Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly

Abstract

The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6–2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.

Introducing site-specific cysteines into nanobodies for mercury labelling allowsde novophasing of their crystal structures

“…The pronounced decay of the anomalous signal inspired us to compare SAD phasing based on the 1-180 data with SIRAS phasing, in which the 180-720 data acted as the native data and the 1-180 data as a derivative with an anomalous signal. This strategy is also known as radiation-damageinduced phasing with anomalous scattering (RIPAS) and has previously been shown to strongly improve the resulting electron density obtained after phasing based on anomalous data with significant decay in the anomalous signal from Cys-Hg derivatives of the YjcF and YidA proteins (Ramagopal et al, 2005). For site identification and phasing we used phenix.autosol and obtained figures of merit (FOMs) of 0.38 and 0.40 for the SAD and SIRAS scenarios, respectively.…”

“…Remarkably, the R meas values for the 1-180 and the 540-720 data were 0.054 and 0.055, respectively. Hence, in line with previous studies of Hgsubstituted cysteine side chains (Ramagopal et al, 2005), we observed a significant selective decay of the anomalous signal from Hg, whereas the overall data quality was preserved during the full rotation range. The pronounced decay of the anomalous signal inspired us to compare SAD phasing based on the 1-180 data with SIRAS phasing, in which the 180-720 data acted as the native data and the 1-180 data as a derivative with an anomalous signal.…”

“…In contrast, the weakest subsite appears to stem from a fraction of molecules in which the Hg atom is only bound to one cysteine side chain since density supporting an alternative conformation of the nearby cysteine is present. A movement of Hg atoms induced by radiation damage was also noticed for the YidA protein (Ramagopal et al, 2005).…”

Stochastic sensing of Angiotensin II with lysenin channels

“…1a), after which the solution in the cis reservoir was exchanged with lysenin-free electrolyte to prevent further insertions. However, more channels may insert after buffer exchange since the formation of a pre-pore attached to the membrane is a condition for channel oligomerization 36 . Although we determined no changes in the characteristic electronic signatures derived from single channel measurements for up to six inserted nanopores (after which the electric noise may become significant and prevent accurate analysis), all translocation experiments on single channels consistently comprised two lysenin nanopores assembled into the lipid membrane.

Figure 1Interaction of Ang II with single lysenin channels inserted into lipid membranes bathed by 1 M KCl solutions buffered with 10 mM Tris and 1 mM EDTA at pH 6.9.

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Advances in structure determination by cryo‐EM to unravel membrane‐spanning pore formation

“…Lysenin is a component of the earthworm's immune system, exerting its function by attacking membranes of parasites . In addition to the X‐ray structure of the lysenin monomer, the 3.1 Å structures of the pore, determined independently using cryo‐EM and X‐ray crystallography, have helped to identify the functional folds and interactions of the three separate domains of the lysenin monomer [Fig. (A,B)].…”