Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution

Abstract

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.Inteins are protein-splicing elements that exist as in-frame fusions with flanking protein sequences called exteins. Inteins are self-splicing at the protein level, with their excision being coupled to extein ligation (1-3). Most of the inteins that have been described are in the 400-to 500-aa range with little absolute sequence conservation among the elements (4, 5). However, Cys or Ser residues are required at the amino termini of both the intein and the second extein, and a His and Asn are present at the carboxy terminus of the intein (Fig. 1A Top). Most inteins contain eight conserved sequence blocks (A-H), two of these being the LAGLIDADG motifs (blocks C and E) that define a family of intron-homing endonucleases (refs. 4 and 5; Fig. 1 A). Consistent with the occurrence of these motifs, several inteins have been shown to have site-specific endonuclease activity (6), and PI-SceI, the VMA1 intein of Saccharomyces cerevisiae, is capable of homing into a cognate inteinless allele (7). The sporadic distribution of inteins in all three biological kingdoms is consistent with their being mobile elements.Endonuclease genes have been assumed to be invasive genetic elements that colonized group I introns, converting them into mobile genetic elements (8-11). Similarly, mobile inteins appear to be derived from invasive endonuclease genes. Recent structural studies indeed suggest that the proteinsplicing and endonuclease domains are separate and that their two activities may have evolved independently. First, the crystal structure of PI-SceI has recently been solved (12). This 454-aa protein is folded into two distinct structural domains. Second, hidden Markov models have been used to define two conserved functional domains of inteins, corresponding to independent endonuclease and splicing modules, separated by nonconserved spacer regions of variable lengths (ref. 13; J.Z.D., A. Klar, M. J. Moser, W. R. H...

Probing the Structure of the PI-SceI-DNA Complex by Affinity Cleavage and Affinity Photocross-linking

“…resembled I-CreI, became associated with a gene that encoded a protein splicing element (9,41). The further addition of the DRR to the splicing domain and its associated or evolved DNA binding activity may have extended the specificity of PI-SceI, thereby permitting it to locate and cleave its recognition sequence within the context of the complex yeast genome.…”

Gelatinase-mediated migration and invasion of cancer cells

“…Furthermore, minimal inteins with full cleavage activity have only 130-160 amino acids (Derbyshire et al, 1997;Mathys et al, 1999;Wood et al, 1999). With a minimal sized fusion partner, the protein expressing cells do not have to consume their metabolic reserves for expressing large unwanted proteins thus maximising the peptide yield.…”

Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity

“…The deduced amino acid sequence of M. tuberculosis recA intervening sequence disclosed the existence of two copies of LAGLIDADG motifs. The protein-splicing ability of PI-MtuI 1 has been studied extensively (22)(23)(24)(25); however, the endonuclease activity has not been demonstrated. To this end, PI-MtuI was cloned, overexpressed, and purified, to explore its biochemical functions.…”