SEA Domain Autoproteolysis Accelerated by Conformational Strain: Energetic Aspects

Abstract

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.

SEA Domain Autoproteolysis Accelerated by Conformational Strain: Mechanistic Aspects

“…7a), which is more than 100-fold slower than the t ½ of ∼ 18 min observed for wild-type MUC1 SEA. 8 The site of cleavage in the 1G mutant was confirmed with mass spectrometry to occur at the GG − 1 S + 1 VVV site (i.e., immediately adjacent to the catalytic serine as in wild-type SEA; data not shown). The methyl NMR spectrum suggests that the structure of the 1G precursor is more similar to that of the cleaved heterodimer than to that of the S1098A precursor (Fig.…”

“…8 It is readily detected by SDS-PAGE analysis of samples of recombinantly produced protein obtained from single-step immobilized metal affinity chromatography (IMAC) purification of the soluble fraction of lysed Escherichia coli cells (Fig. 3b).…”

Native Mutant Huntingtin in Human Brain

“…It is possible that the protease cleaving mutant htt during BNP is released from an internal cellular compartment during the preparation of brain lysates for BNP. Another possibility is that the cleavage is by autoproteolysis through a conformation-dependent event as described for some other proteins (52).…”

Insights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement

“…As discussed above, the Muc1 SEA domain promotes autoproteolysis by applying torsional strain to the scissile bond. This strain raises the pK a of the amide nitrogen sufficiently for protonation at neutral pH, prior to nucleophilic attack (11,37), and was suggested to be a common mechanism for autoprocessing. We note that a grossly strained scissile bond has yet to be observed in a structure of an uncleaved autoproteolytic protein.…”

“…The large size and intrinsic reactivity of most systems precludes labeling schemes and makes nuclear magnetic resonance experiments particularly difficult. These challenges were recently overcome for the 15-kDa Muc1 SEA domain, where it was shown that protein folding introduces torsional strain at the scissile bond, lowering the barrier to cleavage by 7 kcal∕mol (11,12). The Ntn hydrolases were the first structurally characterized cis-autoproteolytic enzymes (13), but their size and complexity hinder similar progress.…”