Structural and kinetic analysis of Escherichia coli signal peptide peptidase A

Secretory proteins contain a signal peptide at their N-terminus. The signal peptide functions to guide proteins to the membrane and is cleaved off by signal peptidase. The remnant signal peptides must be removed from the membrane to prevent their accumulation which can lead to membrane destabilization. Escherichia coli signal peptide peptidase A (SppAEC) has been identified as a major enzyme that processes the remnant signal peptide to smaller fragments. SppAEC, however, had remained uncharacterized such that the structure, catalytic mechanism, and substrate preference were unknown. I have cloned, overexpressed, purified and crystallized an active soluble domain of SppAEC. We have determined the structure of SppAEC by X-ray crystallography, which revealed that: 1) SppAEC has two similar alpha-beta domains (despite limited sequence identity), 2) SppAEC forms a tetramer that results in a dome shaped structure with a hydrophobic interior and hydrophilic exterior, 3) the four active sites within the cavity each utilize a Ser-Lys dyad where the general base (Lys209) arrives from the N-terminal domain and the nucleophile (Ser409) from the C-terminal domain. I have further characterized SppAEC using steady-state kinetics and cocrystallization. Using a series of fluorometric peptide substrates, I discovered that leucine is the most preferred residue at the P1 substrate position. I cocrystallized an SppAEC active site mutant (K209A), in complex with the substrate Z-LLL-MCA. The electron density within the active site of the 1.95 Å resolution structure is consistent with the carbonyl carbon of the substrate’s C-terminal leucine being covalently linked via an ester bond to the Ser409 O, thus revealing an acyl-enzyme complex in SppAEC. This is direct evidence that Ser409 O serves as a nucleophile in the SppAEC catalyzed reaction and confirms the identity of the S1 and S3 substrate specificity pockets. Lastly, I measured the pH dependence of both WT and S431A enzymes (Ser431 Ois hydrogen-bonded to Lys209 Nζ). The pH-rate profiles are consistent with S431 playing a role in lowering the pKa of the lysine general base, which would be critical for activity at physiological pH. We propose that the active site architecture of SppAEC may be best described as a Ser-Lys-Ser triad.