Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage.
Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.
Department of Chemistry, College of the Holy Cross , Worcester, Massachusetts 01610, United States.
Christopher J
CJ
Minteer
Department of Chemistry, College of the Holy Cross , Worcester, Massachusetts 01610, United States.
Nicolle M
NM
Siegart
Department of Chemistry, College of the Holy Cross , Worcester, Massachusetts 01610, United States.
Kathryn M
KM
Colelli
Xinyue
X
Liu
Robert J
0000-0003-2219-5833
RJ
Linhardt
Chunyu
C
Wang
Department of Chemistry, College of the Holy Cross , Worcester, Massachusetts 01610, United States.
Alvin V
AV
Gomez
Department of Chemistry, College of the Holy Cross , Worcester, Massachusetts 01610, United States.
Julie N
JN
Reitter
Department of Chemistry, College of the Holy Cross , Worcester, Massachusetts 01610, United States.
Kenneth V
0000-0001-6052-308X
KV
Mills
D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D003500:Cyclization; D004257:DNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D009154:Mutation; D011499:Protein Processing, Post-Translational; D043903:Pyrococcus abyssi / Q000201:enzymology
D001224:Aspartic Acid; D004257:DNA Polymerase II