Enterotoxigenic Escherichia coli is a common cause of bacterial mediated diarrhoeal disease worldwide and features virulence factor and atypical lipoprotein, CexE, on its cell surface. Transport of CexE to the cell surface is facilitated by a novel composite secretion system termed the Aat system. The Aat system is composed of five proteins (AatPABCD) that incorporates elements of a Type I secretion system (T1SS) and the Lol lipoprotein trafficking pathway. Secretion through this Type I-like system occurs in a two-step manner: (i) CexE is first translocated to the periplasm by the Sec translocon. (ii) the folded CexE substrate is then modified, and its folded form is then recognised by the Aat system for translocation and surface display. Secretion of a folded lipoprotein substrate from the periplasm has not been demonstrated by a T1SS. To better understand this, we used a combination of biochemical, molecular, and structural techniques to determine the molecular rules that govern secretion of CexE by the Aat system. The structure of CexE was solved using NMR, revealing a bullet-shaped protein like that of its homolog dispersin. While there was high structural similarity between CexE homologs, there was limited amino acid conservation. A further 25 structural models of CexE homologs were retrieved from the AlphaFold database and phylogenetic analyses of these protein sequences identified three clades. Between these three clades there are only slight structural variations. From this we hypothesised that the tertiary protein structure is critical for recognition and secretion by the Aat system, rather than its primary linear structure. To investigate the role protein folding and structure in secretion, site directed mutagenesis will be used to interrogate highly conserved residues, including a difsulfide bond that is present in all homologs. Furthermore, the secretion of CexE homologs with low amino acid identity by the ETEC H10407 Aat system will determine whether sequence motifs are required for transport through this system. Understanding the mechanism of CexE secretion could be utilised for biotechnological approaches for protein surface display.