Allosteric Influence of Extremophile Hairpin Motif Mutations on the Protein Splicing Activity of a Hyperthermophilic Intein

Abstract

Protein splicing is a post-translational process mediated by an intein, whereby the intein excises itself from a precursor protein with concomitant ligation of the two flanking polypeptides. The intein that interrupts the DNA polymerase II in the extreme hyperthermophile Pyrococcus abyssi has a β-hairpin that extends the central βsheet of the intein. This β-hairpin is mostly found in inteins from archaea, as well as halophilic eubacteria, and is thus called the extremophile hairpin (EXH) motif. The EXH is stabilized by multiple favorable interactions, including electrostatic interactions involving Glu29, Glu31, and Arg40. Mutations of these residues diminish the extent of Nterminal cleavage and the extent of protein splicing, likely by interfering with the coordination of the steps of splicing. These same mutations decrease the global stability of the intein fold as measured by susceptibility to thermolysin cleavage. 15 N− 1 H heteronuclear single-quantum coherence demonstrated that these mutations altered the chemical environment of active site residues such as His93 (B-block histidine) and Ser166 (F-block residue 4). This work again underscores the connected and coordinated nature of intein conformation and dynamics, where remote mutations can disturb a finely tuned interaction network to inhibit or enhance protein splicing.

An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein

“…We also compared the two inteins by their relative sensitivity to degradation by the thermostable protease thermolysin, which digests proteins in unstructured regions 20 . Thermolysin has been used to measure the stability of minimized versions of the Mycobacterium tuberculosis RecA intein toward thermal unfolding 21 , and we recently used thermolysin analysis to determine the contribution of mutations in the extremophile hairpin (EXH) motif comprised by β strands 4 and 5 to the stability of the fold of the Pab PolII intein 22 . Incubation with thermolysin leaves the Pho PolII intein uncleaved under conditions that lead to controlled digestion of the Pab PolII intein (Figs.…”

“…This is followed by strands β2 and β3 from Arg7 to Ile12 and Thr15 to Thr20, respectively, and then an α-helix from Leu21 to Leu27, with one side packed against the intein β-sheet (Leu21, Leu24, Tyr25) and the other side facing solvent (Lys22, Glu23, and Glu26). Next, strands β4 (Phe28 to Glu34) and β5 (Val37 to Pro44) make up the extremophile hairpin motif 22 . This is followed by a twisted hairpin of β6 (Ile47 to Asn 53) and the first half of β7 (β7a, Lys58 to Ala 70).…”