Structure of the branched intermediate in protein splicing.
Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site.
Frick Laboratory, Department of Chemistry, Princeton University, Princeton, NJ 08544; and.
Zhihua
Z
Liu
Laboratory of Synthetic Protein Chemistry, and.
Silvia
S
Frutos
Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065.
Matthew J
MJ
Bick
Frick Laboratory, Department of Chemistry, Princeton University, Princeton, NJ 08544; and.
Miquel
M
Vila-Perelló
Frick Laboratory, Department of Chemistry, Princeton University, Princeton, NJ 08544; and.
Galia T
GT
Debelouchina
Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065.
Seth A
SA
Darst
Frick Laboratory, Department of Chemistry, Princeton University, Princeton, NJ 08544; and muir@princeton.edu.
Tom W
TW
Muir
D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D027081:DNA Gyrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D047669:Exteins / Q000235:genetics*; D006860:Hydrogen Bonding; D047668:Inteins / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D019154:Protein Splicing; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D000577:Amides; D011506:Proteins; D001216:Asparagine; D027081:DNA Gyrase
expressed; protein semisynthesis