# Chlamydia plasmid-encoded protein Pgp2 is a replication initiator with a unique β-hairpin necessary for iteron-binding and plasmid replication

## TL;DR

The study identifies Pgp2 as a replication initiator in Chlamydia plasmids and shows a unique β-hairpin is essential for its function.

## Contribution

Pgp2 is shown to be a replication initiator with a unique β-hairpin motif critical for plasmid replication.

## Key findings

- AlphaFold predicted a 3-D structure of Pgp2 similar to known plasmid replication initiators.
- A unique β-hairpin motif in Pgp2 is necessary for binding to plasmid iteron sequences.
- Deleting the β-hairpin motif in Pgp2 prevents successful plasmid transformation in Chlamydia.

## Abstract

The virulence plasmid of the obligate intracellular bacterium Chlamydia encodes eight proteins. Among these, Pgp3 is crucial for pathogenicity, and Pgp4 functions as a transcriptional regulator of both plasmid and chromosomal genes. The remaining proteins, Pgp1, Pgp5, Pgp6, Pgp7, and Pgp8, are predicted to play various roles in plasmid replication or maintenance based on their amino acid sequences. However, the function of Pgp2 remains unknown, even though it is required for transformation. In this study, we utilized AlphaFold to predict the three-dimensional (3-D) structure of Chlamydia trachomatis Pgp2. Despite a lack of apparent sequence homology, the AlphaFold structure exhibited high similarity to experimentally determined structures of several plasmid replication initiators. Notably, Pgp2 features a unique β-hairpin motif near the DNA-binding domain, absent in other plasmid replication initiators with overall 3-D structures similar to Pgp2. This β-hairpin motif is also present in AlphaFold models of Pgp2s across all 13 Chlamydia species. To assess its significance, we engineered a plasmid lacking the 11 amino acids constituting the β-hairpin motif in C. trachomatis Pgp2. Although this deletion did not alter the overall structure of Pgp2, the mutated plasmid failed to transform plasmid-free C. trachomatis. These findings reveal that Pgp2 is a plasmid replication initiator, with the β-hairpin motif playing a critical role in binding to its cognate iteron sequences in the replication origin of the chlamydial plasmid.