Analysis of YfgL and YaeT Interactions through Bioinformatics, Mutagenesis, and Biochemistry

In Escherichia coli, YaeT, together with four lipoproteins, YfgL, YfiO, NlpB, and SmpA, forms a complex that is essential for -barrel outer membrane protein biogenesis. Data suggest that YfgL and YfiO make direct but independent physical contacts with YaeT. Whereas the YaeT-YfiO interaction needs NlpB and SmpA for complex stabilization, the YaeT-YfgL interaction does not. Using bioinformatics, genetics, and biochemical approaches, we have identified three residues, L173, L175, and R176, in the mature YfgL protein that are critical for both function and interactions with YaeT. A single substitution at any of these sites produces no phenotypic defect, but two or three simultaneous alterations produce mild or yfgL-null phenotypes, respectively. Interestingly, biochemical data show that all YfgL variants, including those with single substitutions, have weakened in vivo YaeT-YfgL interaction. These defects are not due to mislocalization or low steady-state levels of YfgL. Cysteine-directed cross-linking data show that the region encompassing L173, L175, and R176 makes direct contact with YaeT. Using the same genetic and biochemical strategies, it was found that altering residues D227 and D229 in another region of YfgL from E221 to D229 resulted in defective YaeT bindings. In contrast, mutational analysis of conserved residues V319 to H328 of YfgL shows that they are important for YfgL biogenesis but not YfgL-YaeT interactions. The five YfgL mutants defective in YaeT associations and the yfgL background were used to show that SurA binds to YaeT (or another complex member) without going through YfgL. The discovery of Omp85 in Neisseria meningitidis represents a major piece of the outer membrane biogenesis puzzle (39). Omp85, an essential, highly conserved, integral -barrel outer membrane protein (OMP), is required for the assembly of OMPs (40). Recently, it was shown that the Omp85 homolog in Escherichia coli, YaeT, forms a multiprotein complex comprised of the OMP YaeT (BamA) and four lipoproteins, YfgL (BamB), YfiO (BamC), NlpB (BamD), and SmpA (BamE) (32, 43) (the names in the parentheses are newly standardized designations [22], but the old computer-assigned names are used in this paper due to their prevalence in the literature). Like the N. meningitidis counterpart, the YaeT complex is required for the assembly of OMPs in E. coli (4, 42, 43). The recently solved crystal structure of the N-terminal polypeptide transport-associated (POTRA) domains of YaeT provided a possible clue for the mechanism by which YaeT interacts with OMPs and YfgL (15, 22). OMPs are synthesized in the cytoplasm as precursor polypeptides with N-terminal signal sequences that direct them to the SecYEG complex for translocation across the inner membrane (26). The signal sequences are cleaved during translocation, and the mature polypeptides are released into the periplasm, where they are thought to interact with chaperones, foldases, and lipopolysaccharide (LPS) to avoid aggregation and acquire the proper folding status required for the eventual assembly and insertion into the outer membrane at the YaeT complex site (31).