Background: Mutations of ␣-synuclein (␣Syn) can cause early-onset familial Parkinson disease (PD). Results: The H50Q, H50D, or H50A substitution promotes, whereas the H50R substitution inhibits, ␣Syn aggregation in vitro.
Conclusion:The recently identified PD-causing ␣Syn mutant, ␣Syn(H50Q), accelerates ␣Syn aggregation. Significance: The partial positive charge of His-50 at physiological pH likely plays a role in suppressing ␣Syn aggregation.
IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn-binding motif (1604-1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N-terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C-terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.
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.
Opportunistic pathogens such as Streptococcus pneumoniae secrete a giant metalloprotease virulence factor responsible for cleaving host IgA1, yet the molecular mechanism has remained unknown since their discovery nearly 30 years ago despite the potential for developing vaccines that target these enzymes to block infection. Here we show through a series of cryo-electron microscopy single particle reconstructions how the Streptococcus pneumoniae IgA1 protease facilitates IgA1 substrate recognition and how this can be inhibited. Specifically, the Streptococcus pneumoniae IgA1 protease subscribes to an active-site-gated mechanism where a domain undergoes a 10.0 Å movement to facilitate cleavage. Monoclonal antibody binding inhibits this conformational change, providing a direct means to block infection at the host interface. These structural studies explain decades of biological and biochemical studies and provides a general strategy to block Streptococcus pneumoniae IgA1 protease activity to potentially prevent infection.
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