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The Skaggs Institute
for Chemical Biology


Scientific Report 2006




Structural Biology of a Human Cancer-Related Nucleoside Triphosphatase and of Inteins by Nuclear Magnetic Resonance Spectroscopy


K. Wüthrich, M.S. Almeida, M. Geralt, M.A. Johnson, W.J. Placzek

Structural Genomics of Human Cancer-Related Proteins

Recent advances in DNA microarrays and the completion of the Human Genome Project enabled the identification and characterization of aberrantly expressed mRNAs in cancerous cells and tissues. Structure determination of the protein products of these cancer-related mRNA sequences provides a basis for further biochemical studies and may support attempts to identify the so far unknown functions of these proteins.

In a screen of the Cancer Genome Anatomy Project database, we identified a putative nucleoside triphosphatase (NTPase) with aberrant expression in cancer tissues. The NMR structure of this human cancer-related NTPase (HCR-NTPase) shows a fold with a 9-stranded β-sheet in the core of an α/β sandwich and 5 helices distributed on both sides of this central β-sheet (Fig. 1). The functionally important P-loop (Walker A sequence motif) follows the first β-strand near the N terminus and is close to the Walker B sequence motif. Further spectroscopic and biochemical analysis established that this protein is indeed an active NTPase, with a Walker B motif containing elements of both ATPases and GTPases, and that its 3-dimensional structure is closely related to the 3-dimensional structures of a range of bona fide AAA+ ATPases. The structural classification of HCR-NTPase as a member of the AAA+ class of P-loop NTPases supports its involvement in regulatory mechanisms of cellular control, because proteins of this class are often involved in the regulation of assembly and disassembly of supramolecular protein structures.

Fig. 1. Ribbon diagram of the solution NMR structure of HCR-NTPase.


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.

Fig. 2. Cutaway view of the active site of the M jannaschii KlbA intein precursor. Several of the amino acid residues involved in catalysis are shown in stick representation, and some distances within the active site (in angstroms) are indicated. Parts of the main body of the protein are shown in a ribbon representation.

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.

Publications

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.

 

Kurt Wüthrich, Ph.D.
Cecil H. and Ida M. Green Professor of Structural Biology

Wüthrich Web Site