# Discovery and cryoEM structure of FPM13, a periplasmic metalloprotein unique to Francisella

## TL;DR

Scientists discovered a new metalloprotein in Francisella bacteria using cryoEM, revealing its structure and metal-binding properties.

## Contribution

The discovery of FPM13, a unique Francisella periplasmic metalloprotein, and its structural and functional characterization using cryoID.

## Key findings

- FPM13 forms an 18-mer complex with a central channel and binds iron, copper, and zinc.
- FPM13 catalyzes disulfide bond formation, a function lost in metal-binding mutants.
- Deletion of FPM13 does not affect bacterial growth in vitro or in host cells.

## Abstract

We report the identification and cryoEM structure of the Francisella protein FTN_1118, a previously uncharacterized 13 kDa periplasmic protein unique to the Francisella genus. The protein was serendipitously discovered during purification of Francisella type VI secretion system (T6SS) effector proteins and is hereby designated as FPM13 (Francisella Periplasmic Metalloprotein, 13 kDa) based on its cellular and biochemical properties. Identified by the cryoID approach based on our cryoEM density map, FPM13 exists naturally as a cylindrical 18-mer complex with 9-fold dihedral symmetry, formed by stacking two donut-shaped nonamers head-to-head. Measuring ~8 nm in height and outer diameter with a 3.5 nm central channel, the complex features a double-layered wall comprising an inner β-sheet core and an outer α-helical shell. Each FPM13 monomer adopts a compact fold comprising an N-terminus β-strand, an α-helix and two additional β strands at the C-terminus. Inter-ring loop interactions, hydrophobic contacts, and electrostatic interactions between adjacent subunits stabilize the assembly. Biochemical analyses, including APEX-biotinylation and Triton X-114 phase partitioning, confirmed FPM13 as a soluble periplasmic protein. Inductively coupled plasma mass spectrometry (ICP-MS) revealed FPM13 binds iron, copper, and zinc, with alanine substitution of predicted metal-binding cysteine and histidine residues abolishing this capability. Biochemical assays further revealed that wild-type FPM13 catalyzes disulfide bond formation and rescues alkaline phosphatase from reductive inactivation, indicating a role in maintaining periplasmic disulfide bonds. The metal-binding disruption mutant loses this oxidation activity. Deletion of FPM13 in Francisella novicida caused no growth defects in vitro, in macrophages, or in mice under tested conditions, suggesting functional redundancy may compensate for its absence. This study unveils a novel metalloprotein and demonstrates the power of cryoID in identifying uncharacterized proteins directly from structural data, offering new insights into Francisella biology.

Francisella tularensis, the cause of tularemia, is a highly infectious bacterium and a Tier 1 U.S. Federal Select Agent with sophisticated survival strategies. While studying Francisella novicida, a model organism closely related genetically to the biosafety level 3 agent Francisella tularensis, we discovered FPM13, a previously unknown 13 kDa protein unique to this genus. Found unexpectedly during efforts to isolate components of the bacterium’s type VI secretion system, a key virulence factor, FPM13 was visualized using cryogenic electron microscopy and identified via the innovative cryoID method. This revealed a ring-shaped complex of 18 subunits capable of binding metals like iron, copper, and zinc, a function reliant on specific amino acid residues. Our in vitro biochemical assays show that FPM13 catalyzes disulfide bond formation. The metal-binding disruption mutant loses this oxidation activity. However, deletion of FPM13 did not impair bacterial growth in lab cultures, immune cells, or animal models, hinting at compensatory mechanisms. This discovery underscores the power of advanced structural biology to uncover hidden microbial components and highlights cryoID as a transformative tool for identifying novel proteins in pathogens.