Structural and Functional Interrogation of Active Streptococcus pneumoniae Sortase A

Mutation-induced heterogeneity of the β7-β8 loop of the Staphylococcus aureus class A sortase leading to enhanced catalytic efficiency characterized by NMR and enzyme kinetics

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the β7-β8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the β7-β8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

A generalisable method for the purification and biophysical characterisation of bacterial membrane receptors

Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterisation of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in E. coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilisation tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.

Structural insight into the role of the periplasmic chaperone Skp in pathogenic Neisseria.

It is postulate that Skp in Neisseria must utilize a different mechanism than in E. coli to stabilize substrates by expanding and contracting along its long axis to accommodate substrates of differing sizes.

Crystal structure of Legionella pneumophila glycosidase effector LegY.

Legionella pneumophila translocates approximately 330 effectors into host cells via its type IVB secretion system. These effectors mediate a diverse array of post-translational modifications, among which reversible glycosylation is closely associated with bacterial virulence. Although several glycosyltransferase effectors have been identified that glycosylate host proteins to subvert host cellular processes, no glycosidase effector has been reported to date. Here, we report the crystal structure of LegY, a putative glycosidase effector from L. pneumophila, determined to 1.46 Å resolution (PDB entry 27xj). LegY adopts a single-domain (α/α)6-barrel fold and is structurally assigned as a member of glycoside hydrolase family 15 (GH15). LegY structurally shows high similarity to fungal glucoamylases but shares lower homology with known prokaryotic counterparts. Phylogenetic analysis clusters LegY within the fungal clade, suggesting possible horizontal gene transfer or convergent evolution. This study provides structural insights into LegY, laying a foundation for future uncovering of its putative function during L. pneumophila infection.

Rescue of ribosomal protein bL27 in Streptococcus pneumoniae TIGR4 by an alternate protease

Structural determinants of endopilus assembly, stability, and functional specificity in bacterial type II secretion.

Key determinants of secretion specificity and endopilus stability are identified, revealing how minor sequence variations in conserved nanomachines drive functional adaptation to diverse environments.