Intein Structure and Function
Posttranslational modifications of proteins occur widely and greatly increase the diversity of protein functions in living organisms. We are investigating a particularly interesting type of modification: protein splicing. Protein splicing refers to the cleavage and ligation of protein fragments from precursors in a series of coordinated reactions catalyzed by a single protein domain known as an intein. Inteins occur as insertions in a variety of different genes in unicellular organisms. They can catalyze their own excision from the precursor protein and the concomitant ligation of the flanking regions of the precursor (exteins). This multistep reaction includes the cleavage of 2 peptide bonds at the intein-extein junctions and the formation of a new peptide bond linking the 2 exteins. In this research, a collaboration with F.B. Perler, New England BioLabs, Ipswich, Massachusetts, we aim to determine the structural factors that allow a single protein domain to catalyze such a complex process.
Because they occur only in microorganisms, inteins may be good targets for new antibiotic or antifungal therapies. The proteins have also been used in biotechnology, such as in the synthesis of artificial proteins with chemical labels or proteins with specific isotope-labeling patterns for NMR studies. This project thus relates also directly to biomacromolecular NMR, a key technique used in the Skaggs Institute.
In continuation of previous work, we refined the NMR structure of the intein KlbA from the hyperthermophilic archaeon Methanococcus jannaschii. For the structural studies, we used a modified construct with 2 amino acid replacements that prevented splicing and yielded a precursor protein that is stable in solution. In the scaffold of the horseshoe-shaped intein fold, which includes 14 β-strands, 1 major α-helix, and 2 310-helical turns, we could now identify detailed local features with direct relevance for intein function (Fig. 2).
The intein-extein junction sites occur on extended strands that pass through the center of the horseshoe and contribute to the formation of the active site together with residues from other β-strands. Two residues of a conserved sequence motif, T100 and H103, are found near the N-terminal scissile peptide bond and most likely help catalyze N-terminal cleavage (Fig. 2). The residue D154 is also positioned near the N terminus, where it may protonate the carbonyl oxygen of the scissile bond, thus promoting cleavage.
More definite information on the roles of individual residues was obtained from recombinant generation of variant proteins. Thus, replacement of D154 with different amino acids resulted in greatly reduced splicing efficiency, indicating that this residue is important in the splicing mechanism. In contrast, the hydroxyl group of the residue Y163 could be eliminated by a tyrosine-to-phenylalanine substitution without loss of catalytic activity. The side chain of S176, which acts as the nucleophile in the N-terminal cleavage reaction, is located more than 8 Å from the N-terminal scissile bond between the residues G7 and A8. The NMR structure therefore implies that the polypeptide segment including this residue must undergo a conformational change to enable the intein reaction to occur.
Johnson, M.A., Southworth, M.W., Perler, F.B., Wüthrich, K. NMR assignment of a KlbA intein precursor from Methanococcus jannaschii. J. Biomol. NMR, in press.
Placzek, W.J., Almeida, M.A., Wüthrich, K. NMR assignment of a human cancer-related nucleoside triphosphatase. J. Biomol. NMR 36(Suppl. 5):59, 2006.
Placzek, W.J., Almeida, M.A., Wüthrich, K. NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase. J. Biomol. NMR, in press.