High-Resolution Single-Particle Cryo-EM Hydrated Structure of Streptococcus pyogenes Enolase Offers Insights into Its Function as a Plasminogen Receptor

Abstract

Cellular plasminogen (Pg) receptors (PgRs) are utilized to recruit Pg; stimulate its activation to the serine protease, plasmin (Pm); and sterically protect the surface Pm from inactivation by host inhibitors. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of Streptococcus pyogenes enolase (Sen) to function in this manner. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from S. pyogenes was modeled at 2.6 Å as a stable symmetrical doughnut-shaped homooctamer with point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ∼49 kDa. Binding sites for hPg were reported in other studies to include an internal K 252,255 and the COOH-terminal K 434,435 residues of Sen. However, in native Sen, the latter are buried within the minor interfaces of the octamer and do not function as a Pg-binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K 252,255,434,435 . PgRs devoid of COOH-terminal lysine utilize lysine isosteres comprising a basic residue, "i", and an anionic residue at "i + 3" around one turn of an α-helix. We highlight a number of surface-exposed potential hPg-binding lysine isosteres and further conclude that while the octameric structure of Sen is critical for hPg binding, disruption of this octamer without dissociation exposes hPg-binding epitopes.

A high-resolution cryo-EM structure of a bacterial M-protein reveals a compact structure that diverges from related M-proteins

“…The protocol for sample preparation of PAM-LVP was adapted from our previous publication (37) with the following changes. All image processing was performed on CryoSPARC v3.3.1 (http://www.cryosparc.com).…”

“…Initial model building from the PAM cryo-EM map is similar to that previously described (37). Phenix was used to generate the initial model using map-to-model function with the known PAM sequence.…”

“…The protocol for sample preparation of PAM-LVP was adapted from our previous published work (37) with the following changes. On the day of the cryo-EM experiments, the PAM-LVP concentrate was thawed at room temperature, after which recombinant hPg (3 µL of 0.6 mg/mL in 50 mM PBS, pH 7.4) was added to the 15 µL of concentrated PAM-LVP and the mixture was pipetted onto glow-discharged CF-1.2/1.3 Gold Ultrafoil® 300 mesh grids (Quantifoil, Jena, Germany).…”

The Recruitment and Activation of Plasminogen by Bacteria—The Involvement in Chronic Infection Development

“…The analysis performed for the model of octameric enolase demonstrated that the C-terminal lysine residues are located near the internal lysine residues, suggesting a common concerted function in plasminogen binding [ 109 ]. Thus, it is somewhat surprising that, recently, the involvement of C-terminal lysines and lysines present in the internal motif of S. pyogenes enolase in the binding of the solution-phase plasminogen has not been confirmed with site-directed mutagenesis [ 122 ]. Interestingly, as in previous in vitro studies [ 108 , 109 ], binding was observed when enolase adhered to different surfaces; however, this interaction was independent of C-terminal lysines [ 122 ].…”

RopB-regulated SpeB cysteine protease degrades extracellular vesicles-associated streptolysin O and bacterial proteins from group A Streptococcus

“…Further, enolase (also known as streptococcal surface enolase), which was resistant to SpeB degradation, could be utilized as the internal control protein for GAS EV analyses. SpeB and enolase are virulence-associated proteins [ 21–25 ]. This study reveals that SpeB and enolase are EV-associated proteins and might contribute to GAS pathogenesis during infection.…”