Direct observations of the Transpeptidation activity of Sortase in a DNA nanostructure

Session

LSA.4 - Applications of AFM in geological and biological context

Authors

Dr Andrew Lee (1), Dr Zoe Arnott (1), Dr Mike Webb (1), Prof. Giles Davies (1), Prof. Christoph Wälti (1)

Affiliations

1. University of Leeds

Keywords

DNA origami, High Speed AFM, Sortase

Abstract text

High Speed Atomic Force Microscopy (HS-AFM) provides direct access to the interactions of single biological entities within physiologically relevant aqueous environments. This approach is dramatically enhanced where it is coupled with DNA-based nanostructures capable of organising and contextualising the interacting species [1].

 

DNA-based nanostructures provide a support surface upon which peptide and nucleic acid enzyme substrates can be organised, present alongside inbuilt positional and orientational markers and heterogenous controls. As such, this combined approach provides excellent tools to directly investigate the dynamic interactions of single biological entities, inaccessible to any other approach. 

 

Here, we apply this approach to study the commercially important and clinically relevant transpeptidation reaction of the bacterial protein, Sortase. This process is central to the attachment and presentation of proteins at the external cell wall surface of Gram-positive bacteria and as such is key to controlling the virulence of these pathogens. Consequently, Sortase enzymes are considered an important universal antibiotic target [2].

 

The action of Sortase is analogous to the combined action of nucleic acid restriction enzymes and ligases, with three main stages; recognition of an amino acid sequence, cleavage of a peptide bond and the formation of ade novopeptide bond with a new amino acid chain.

 

By employing a novel nucleic acid-peptide fusion, which includes the Sortase recognition sequence, we position a globular protein (MBP) within a DNA nanostructure as a fiducial marker of Sortase activity. Not only do we observe the transition between two fiducial markers (MBP & MupB) of different sizes, but we are able to dissect the various states of the Sortase interaction (figure 1) in situ and in real time. 

 

Interestingly we observe the existence of a bistable Sortase population and demonstrate that only the larger observed Sortase species orchestrates successful transpeptidation activity. The proposed existence of Sortase as a dimer has previously been described by others [3–5], however to date only limited biochemical evidence exists in support. 

 

We propose that the observations in our study represent the first direct evidence of an active Sortase dimer, potentially revealing a critical part of the Sortase pathway for disruption with novel antibiotics, for which further work is required to exploit. This novel insight further demonstrates the unique advantages of contextualised direct single entity observations using DNA nanostructures and HS-AFM over traditional indirect ensemble approaches.

Figure 1. The observed states of Sortase-mediated transpeptidation activity within a DNA nanostructure. Schematic diagrams are depicted alongside representative HS-AFM micrographs. A) an empty DNA nanostructure. B) initial protein substrate (MBP). C) Unbound Sortase with protein substrate (MBP). D) Substrate (MBP) bound Sortase complex. E) Cleaved protein substrate (MBP). F) De novo protein attachment (MupB). Scale bar = 40 nm. Insets = 40 nm. 

References

[1]        A. J. Lee et al,  ACS Nano (2018) 12, 272–278.

[2]        A. Maresso et al, Pharmacol. Rev. (2008) 60, 128–141.

[3]        C. Lu et al, Biochemistry (2007) 46, 9346–9354.

[4]        J. Zhu et al, Biochemistry (2008) 47, 1667–1674.

[5]        J. Zhu et al, Exp. Biol. Med. (2016) 241, 90–100.