SrcA interacts with the SPI-2 T3SS ATPase 
An emerging function for T3SS chaperones is delivery of cargo to the base of the apparatus through interactions with an ATPase.
This was shown for the flagellar T3SS [17] and later in the virulence-associated T3SS in E. coli [16],[27] and the SPI-1 T3SS in Salmonella [15].
However, analogous interactions have not been described for the SPI-2 T3SS.
Since srcA expression was co-regulated with genes in SPI-2, we hypothesized that it had a functional role in this system.
To address this biochemically we purified SrcA and the predicted ATPase for the SPI-2 T3SS, SsaN, and performed binding experiments and gel filtration chromatography of the protein mixtures.
SsaN contains conserved amino acid residues characteristic of Walker-A and Walker-B motifs of P-loop nucleoside triphosphate hydrolases, as well as a number of residues shown to contribute to ATP binding or ring stacking with the adenine base of ATP in the E. coli orthologue, EscN, (Q412, E191, R366) (Fig. S1).
Since SsaN had not been characterized biochemically we first verified that our purified protein had ATPase activity (Fig. S1).
We then mixed SrcA and SsaN proteins and resolved the protein complexes by gel filtration chromatography.
By itself, SrcA existed as a dimer in solution (Fig. 4A) with no higher oligomers present, substantiating the stoichiometry obtained from our crystal data.
SsaN existed as a monomer with a minor population eluting in a volume consistent with a probable dimer (Fig. 4B).
When SrcA was mixed with SsaN, a new protein complex of high molecular weight was observed, along with diminished peaks corresponding to the SrcA dimer and SsaN monomer (Fig. 4C).
This new complex elutes with a Stokes radius consistent with an apparent molecular mass of approximately600 kDa.
We verified the identities of protein originating from each peak by western blot (Fig. 4D) and LC-MS/MS, which showed the new complex was comprised of both SsaN and SrcA.
