Insights Into Cis-Autoproteolysis Reveal a Reactive State Formed Through Conformational Rearrangement
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Insights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement Andrew R. Bullera, Michael F. Freemanb,1, Nathan T. Wrightc,2, Joel F. Schildbachc, and Craig A. Townsendb,3 aDepartments of Biophysics, bChemistry, and cBiology, Johns Hopkins University, 3400 North Charles Street, Baltimore MD 21218 Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved December 20, 2011 (received for review August 19, 2011) ThnT is a pantetheine hydrolase from the DmpA/OAT superfamily for the 15-kDa Muc1 SEA domain, where it was shown that involved in the biosynthesis of the β-lactam antibiotic thienamycin. protein folding introduces torsional strain at the scissile bond, We performed a structural and mechanistic investigation into the lowering the barrier to cleavage by 7 kcal∕mol (11, 12). The Ntn cis-autoproteolytic activation of ThnT, a process that has not pre- hydrolases were the first structurally characterized cis-autopro- viously been subject to analysis within this superfamily of enzymes. teolytic enzymes (13), but their size and complexity hinder similar Removal of the γ-methyl of the threonine nucleophile resulted in a progress. Enzymes from this superfamily are known to utilize all rate deceleration that we attribute to a reduction in the population three possible nucleophiles (Thr, Ser, or Cys); however, the of the reactive rotamer. This phenomenon is broadly applicable and mechanistic constraints imparted by a given residue are entirely constitutes a rationale for the evolutionary selection of threonine unknown. Once described as belonging to the Ntn superfamily, nucleophiles in autoproteolytic systems. Conservative substitution the autoactivation of the less numerous DmpA/OAT (D/O) of the nucleophile (T282C) allowed determination of a 1.6-Å proen- superfamily has not been studied (14). Understanding the auto- zyme ThnT crystal structure, which revealed a level of structural proteolytic protein scaffold is of significant practical interest be- flexibility not previously observed within an autoprocessing active cause enzymes from the D/O or Ntn superfamiles are implicated site. We assigned the major conformer as a nonreactive state that in the biosynthesis of all classes of the naturally occurring bicyclic is unable to populate a reactive rotamer. Our analysis shows the β-lactam antibiotics (15–19). system is activated by a structural rearrangement that places the Here, we describe our investigation into the autoproteolytic scissile amide into an oxyanion hole and forces the nucleophilic re- activation of a D/O enzyme, ThnT, a pantetheine hydrolase from sidue into a forbidden region of Ramachandran space. We propose Streptomyces cattleya involved in the biosynthesis of the β-lactam that conformational strain may drive autoprocessing through the antibiotic thienamycin (19). We identify specific structural con- destabilization of nonproductive states. Comparison of our data straints imparted by the γ-methyl of a Thr nucleophile, providing with previous reports uncovered evidence that many inactivated a common mechanistic link between the Ntn and D/O enzymes structures display nonreactive conformations. For penicillin and and a rationale for the evolutionary selection of this residue. The cephalosporin acylases, this discrepancy between structure and func- X-ray crystal structure of an uncleaved ThnT precursor showed tion may be resolved by invoking the presence of a hidden confor- unusual structural flexibility in the active site, allowing inter- mational state, similar to that reported here for ThnT. conversion between inactive and active states. Evaluation of pre- viously reported uncleaved Ntn structures found that they often crystallography ∣ mechanism ∣ reactive rotamer effect ∣ self-cleavage contain comparable active site geometries to the inactive state of ThnT, and they are unlikely to promote the requisite chemistry. n stark contrast to the evolutionary selection for chemically Application of the insights gleaned from analysis of ThnT may be Istable polypeptides, there exist several classes of intrinsically used to address these deficiencies, providing a strengthened basis reactive proteins that cleave an internal peptide bond as part of for continued research into autoprocessing. their cellular function (1). Many cis-autoprocessing events proceed through an N-O(S) acyl shift, where a threonine (Thr), serine (Ser), Results and Discussion