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

Abstract

ThnT is a pantetheine hydrolase from the DmpA/OAT superfamily involved in the biosynthesis of the β-lactam antibiotic thienamycin. We performed a structural and mechanistic investigation into the cis-autoproteolytic activation of ThnT, a process that has not previously been subject to analysis within this superfamily of enzymes. Removal of the γ-methyl of the threonine nucleophile resulted in a rate deceleration that we attribute to a reduction in the population of the reactive rotamer. This phenomenon is broadly applicable and constitutes a rationale for the evolutionary selection of threonine nucleophiles in autoproteolytic systems. Conservative substitution of the nucleophile (T282C) allowed determination of a 1.6-Å proenzyme ThnT crystal structure, which revealed a level of structural flexibility not previously observed within an autoprocessing active site. We assigned the major conformer as a nonreactive state that is unable to populate a reactive rotamer. Our analysis shows the system is activated by a structural rearrangement that places the scissile amide into an oxyanion hole and forces the nucleophilic residue into a forbidden region of Ramachandran space. We propose that conformational strain may drive autoprocessing through the destabilization of nonproductive states. Comparison of our data with previous reports uncovered evidence that many inactivated structures display nonreactive conformations. For penicillin and cephalosporin acylases, this discrepancy between structure and function may be resolved by invoking the presence of a hidden conformational state, similar to that reported here for ThnT.

Fig. 1.

General mechanism of autoproteolysis. The nucleophilic residue attacks into its N-terminal amide bond. The resultant 5-membered oxazolidine ring collapses with concomitant protonation of the nitrogen leaving group. The nascent amine then acts as a general base to activate a water molecule, which hydrolyzes the ester intermediate. The new C-terminal residue dissociates, exposing the catalytic N-terminal nucleophile. “A-H” and “B:” represent a general acid and general base, respectively. A comparable mechanism also occurs for autoproteolytic systems that utilize Cys and Ser nucleophiles.

Fig. 2.

The reactive rotamer effect in autoproteolysis. (A) Time course of the autoproteolysis reactions of ThnT and T282S shown on 15% SDS-PAGE. As the reaction progresses, there is a decrease in the intensity of the uncleaved (π) band and a corresponding increase in the intensity of the two cleavage products (α and β). Quantification with first-order kinetics shows a 4.3-fold rate deceleration caused by removal of the γ-methyl. (B) Hypothetical reaction coordinate diagram showing how threonine accelerates nucleophilic attack into the N-terminal amide bond by minimizing the number of gauche interactions for the reactive rotamer. This effect is specific to the 3R stereochemistry of the β-carbon and is also influenced by the geometry of the local protein backbone.

Fig. 3.

Representative autoproteolytic proteins from distinct folds. Autoproteolysis sites (black arrows) separate the N-terminal domain (silver) from the C-terminal domain (red). In each system, the autoproteolysis site is found at the terminus of a β-strand. The Ntn hydrolases (A) and the D/O hydrolases (D) share similar αββα architecture, but the connectivity between the secondary structure elements is different, indicating their relationship through convergent evolution. Protein Data Bank ID: (A) 2A8I, hTaspase1; (B) 2Q5X, hNup98; (C) 2ACM, Muc1 SEA; (D) 3S3U, ThnT. For clarity, a single monomer of hTaspase1 and ThnT is shown.

Fig. 4.

Dual occupancy at the active site residues N281, C282, and T283. (A) The π₂ subunit with states A and B modeled with a 55∶45 ratio, 2mF_o - DF_c electron density map (blue) calculated at 1.0 σ. (B) The π₁ subunit with a single conformer, state B, modeled. (C) Modeling state B alone into π₂ produces poor F_o - F_c electron density at 4.0 σ with negative (red) and positive (green) density corresponding to the omitted conformer (red residues, water as asterisks). (D) Modeling state A alone into π₂ clearly shows that the missing density may be satisfied by the partial occupancy of state B, identified in π₁.

Fig. 5.

Active and inactive states of ThnT. (A) Model of the inactive state B of ThnT. Hydrogen bonds are shown as green dashes. The 2.7-Å steric clash between T282 and N217 is shown with black dashes. A yellow arrow indicates the scissile bond. (B) Model of the cleavage-competent state of ThnT where the steric clash and twisted amide of T282 have been removed. The direction of attack is indicated with red dashes. (C) Alignment of T282C-state B with precursor Ntn hydrolases (2X1C, teal; 2IWM, pink; 1OQZ, slate; 1KEH, green) using the two oxyanion hole nitrogen atoms, oxygen of the scissile bond, and Cβ atoms (blue, red, and gray, respectively). This alignment reveals significant structural similarity between each active site and the occupation of the oxyanion hole with a crystallographic water molecule, corresponding to Wat2 in (A). For these Ntn hydrolases, a hidden rearrangement that displaces the bound water and places the scissile carbonyl in the oxyanion would activate the system for autoproteolysis.

Fig. 6.

Proposed autoproteolytic mechanism of ThnT. (A) The precleavage conformation shown in Fig. 5B. Dashed lines represent hydrogen bonds critical to activation of the system. A fourth hydrogen bond exists for Wat4, enabling it to shuttle the labile proton on T282 into solvent. (B) The amine leaving group is protonated by Wat3 and expelled, generating a planar ester. (C) The nucleophilic Wat3 is activated by the nascent amine and hydrolyzes the ester.