Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing.
Several genes from prokaryotes and lower eukaryotes have been found to contain an in-frame open reading frame, which encodes for an internal protein (intein). Post-translationally, the internal polypeptide auto-splices and ligates the external sequences to yield a functional external protein (extein) and an intein. Most, but not all inteins, contain, apart from a splicing domain, a separate endonucleolytic domain that enables them to maintain their presence by a homing mechanism. We report here the crystal structure of an intein found in the gyrase A subunit from Mycobacterium xenopi at 2.2 A resolution. The structure contains an unusual beta-fold with the catalytic splice junctions at the ends of two adjacent antiparallel beta-strands. The arrangement of the active site residues Ser 1, Thr 72, His 75, His 197, and Asn 198 is consistent with a four-step mechanism for the cleavage-ligation reaction. Using site-directed mutagenesis, the N-terminal cysteine, proposed as the nucleophile in the first step of the splicing reaction, was changed to a Ser 1 and Ala 0, thus capturing the intein in a pre-spliced state.
Klabunde
T
T
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Sharma
S
S
Telenti
A
A
Jacobs
W R
WR
Sacchettini
J C
JC
D000595:Amino Acid Sequence; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000378:metabolism* / Q000648:ultrastructure; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism / Q000648:ultrastructure; D029721:Drosophila Proteins; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism; D053823:Hedgehog Proteins; D019476:Insect Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D019912:Mycobacterium xenopi; D011498:Protein Precursors / Q000378:metabolism; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D019476:Insect Proteins; D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; C077245:hh protein, Drosophila; D004720:Endonucleases; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II