6W0V
The Crystal Structure of the Mutant Nuclease Domain of Pyocin S8 with its Cognate Immunity Protein
Summary for 6W0V
| Entry DOI | 10.2210/pdb6w0v/pdb |
| Descriptor | Pyocin S8, Bacteriocin immunity protein (3 entities in total) |
| Functional Keywords | protein antibiotic, h-h-n dnase, toxin, antimicrobial protein |
| Biological source | Pseudomonas aeruginosa More |
| Total number of polymer chains | 2 |
| Total formula weight | 23097.79 |
| Authors | Turano, H.,Gomes, F.,Domingos, R.M.,Netto, L.E.S. (deposition date: 2020-03-03, release date: 2020-09-02, Last modification date: 2023-10-11) |
| Primary citation | Turano, H.,Gomes, F.,Domingos, R.M.,Degenhardt, M.F.S.,Oliveira, C.L.P.,Garratt, R.C.,Lincopan, N.,Netto, L.E.S. Molecular Structure and Functional Analysis of Pyocin S8 from Pseudomonas aeruginosa Reveals the Essential Requirement of a Glutamate Residue in the H-N-H Motif for DNase Activity. J.Bacteriol., 202:-, 2020 Cited by PubMed Abstract: Multidrug resistance (MDR) is a serious threat to public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by strains to kill closely related cells during intraspecific competition. Here, we report an in-depth biochemical, microbicidal, and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (ImS8) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. The integrity of the H-N-H motif is probably essential in the killing activity of S8, as Glu100 is a highly conserved residue of this motif. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni strongly induced this DNase activity, whereas Mn and Mg exhibited moderate effects and Zn was inhibitory. Additionally, S8 bound Zn with a higher affinity than Ni and the Glu100Ala mutation decreased the affinity of S8 for these metals, as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala S8 DNase-ImS8 complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possibility of using pyocin S8 as an alternative therapy for infections caused by MDR strains, while leaving commensal human microbiota intact. Pyocins are proteins produced by strains that participate in intraspecific competition and host-pathogen interactions. They were first described in the 1950s and since then have gained attention as possible new antibiotics. However, there is still only scarce information about the molecular mechanisms by which these molecules induce cell death. Here, we show that the metal-dependent endonuclease activity of pyocin S8 is involved with its antimicrobial action against strain PAO1. We also describe that this killing activity is dependent on a conserved Glu residue within the H-N-H motif. The potency and selectivity of pyocin S8 toward a narrow spectrum of strains make this protein an attractive antimicrobial alternative for combatting MDR strains, while leaving commensal human microbiota intact. PubMed: 32817098DOI: 10.1128/JB.00346-20 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.381 Å) |
Structure validation
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