Fig. 2.

Proposed topology models. a The earliest proposed topology was based on a bioinformatics investigation, which concluded that the enzyme had seven membrane helices, a cytoplasmic N-terminus and an active site facing the cytoplasm⁶¹. The putative active site included the BacA2 sequence motif while the conserved BacA1 region mapped to the first membrane helix and was proposed to be involved in catalysis and in substrate specificity. b The second proposed model relied on bioinformatics, modeling, and functional and site-directed mutation work employing constructs of BacA fused with bacteriorhodopsin and green fluorescent protein¹⁹. This led to the conclusion that the protein had eight membrane helices with both N- and C-termini in the cytoplasm and an outward facing active site. c The third model involved an experimental investigation focussed on biochemistry with functional and mutational studies employing hybrids prepared from C-terminal truncations of BacA fused to β-lactamase¹⁸. This facilitated in vivo screening based on ampicillin resistance or susceptibility depending on which side of the membrane that β-lactamase resided. The model to emerge from this study had seven membrane helices with the N-terminus in the cytoplasm and the active site facing the periplasm. d The latest model was based on residue-residue co-evolution constraints applied in the Rosetta structure prediction program to generate a structure of BacA²⁴. The modeled protein includes a pair of inverted helices and has twofold pseudosymmetry as observed in the structure determined experimentally in the current study. The membrane orientation in d was not defined. Putative catalytic residues Ser27 and R174 are indicated by red and blue dots, respectively