Structural and biophysical analysis of nuclease protein antibiotics

Abstract

Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years, especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here, we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa, known as E-type colicins and S-type pyocins, respectively, bound to their specific immunity (Im) proteins. First, we report the 3.2 Å structure of the DNase colicin ColE9 in complex with its ultra-high affinity Im protein, Im9. In contrast with Im3, which when bound to the ribonuclease domain of the homologous colicin ColE3 makes contact with the translocation (T) domain of the toxin, we find that Im9 makes no such contact and only interactions with the ColE9 cytotoxic domain are observed. Second, we report small-angle X-ray scattering data for two S-type DNase pyocins, S2 and AP41, into which are fitted recently determined X-ray structures for isolated domains. We find that DNase pyocins and colicins are both highly elongated molecules, even though the order of their constituent domains differs. We discuss the implications of these architectural similarities and differences in the context of the translocation mechanism of protein antibiotics through the cell envelope of Gram-negative bacteria.

A Colicin M-Type Bacteriocin from Pseudomonas aeruginosa Targeting the HxuC Heme Receptor Requires a Novel Immunity Partner

“…4D to F). These observations are in line with the generally high specificity and selectivity of immunity partners in silencing toxin functions (14,(44)(45)(46).…”

Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models

“…The present analysis places these NBs in group I, which also includes NBs from Klebsiella pneumoniae , Serratia marcescens , Shigella sonnei , Enterobacter cloacae , Xenorhabdus , and Citrobacter ( Fig 3 ). Structures for both the rRNase NB ColE3 and DNase NB ColE9 from this group have been reported [ 56 ], plus there have been numerous biophysical and structural studies of other NBs from this group [ 57 ]. The main structural features of group I NBs are a T-domain composed of a disordered N-terminal region adjoining a folded domain, the latter identified in the PFAM database as PFAM 03515, and a central coiled-coil region which constitutes the R-domain.…”

“…[ 20 ] previously highlighted a similar region in a few nuclease pyocins and colicins the deletion of which abolished cytotoxic activity [ 74 ]. The structures of three NB T-domains are known (annotated in the PFAM database as PFAM 03515); colicin E3 (2B5U), colicin E9 (5EW5 [ 56 ]) and the S-type pyocin domain from Erwinia carotovora (3MFB) as well as the T-domain of the pore forming colicin B (1RH1) which shares high sequence identity to the T-domain of the NB colicin D. We performed structure-based sequence alignments and found that the DPY motif is integral to a much larger segment spanning the C-terminal half (~140 amino acids) of the T-domain ( Fig 4b ). This segment (coloured blue in Fig 4b ) is identified in the PFAM database as the pyocin_s domain (PFAM 06958) [ 42 ].…”

Systematic classification of the His-Me finger superfamily

“…Similarly, Vibrio vulnificus nuclease (Vvn) ( 21 ) and endonuclease I ( 22 ) retain a big α-helical domain located at the C-terminus sheltering the central His-Me finger nuclease (Figure 3C ), whereas toxins (e.g. Colicin E9 ( 40 ), Pyocin S2 ( 41 )) possess expanded structural decorations at the N-terminus.…”