename: Julie N
authors.initials: JN
authors.lastname: Reitter
authors.affiliation: Department of Chemistry, College of the Holy Cross, Worcester, Massachusetts 01610. Electronic address: kmills@holycross.edu.
authors.forename: Kenneth V
authors.initials: KV
authors.lastname: Mills
authors.affiliation: Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180. Electronic address: wangc5@rpi.edu.
authors.forename: Chunyu
authors.initials: C
authors.lastname: Wang
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D004257:DNA Polymerase II / Q000737:chemistry*; D047668:Inteins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D043903:Pyrococcus abyssi / Q000201:enzymology*
chemical_list: D011506:Proteins; D004257:DNA Polymerase II