or cysteine (Cys) attacks the peptide bond of the preceding residue The Reactive Rotamer Effect Accelerates the N-O Acyl Shift. ThnT (Fig. 1). Collapse of the resultant (thia)oxazolidine exchanges the matures with a half-life ðt1∕2Þ¼43 min at 37 °C, as measured by amide for a reactive (thio)ester that undergoes different chemical a kinetic SDS-PAGE assay. Conservative mutation of the nucleo- reactions, depending on biological context. This chemistry has been phile, T282S, resulted in a 4.3-fold reduction in the self-cleavage implicated in such diverse phenomena as protein folding, nucleo- rate (Fig. 2A). An extensive review of the literature revealed that porin biogenesis, Hedgehog and G-protein coupled receptor sig- comparable Thr → Ser mutations always impede autoproteolysis, naling, protein splicing, and apoptosis (2–6). Additionally, cis-auto- suggesting a common cause underlies this phenomenon (20–27). processing is necessary for maturation of many enzymes including Temperature dependent analysis of the autoproteolysis of the the proteasome β-subunit, pyruvoyl-dependent decarboxylases, and the industrially used penicillin and cephalosporin acylases (PA and CA, respectively) (1, 7, 8). Enzymes use the N-terminal nucleophile Author contributions: A.R.B., M.F.F., J.F.S., and C.A.T. designed research; A.R.B., M.F.F., and N.T.W. performed research; A.R.B. and M.F.F. contributed new reagents/analytic tools; (Ntn) liberated by autoproteolysis as a noncanonical catalytic diad A.R.B., N.T.W., J.F.S., and C.A.T. analyzed data; and A.R.B., M.F.F., N.T.W., J.F.S., and C.A.T. (9). Central to these systems is the challenge of hydrolyzing an in- wrote the paper. ternal peptide bond, whose half-life at 25 °C and neutral pH is ap- The authors declare no conflict of interest. proximately 600 y, with no assistance from an external catalyst (10). This article is a PNAS Direct Submission. Although the chemical reactions underlying self-cleavage have Data deposition: The atomic coordinates and structure factors have been deposited in the been appreciated for the past decade, understanding how these Protein Data Bank, www.pdb.org (PDB ID code 3S3U). reactions are facilitated is hampered by the inapplicability of 1Present address: Kekulé Institute of Organic Chemistry and Biochemistry, University of many enzymological techniques. Because the active site is not Bonn, 53223 Bonn, Germany. returned to its original state, autoprocessing is not formally cat- 2Present address: Department of Chemistry, James Madison University, Harrisonburg, alytic; this limits the utility of traditional kinetic assays. The large VA 22807. size and intrinsic reactivity of most systems precludes labeling 3To whom correspondence should be addressed. E-mail: [email protected]. schemes and makes nuclear magnetic resonance experiments This article contains supporting information online at www.pnas.org/lookup/suppl/ particularly difficult. These challenges were recently overcome doi:10.1073/pnas.1113633109/-/DCSupplemental. 2308–2313 ∣ PNAS ∣ February 14, 2012 ∣ vol. 109 ∣ no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1113633109 Downloaded by guest on September 29, 2021 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. Ntn-hydrolase glycosylasparaginase (GA) showed the Thr → Ser We identified crystallization conditions in which crystals of rate deceleration is caused by an increase in the activation entro- T282C grew overnight, before measurable autoproteolysis could py; however, the molecular interaction giving rise to this effect occur. These crystals diffracted to high resolution and we report remained elusive (22). here the 1.6-Å X-ray crystal structure of this mutant, which al- The N-O acyl shift proceeds through a sterically disfavored lowed for the analysis of a D/O enzyme in its uncleaved form. gauche-rotamer of the nucleophile (7, 28). The reactive g− rota- ThnT is dimeric both by native PAGE (19) and within the asym- mer of Thr offsets this instability by placing the γ-methyl in its metric unit of the crystal. Like other D/O and Ntn proteins, ThnT anti-position (Fig. 2B). The rotamer distribution of the nucleo- has an αββα architecture (Fig. 3). The connectivity of the second- phile is also affected by interactions with the local backbone. Such ary structural elements differs between these superfamilies, indi- interactions specifically accelerate autoproteolysis with Thr when cating they are related through convergent evolution (14). We the γ-methyl forms unfavorable steric interactions in nonproduc- compared the architecture