The Autocatalytic Cleavage Domain Is Not Required for the Activity of ScpC, a Virulence Protease from Streptococcus pyogenes: A Structural Insight.

Group A Streptococcus (GAS, or Streptococcus pyogenes) is a leading human bacterial pathogen with diverse clinical manifestations, ranging from mild to life-threatening and to severe immune sequela. These diseases, combined, account for more than half a million deaths per year, globally. To accomplish its vast pathogenic potential, GAS expresses a multitude of virulent proteins, including the pivotal virulence factor ScpC. ScpC is a narrow-range surface-exposed subtilisin-like serine protease that cleaves the last 14 C-terminal amino acids of interleukin 8 (IL-8 or CXCL8) and impairs essential IL-8 signaling processes. As a result, neutrophil migration, bacterial killing, and the formation of neutrophil extracellular traps are strongly impaired. Also, ScpC has been identified as a potential vaccine candidate. ScpC undergoes an autocatalytic cleavage between Gln244 and Ser245, resulting in two polypeptide chains that assemble together forming the active protease. Previously, we reported that the region harboring the autocatalytic cleavage site, stretching from Gln213 to Asp272, is completely disordered. Here, we show that a deletion mutant (ScpCΔ60) of this region forms a single polypeptide chain, whose crystal structure we determined at 2.9 Å resolution. Moreover, we show that ScpCΔ60 is an active protease capable of cleaving its substrate IL-8 in a manner comparable to that of the wild type. These studies improve our understanding of the proteolytic activity of ScpC.

Bacterioferritin nanocage structures uncover the biomineralization process in ferritins.

Iron is an essential element involved in various metabolic processes. The ferritin family of proteins forms nanocage assembly and is involved in iron oxidation, storage, and mineralization. Although several structures of human ferritins and bacterioferritins have been solved, there is still no complete structure that shows both the trapped Fe-biomineral cluster and the nanocage. Furthermore, whereas the mechanism of iron trafficking has been explained using various approaches, structural details on the biomineralization process (i.e. the formation of the mineral itself) are generally lacking. Here, we report the cryo-electron microscopy (cryo-EM) structures of apoform and biomineral bound form (holoforms) of the Streptomyces coelicolor bacterioferritin (ScBfr) nanocage and the subunit crystal structure. The holoforms show different stages of Fe-biomineral accumulation inside the nanocage, in which the connections exist in two of the fourfold channels of the nanocage between the C-terminal of the ScBfr monomers and the Fe-biomineral cluster. The mutation and truncation of the bacterioferritin residues involved in these connections significantly reduced the iron and phosphate binding in comparison with those of the wild type and together explain the underlying mechanism. Collectively, our results represent a prototype for the bacterioferritin nanocage, which reveals insight into its biomineralization and the potential channel for bacterioferritin-associated iron trafficking.