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Structural Characteristics of Pneumolysin and Its Domains in a Biomimetic Solution.

ABSTRACT: Pneumolysin (PLY) and its truncated fragments, domains 1-3 (D1-3), and domain 4 (D4), were purified as recombinant proteins after being cloned and over-expressed in Escherichia coli. The three-dimensional structures of these proteins were quantitatively investigated in a biomimetic condition, phosphate buffered saline (PBS) by synchrotron X-ray scattering. X-ray scattering analysis revealed important structural features including structural parameters. PLY was present as a monomeric form in PBS. The monomeric form resembled its crystallographic structure with a discrepancy of only 6.3%, confirming that PLY forms a stable structure and, thus, retains its structure in the crystalline state and even in PBS solution. D4 was also present as a monomeric form, but its structure was very different from that of the corresponding part in the crystallographic PLY structure; the discrepancy was 92.0%. Such a dissimilar structure might originate from a less folded-chain conformation. This result suggested that the structure of D4 is highly dependent on the crystalline or solution state and further on the presence or absence of the D1-3 unit. In contrast, D1-3 was dimeric rather than monomeric. Its structure was close to the most probable dimeric form of the corresponding part in the crystallographic PLY structure with 13.1% discrepancy. This fact indicated that the D1-3 unit forms a stable structure and, indeed, such structure is well maintained in the crystalline state as well as in PBS although presented as a dimer. This result further supported that the whole structural stability of PLY is mainly attributed to the structure of D1-3. All of PLY, D1-3, and D4 revealed aggregation tendencies during purification and storage. Overall, the structural characteristics of PLY and its domains in PBS may correlate to the PLY oligomer formation yielding large pore structures for the penetration of cell membranes.

SUBMITTER: Lee J

PROVIDER: S-EPMC6644606 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Structural Characteristics of Pneumolysin and Its Domains in a Biomimetic Solution.

Lee Jongchan J Suh Eunae E Byambabaatar Sumiya S Lee Soomin S Kim Heesoo H Jin Kyeong Sik KS Ree Moonhor M

ACS omega 20180820 8

Pneumolysin (PLY) and its truncated fragments, domains 1-3 (D1-3), and domain 4 (D4), were purified as recombinant proteins after being cloned and over-expressed in Escherichia coli. The three-dimensional structures of these proteins were quantitatively investigated in a biomimetic condition, phosphate buffered saline (PBS) by synchrotron X-ray scattering. X-ray scattering analysis revealed important structural features including structural parameters. PLY was presen ...[more]

PMID: 31459080

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Project description:Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433-->Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-->Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-->Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-->Phe. Electron microscopy revealed that Trp-433-->Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-->Phe variant. We conclude that the change Trp-433-->Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.

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Structural Characteristics of Pneumolysin and Its Domains in a Biomimetic Solution.

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Structural Characteristics of Pneumolysin and Its Domains in a Biomimetic Solution.

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