Streptococcus pneumoniae G5 domains bind different ligands

Abstract

Many bacterial pathogens express small G5 domains that exist in the context of various membrane‐anchored proteins and these G5 domains have been associated with colonization, cellular adhesion, and biofilm formation. However, despite over a decade since the computational prediction of these G5 domains, many remain uncharacterized, particularly those from Streptococcus pneumoniae. Of five previously predicted G5 domains we found that four of these, all derived from S. pneumoniae, are independently folded modules. As one of these exhibits extreme line broadening due to self‐association, we were able to use NMR solution studies to probe the potential ligand interactions of the remaining three G5 domains. None of these G5 domains engage N‐acetylglucosamine (NAG) as previously predicted but do interact with other small molecules that may modulate adherence to both bacteria and host cells. Specifically, while all G5 domains tested engage Zn, only one of these G5 domains engage heparin. NMR solution structural studies of the IgA1 Protease G5 (IgA1P‐G5) and endo‐beta‐N‐acetylglucosaminidase‐D G5 (ENDD‐G5) also facilitated identification of the ligand binding sites and confirm the typical G5 fold that comprises two connected β‐sheets with no canonical core. NMR relaxation experiments indicate flexibility on both ends and within the connecting regions between the β‐sheets. Our studies thus establish a basis for future biological experiments to test whether the ligands presented here are involved in bacterial adherence, either to bacteria or to host cells.

Mechanism and inhibition of Streptococcus pneumoniae IgA1 protease

“…The number of interactions between the IgA1P NTD and MD are diminished upon this rearrangement, providing for an energetic rationale for why the enzyme "snaps" back after product release. Such findings also led us to hypothesize that the IgA1P NTD may be soluble alone, similar to its G5 domain and CTD that have been previously shown to be independently folded 21,22 . Indeed, we were able to engineer an NTD construct that gives rise to a well-dispersed 15 N-HSQC analogous to the independently folded IgA1P CTD and the G5 domain (Supplementary Fig.…”

“…The high-resolution structure of the IgA1P catalytic region. In order to identify the S. pneumoniae IgA1P catalytic domain, we engineered several constructs based on our previous results of limited proteolysis on the full, mature IgA1P (residues 154-1963, UniProt accession Q59947; NCBI accession WP_000417171 that corresponds to the common D39 and R6 strains) 21,22 . Only constructs that began at or prior to residue 665 were accessible to enzymatic cleavage of the MBP tag by thrombin ( Supplementary Fig.…”

“…Finally, amylose-resin flow through was concentrated and applied onto an S200 size exclusion column (120 ml, GE Healthcare) equilibrated in 20 mM HEPES, pH 7.4, and 150 mM NaCl. The IgA1P G5 domain and CTD were expressed and purified as previously described 22 . The IgA1P NTD was engineered into the pJ401 plasmid and expressed and purified using the same method previously described 21,22 : an N-terminal 6xHis tag was followed by a thrombin cleavage site and IgA1P residues 665-1010.…”

“…All NMR data were collected on a Varian 900 equipped with a cryoprobe. Samples were produced as previously described using 15 N M9 minimal media for the S. pneumoniae IgA1P G5 domain (residues 312-393) 22 and 15 N, 2 H M9 minimal media for both the CTD (residues 1611-1963) 21 and the NTD (residues 665-1010). Final size exclusion was performed in NMR buffer (50 mM Na 2 HPO 4 , pH 6.5, and 150 mM NaCl).…”

“… a The family of IgA1Ps cleave host IgA1 at its hinge region, separating the IgA1 Fc from its Fab and effectively masking bacterial cells with host IgA1 Fab. The general domain architecture of mature S. pneumoniae IgA1P consists of a flexible N-terminal region (residues 154–664) attached to the bacterial cell wall, which includes a small G5 domain followed by a large C-terminal catalytic region (residues 665–1963) 21 , 22 . b S. pneumoniae IgA1P cleavage is similar between the mature IgA1P 154–1963 and isolated catalytic region of IgA1P 665–1963.…”