Cholesterol Promotes Cytolysin a Activity by Stabilizing the Intermediates during Pore Formation

Abstract

Biological processes performed by proteins interacting with and processing DNA and RNA are key to cell metabolism and life. Detailed insights into these processes provide essential information for understanding the molecular basis of life and the pathological conditions that develop when such processes go awry. The next scientific breakthrough consists in the actual, direct, real-time observations and measurements of the individual mechanisms involved, in order to validate and complete the current biological models. Single-molecule technologies offer an exciting opportunity to meet these challenges and to study protein function and activity in real-time and at the single-molecule level. Here, we present our efforts for further enabling discoveries in the field of biology and biophysics using both the combination of optical tweezers with singlemolecule fluorescence microscopy (C-Trap TM ). We show the latest applications of these technologies that can enhance our understanding not only in the field of DNA/RNA-protein interactions but also in the fields of molecular motors, protein folding/unfolding, cell membranes and genome structure and organization. These experiments show that the technological advances in hybrid single-molecule methods can be turned into an easy-to-use and stable instrument that has the ability to open up new venues in many research areas.

AnIn silicoAlgorithm for Identifying Amino Acids that Stabilize Oligomeric Membrane-Toxin Pores through Electrostatic Interactions

“…For characterizing proteins, while ClyA nanopores were advantageous compared to β -barrel pores such as AHL because of their larger pore lumen, ClyA pores were also more unstable, necessitating directed evolution to produce mutants that could form more stable nanopores (Soskine et al, 2013). This is consistent with oligomeric α -helical protein assemblies such as the ClyA pore being less stable than β -PFT pores (Gouaux, 1998; Woolfson et al, 2012), especially because their membrane-inserted α helices are significantly stabilized by surrounding lipids and cholesterol (Sathyanarayana et al, 2018; Tanaka et al, 2015; Kristan et al, 2009), whereas the rigid β -barrels of β -PFTs are mainly stabilized by inter-strand interactions of the membrane inserted β barrels (Gouaux, 1998). Hence, our study of the inter-protomer interfacial interactions which may either stabilize or destabilize the large α -PFT ClyA pore may yield insights into general factors that could stabilize higher order α -helical assemblies.…”

“…Our screening protocol illustrates that Y27 present on the membrane inserted α -helix of ClyA ( α A1 in Figure 6 c ) forms a hydrogen bond with A179 present on the membrane-inserted hydrophobic motif, which is in close proximity to the helix α A1. Recent work from our group has shown that the mutation Y27A reduced the lytic activity of ClyA by 24-fold (Sathyanarayana et al, 2018) and the mutation Y27F completely abolished lytic activity. Further, Y27 was associated with a putative cholesterol recognition and consensus motif (CRAC) located on the α helix of ClyA and the neighbouring residues D25 and K29 were associated with strong cholesterol binding sites (Giri Rao et al, 2016; Sathyanarayana et al, 2018).…”

“…Recent work from our group has shown that the mutation Y27A reduced the lytic activity of ClyA by 24-fold (Sathyanarayana et al, 2018) and the mutation Y27F completely abolished lytic activity. Further, Y27 was associated with a putative cholesterol recognition and consensus motif (CRAC) located on the α helix of ClyA and the neighbouring residues D25 and K29 were associated with strong cholesterol binding sites (Giri Rao et al, 2016; Sathyanarayana et al, 2018). The second set of residues implicated as potential targets (Figure 6 a, c ) are associated with K206 and D171 located on neighbouring membrane inserted β tongues at the membrane-water interface.…”

“…The second set of residues implicated as potential targets (Figure 6 a, c ) are associated with K206 and D171 located on neighbouring membrane inserted β tongues at the membrane-water interface. Interestingly, K206 was found to have strong interactions with cholesterol (Sathyanarayana et al, 2018) located in the pocket formed by adjacent β tongues with K206 and D171 present on neighbouring protomers that formed the pore. In addition to the previously implicated role played by cholesterol, this study suggests a complex interplay of interactions between α helices that contribute to the emergence of stability of the ClyA pore.…”

“…Direct visualization of the pore-forming process in molecular detail, especially the membrane-bound protomer and pore-intermediate conformations, is a formidable issue since pores lie within or at the edge of the optical resolution limit. Additionally, temporal resolution of pore formation is tricky since it is a rapid process that typically finishes within time scales of seconds (Thompson et al, 2011; Sathyanarayana et al, 2018). For these reasons, experimentally solved structures of pore intermediates are not available, which precludes direct structure-based inhibitor design against protomers and pore-intermediates.…”