Discerning the catalytic mechanism of Staphylococcus aureus sortase A with QM/MM free energy calculations

Highlights

• The binding and catalysis of two substrate conformations by sortase A is explored.

• Conventional MD simulations show a slight preference for the “Threonine In” state.

• Also, QM/MM metadynamics calculations show lower free energy barriers in this state.

Abstract

Sortases are key virulence factors in Gram-positive bacteria. These enzymes embed surface proteins in the cell wall through a transpeptidation reaction that involves recognizing a penta-peptide “sorting signal” in a target protein, cleaving it, and covalently attaching it to a second substrate that is later inserted into the cell wall. Although well studied, several aspects of the mechanism by which sortases perform these functions remains unclear. In particular, experiments have revealed two potential sorting signal binding motifs: a “Threonine-Out” (Thr-Out) structure in which the catalytically critical threonine residues protrudes into solution, and a “Threonine-In” (Thr-In) configuration in which this residue inserts into the binding site. To determine which of these is the biologically relevant state, we have performed a series of conventional and hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of the Staphylococcus aureus sortase A (SrtA) enzyme bound to a sorting signal substrate. Through the use of multi-dimensional metadynamics, our simulations were able to both map the acylation mechanism of SrtA in the Thr-In and Thr-Out states, as well as determine the free energy minima and barriers along these reactions. Results indicate that in both states the catalytic mechanisms are similar, however the free energy barriers are lower in the Thr-In configuration, suggesting that Thr-In is the catalytically relevant state. This has important implications for advancing our understanding of the mechanisms of sortase enzymes, as well we for future structure based drug design efforts aimed at inhibiting sortase function in vivo.

Introduction

Bacterial cell walls are decorated with surface proteins that play key roles in several pathogenic activities including cellular adhesion, host invasion, and evasion of the immune response [1], [2]. In Gram-positive bacteria, these proteins are covalently attached to the cell wall through a mechanism that is dependent on sortase enzymes [3], [4]. The functioning of sortases is therefore directly linked to bacterial virulence, and indeed knockout strains of Staphylococcus aureus, Streptococcus pneumonia, and Listeria monocytogenes have been shown to be less pathogenic than their wild type counterparts [5], [6], [7]. These observations have motivated recent drug-discovery efforts that focus on inhibiting sortases, as antimicrobial compounds that reduce pathogenicity may be more resistant to selective pressures than traditional therapeutics [8], [9], [10]. However, before the potential of these efforts can be fully realized, a more complete understanding of the structure/function relationship in sortases is required.

The prototypical sortase is the Staphylococcus aureus class A sortase (SrtA) enzyme. SrtA recognizes an LPXTG “sorting signal” (SS) motif in the protein destined to be embedded in the cell wall (the first substrate of catalysis) and then catalyzes a transpeptidation reaction that transfers the target protein to a lipid II molecule (the second substrate of catalysis) that is later incorporated into the cell wall [11], [12], [13]. The physical basis for this transpeptidation mechanism follows two steps. First, the target protein binds to the sortase through its sorting signal, and in the acylation step the SS is cleaved between the threonine and glycine residues and the target protein is covalently attached to SrtA. Second, a deacylation step occurs in which the target protein is transferred to the N-terminal glycine chain of a lipid II molecule (Fig. 1a). Mutagenesis and biochemical experiments have indicated that SrtA uses the catalytic triad of His120, Cys184, and Arg197 in a reverse protonation mechanism to perform this function [11], [14], [15], [16], [17], [18]. In this mechanism, His120 and Cys184 typically exist in a neutrally charged/inactive state, however the system transiently samples an active configuration that has both a deprotonated/negatively charged Cys184 and a protonated/positively charged His120 residue [17], [18]. When this occurs, the cysteine thiolate anion attacks the carbonyl of the SS ThrGly bond, whereas the histidine protonates the SS leaving group.

Significant insight into the structural basis for the SrtA transpeptidation mechanism was provided by Suree et al. when they reported the NMR-derived solution structure of SrtA covalently bound to a sorting signal analog [19]. By examining the acyl-enzyme intermediate, they were able to show that sortases function by an “induced fit” mechanism that involves an opening of the β 7/β 8 loop to accommodate the SS in the active site, along with a closing of the β 6/β 7 loop to form a hydrophobic pocket that recognizes the leucine and proline SS residues. This structure was the first reported sortase/substrate bound structure that could explain the roles of each residue in the catalytic triad. Molecular dynamics (MD) simulations based on this model demonstrated that the substrate bound states of the β 6/β 7 and β 7/β 8 loops are highly stable on the hundreds of nanoseconds timescale and do not sample conformations observed in the substrate free crystal structure [21], even when the sorting signal is removed [22].

More recently, Jacobitz et al. solved the crystal structure of a similar sortase enzyme, Staphylococcus aureus sortase B (SrtB), bound to an analog of its NPQTN sorting signal substrate [20]. Although many of the same induced fit characteristics were observed in SrtB as in SrtA, there was an intriguing difference in the SS conformation. In SrtA, the sidechain of the SS threonine residue protruded into solution, while the alanine sidechain was inserted into the active site pocket (see Fig. 1b). However, in SrtB the threonine sidechain was inserted into the active site, whereas the sidechain of the third SS residue, which in SrtB is a glutamine, was pointed into solution (Fig. 1c). Multiple lines of evidence lead to the hypothesis that the “Thr-In” state of SrtB is the more catalytically active configuration than the previously solved “Thr-Out” state observed in SrtA. First, the Thr-In configuration creates hydrogen bonds that help stabilize the position of the catalytically important arginine residue such that it creates an oxyanion hole. Second, insertion of the threonine sidechain into the sortase active site creates a mechanism for recognition of the threonine residue, which has been shown to be critical for substrate binding [23]. Finally, positioning the sidechain of the third SS residue in solution provides an explanation for the lack of specificity observed at this position in SS recognition by SrtA [23].

To address whether the Thr-In position is the catalytically active state in SrtA, we previously performed MD simulations using an umbrella sampling protocol to determine the free energy differences between Thr-In and Thr-Out in both SrtA and SrtB [20]. The results revealed that in the acyl-enzyme intermediate, SrtA can adopt both a Thr-In and Thr-Out state, however SrtB could only accommodate a SS in the Thr-In state. Furthermore, although the free energy barriers between the Thr-In and Thr-Out states were relatively low for a sorting signal substrate (on the order of a few kcal/mol), the path between these states suggested that these barriers would be significantly increased in a full-length protein substrate. Therefore, we expect the interconversion rate between these two states to be significantly slower than the transpeptidation reaction in vivo. Taken together, although it has not been experimentally observed, the evidence suggests that the Thr-In state is also present in SrtA and it may be the more biologically active SS configuration.

To test the hypothesis that the Thr-In state is more catalytically active than Thr-Out in SrtA, we performed a series of conventional and hybrid quantum mechanics/molecular mechanics (QM/MM) MD simulations of the SS/SrtA complex in both states. Our protocol involved multiple steps that were repeated for both states, including: (1) generating and equilibrating initial models of the non-covalently bound SS/SrtA complexes, (2) deriving several approximate reactant (noncovalently bound SS/SrtA complexes) and product (acyl-enzyme intermediate) configurations, (3) computing multiple approximate reaction pathways, and (4) performing extensive metadynamics calculations to compute the potentials of mean force (PMFs) for the acylation reaction with semiempirical QM calculations. Results indicate that the reaction mechanisms for Thr-In and Thr-Out are similar, however the free energy barriers are lower in Thr-In, suggesting this is the catalytically relevant state.