Crystal Structure of the Type VI Secretion System Accessory Protein TagF from Pseudomonas Aeruginosa
Authors: Ok Chang-Kyu , Chang Jeong Ho 
Published: January 2019

Background:

Type VI Secretion System (T6SS) has been found in approximately onequarter
of the gram-negative bacterial species, and its structural characteristics appear to slightly
differ from species to species. The genes encoding T6SS are designated as type six secretion A–M
(tssA–M). The expression of the tss gene cluster is regulated by various accessory genes, designated
as type VI-associated genes A–P (tagA–P). Tag family proteins have been commonly found
in bacteria expressing T6SS but not in all bacterial species. For instance, the tag gene cluster is
well-conserved in Pseudomonas aeruginosa (Pa). The PaTagF protein has large homology with
ImpM in Rhizobium leguminosarum and SciT in Salmonella enterica. The overexpression of
PaTagF represses T6SS complex accumulation and suppresses T6SS antibacterial activity. Thus,
the functions of TagF are mediated through direct interactions with the forkhead-associated protein
Fha, as evident from the results of the yeast-two hybrid assays. Fha is involved in recruiting a
membrane-associated complex either in threonine phosphorylation pathway-dependent or -
independent manner. However, functional reports of the tag gene cluster are still limited.
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Objective: In this article, our motivation is to understand the molecular mechanism underlying the
regulation of expression of the type VI secretion system complex.

Methods:

In this article, we start with obtaining the gene encoding PaTagF protein by polymerase
chain reaction (PCR). Subsequently, the cloned gene is applied to overexpress of PaTagF protein in
Escherichia coli, then purify the recombinant PaTagF protein. Thereafter, the protein is crystallized
in a condition of 2.5 M NaCl, 0.1 M imidazole (pH 8.0), 3.2 M NaCl, 0.1 M BIS-TRIS propane (pH
7.0) and diffraction datasets of the PaTagF crystals are collected at the Pohang Accelerator Laboratory
(PAL). The molecular structure of PaTagF protein is determined by molecular replacement
using the uncharacterized protein PA0076 (PDB code:2QNU) as an initial search model by
PHENIX crystallographic software package. Model building of PaTagF structure is performed using
Coot program. Finally, the structural model is validated using phenix.refine program.

Results:

PaTagF exists as a tetramer in the asymmetric unit, and the overall fold of each monomer
is composed of continuous beta-sheets wrapped by alpha-helices. Each monomer has variable conformations
and lengths of both the N- and C-termini. Twelve residues, including the His6 tag from
the N-terminus of a symmetry-related molecule, have been found in two of the tetrameric PaTagF
structures. A structural homology search revealed that PaTagF was similar to the α-β-α sandwichlike
structure of the longin domain on the differentially expressed in normal and neoplastic (DENN)
superfamily, which is commonly found in proteins related to trafficking.

Conclusion:

The tetrameric structure of PaTagF comprises varied N- and C-terminal regions in
each subunit and may be stabilized by a symmetry-related molecule. This feature was also shown in
the TssL structure from V. cholerae. Furthermore, our study showed that the overall fold of PaTagF
is homologous to the longin domain of the DENN family. Therefore, further studies are warranted
to elucidate the structure-based evolutionary relationship between protein transport systems from
the bacteria and eukaryotic cells.