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SEA domain autoproteolysis accelerated by conformational strain: energetic aspects.

Journal article
Authors Anders Sandberg
Denny Johansson
Bertil Macao
Torleif Härd
Published in Journal of molecular biology
Volume 377
Issue 4
Pages 1117-29
ISSN 1089-8638
Publication year 2008
Published at Swedish NMR Centre at Göteborg University
Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 1117-29
Language en
Keywords Energy Metabolism, Humans, Kinetics, Mucin-1, chemistry, genetics, metabolism, Mutagenesis, Site-Directed, Mutant Proteins, chemistry, Protein Denaturation, Protein Folding, Protein Processing, Post-Translational, Protein Structure, Tertiary, physiology, Receptors, G-Protein-Coupled, chemistry, metabolism
Subject categories Biophysical chemistry, Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)


A subclass of proteins with the SEA (sea urchin sperm protein, enterokinase, and agrin) domain fold exists as heterodimers generated by autoproteolytic cleavage within a characteristic G(-1)S+1VVV sequence. Autoproteolysis occurs by a nucleophilic attack of the serine hydroxyl on the vicinal glycine carbonyl followed by an N-->O acyl shift and hydrolysis of the resulting ester. The reaction has been suggested to be accelerated by the straining of the scissile peptide bond upon protein folding. In an accompanying article, we report the mechanism; in this article, we provide further key evidence and account for the energetics of coupled protein folding and autoproteolysis. Cleavage of the GPR116 domain and that of the MUC1 SEA domain occur with half-life (t((1/2))) values of 12 and 18 min, respectively, with lowering of the free energy of the activation barrier by approximately 10 kcal mol(-1) compared with uncatalyzed hydrolysis. The free energies of unfolding of the GPR116 and MUC1 SEA domains were measured to approximately 11 and approximately 15 kcal mol(-1), respectively, but approximately 7 kcal mol(-1) of conformational energy is partitioned as strain over the scissile peptide bond in the precursor to catalyze autoproteolysis by substrate destabilization. A straining energy of approximately 7 kcal mol(-1) was measured by using both a pre-equilibrium model to analyze stability and cleavage kinetics data obtained with the GPR116 SEA domain destabilized by core mutations or urea addition, as well as the difference in thermodynamic stabilities of the MUC1 SEA precursor mutant S1098A (with a G(-1)A+1VVV motif) and the wild-type protein. The results imply that cleavage by N-->O acyl shift alone would proceed with a t((1/2)) of approximately 2.3 years, which is too slow to be biochemically effective. A subsequent review of structural data on other self-cleaving proteins suggests that conformational strain of the scissile peptide bond may be a common mechanism of autoproteolysis.

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