# A new family of small ArdA proteins reveals antirestriction activity

## TL;DR

Researchers discovered a new family of small proteins called sArdA that can protect genetic material from host defenses, similar to larger proteins but with unique properties.

## Contribution

The discovery of a new family of small antirestriction proteins (sArdA) and their distinct interaction mechanisms with restriction-modification systems.

## Key findings

- sArdA proteins are about one-third the size of full ArdA proteins and can be split into two subgroups, sArdN and sArdC.
- Both sArdN and sArdC can independently interact with RM systems and trigger similar intermediate closed states in EcoKI.
- sArdN and sArdC showed different specificities against various RM systems and exhibited antimethylation activity against EcoKI.

## Abstract

Antirestriction proteins protect mobile genetic elements from the
host’s restriction-modification (RM) systems. In our study, we
identified a new family of small proteins, which we named sArdA. The sArdA
proteins are homologous to DNA-mimicking ArdA proteins but differ in size,
being approximately one-third the length of full ArdAs. Moreover, the sArdA
family contains two subgroups, one of which is structurally similar to the
N-terminal end of ArdA, whereas the other one matches the C-terminal end.
Both the N-terminal and C-terminal domains of ArdA appear capable of
independent expression. Phylogenetic analysis demonstrated that genes
encoding these proteins evolved into evolutionarily stable subfamilies,
named sArdN and sArdC, respectively. AlphaFold structure prediction of sArdA
interaction with RM systems revealed four states of EcoKI, which differ in
the angle between its two M-subunits while interacting with different ArdAs
or DNA. Interestingly, both sArdN and sArdC triggered the same intermediate
closed state of EcoKI, indicating possible new interaction pathways of Ards
with RM systems. For phenotypic studies in Escherichia coli
cells, we cloned the sardN gene from the chromosome of
Corynebacterium pilbarense and the
sardC gene from Lactococcus cremoris.
Both genes protected λ phage DNA from restriction by the type I RM
system. However, they revealed specificities to different
restriction-modification systems. Specifically, sArdC was
more effective against EcoR124II, whereas sArdN was more
potent against EcoKI. Furthermore, both genes demonstrated antimethylation
activity against EcoKI. Our current findings suggest the idea that the
binding specificity of DNA-mimicking proteins to their targets could also be
achieved by very short proteins.

Our current findings suggest that the binding specificity of
DNA-mimicking proteins to their targets could also be achieved by very
short proteins. The ability of these DNA-mimicking proteins to
specifically inhibit different DNA-binding proteins makes them a
promising tool for regulating a range of intracellular processes,
including gene expression.