Structural and Mutational Studies of a Hyperthermophilic Intein from DNA Polymerase II of Pyrococcus abyssi

Zhenming Du,Jiajing Liu,Clayton D Albracht,Alice Hsu,Wen Chen,Michelle D Marieni,Kathryn M Colelli,Jennie E Williams,Julie N Reitter,Kenneth V Mills,Chunyu Wang

doi:10.1074/jbc.m111.290569

Zhenming Du, Jiajing Liu + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m111.290569

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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.

Highlights

The PolII intein from the hyperthermophilic Pyrococcus abysii only splices at very high temperature
The Pab PolII intein [23] is only the second intein demonstrated to splice with a C-terminal glutamine, after the Chiloiridescent virus (CIV) RNR intein [21, 22]
Protein Overexpression, Purification, and NMR Sample Preparation—The Pab PolII intein gene cloned into pETM-44 vector ppC1Q185 expresses a fusion protein with an N-terminal (His)6 tag and maltose-binding protein (MBP)

Summary

Background

The PolII intein from the hyperthermophilic Pyrococcus abysii only splices at very high temperature. The Pyrococcus abyssi DNA polymerase II intein, abbreviated as the Pab PolII4 intein, is found in a hyperthermophilic organism that lives near deep sea thermal vents, with an optimal growth temperature of 96 °C [9] This suggests that one could isolate a stable precursor with native intein sequence for structural studies. There are four steps of protein splicing in canonical inteins (Fig. 1A): step 1, N-X acyl shift (X ϭ S or O); step 2, transesterification and the formation of a branched intermediate; step 3, asparagine cyclization coupled with C-terminal cleavage; and step 4, X-N acyl shift and succinimide hydrolysis [10, 11] These steps are catalyzed by active site residues in conserved blocks in intein sequences.

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION