Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing.
We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-A resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the N- and C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain Ndelta atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S --> O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.
Ding
Yi
Y
Laboratory of Structural Biology and the Ministry of Education Laboratory of Protein Science, School of Life Science and Engineering, Tsinghua University, Beijing 100084, People's Republic of China.
Xu
Ming-Qun
MQ
Ghosh
Inca
I
Chen
Xuehui
X
Ferrandon
Sebastien
S
Lesage
Guillaume
G
Rao
Zihe
Z
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics*; D053488:DnaB Helicases; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D008956:Models, Chemical; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D010455:Peptides; D014867:Water; D001224:Aspartic Acid; D006639:Histidine; D001216:Asparagine; D002244:Carbon; D004265:DNA Helicases; D053488:DnaB Helicases; D010100:Oxygen