Outer membrane protein folding from an energy landscape perspective

Abstract

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding.

Fig. 1

Crystal structures of OMPs from Gram-negative bacteria. Top row: OmpX (2MO6) [222]; PagP (3GP6) [73]; tOmpA (transmembrane domain of OmpA; 1QJP) [223]; OmpW (2F1V) [75]; OmpT (1 L78) [224]; EspP (β-domain) (2QOM) [225]. Middle row: OmpLA (1QD5) [226]; OmpG (2IWV) [227]; FadL (1T1L) [228]; OmpF (1OPF) [229]; BamA (4K3B) [76]. Bottom row: LamB (1MAL) [230]; FhuA (1BY3) [231]; FimD (3OHN) [232]; LptD (4Q35) [233]. All structures are from E. coli with the exceptions of BamA (Neisseria gonorrhoeae) and LptDE (Shigella flexneri). The LptD barrel has an associated lipoprotein subunit (LptE) in the functional complex (see main text). LptD and LptE are shown in orange and cyan, respectively. Approximate location of the membrane is shown in yellow, with the periplasmic face to the lower side of each structure. Note that OmpF and LamB are shown in their native trimeric forms

Fig. 2

Requirements for OMPs to fold to a stable and functional state. Clockwise from top: (i) OmpX (2MO6 [222], grey)—hydrophobic residues are shown as orange sticks; (ii) OmpLA (1QD6 [226], blue) in a dimyristoylphosphatidylethanolamine (DMPE; diC_14:0PE) bilayer (from [234]); (iii) alignment of BamA β-barrel structures in the ‘lateral open’ (5D0Q [78], green) and ‘lateral closed’ (5D0O [78], yellow) states; (iv) OmpF (2OMF [229])—monomers in the trimeric structure are shown in red, yellow and blue; (v) FhuA (1FI1 [235], pink)—bound LPS is shown as yellow sticks; (vi) OmpT (1I78 [224], green cartoon)—regions of red and blue represent areas of electronegative and electropositive surface potential (−1 kT/e to +1 kT/e) and were created using the APBS plugin for PyMOL [236]; (vii) PagP (3GP6 [237], orange)—conserved residues important in enzymatic function, H33, D76 and S77 (pink, green and cyan, respectively), are highlighted; (viii) free energy diagram showing the difference in stability of the folded (F) and unfolded (U) states; (ix) LptD (4N4R [37], lime green)—Trp residues are shown as red sticks. The central image shows the transmembrane domain of OmpA (1QJP [223], with mutated residues in the structure replaced with wild-type residues and missing residues in the loops built in using MODELLER [238]) in a DMPE bilayer (taken from [234])

Fig. 3

Hypothetical energy landscape for unassisted OMP folding. The green surface on the left depicts OMP conformations that may be formed en route to the native state. Non-native intramolecular interactions, and/or interactions with the membrane, may lead to ruggedness in the landscape. The surface on the right shows some possible conformations of self-associated OMPs, which may lead to the formation of ordered amyloid-like or disordered aggregates. How folding factors in the cell influence this landscape remains an open question. The OMP polypeptide chain and the membrane are shown in red and blue, respectively

Fig. 4

OMP assembly pathway across the periplasm. OMPs are secreted into the periplasm by SecYEG (purple, 5ABB [239]), where they are recognised by chaperones, of which the most important in E. coli are SurA (red, 1M5Y [148], with missing residues built using MODELLER [238]), and Skp (blue, 1U2M [240], missing residues built using PyMOL [241]). OMP sequences must also contain information for targeting to the BAM complex (5LJ0 [79]), which catalyses the final folding and insertion step into the OM. Misfolded OMPs trigger stress responses (e.g. DegS/RseA pathway [242, 243] (not shown)), and are degraded by the periplasmic protease DegP (shown with multiple colours highlighting the subunits of this dodecameric complex (2ZLE [244])). An example of a folded OMP in the OM (FhuA, 1BY3 [231]) is shown in orange. Note that LPS and the peptidoglycan layer are omitted from this schematic. The inner and outer membranes are shown approximated to the width of a di-oleoylphosphatidylethanolamine (diC_18:1PE, DOPE) bilayer (generated in VMD [245]). The distance between the inner and the outer membranes can be inferred from the structures of machineries that span the periplasm, giving estimates in the range ~ 165–170 Å [179, 246] to ~ 190–210 Å [247]. Here, the latter distance (210 Å) is used, consistent with the dimensions of the periplasm observed by cryo-electron microscopy [248]. The question mark highlights the fact that whether Skp can deliver OMPs to BAM in vivo remains unknown, although BamA-containing membranes can promote folding of OMPs from their complexes with Skp in vitro [65, 126]

Fig. 5

Possible, currently hypothetical, mechanisms for OMP assembly by the BAM complex. a BamA-assisted. OMPs may fold via a pathway similar to that observed in vitro, with BamA acting as a membrane ‘disruptase’ to assist folding. b BamA-budding. OMP assembly involves the formation of a hybrid barrel by sequential insertion of β-strands templated by the β1/β16 strands of BamA. When the final substrate β-strand has been inserted, the nascent OMP buds off from the BamA barrel to complete folding. c Barrel-elongation. Interaction of the nascent OMP with the periplasmic BAM region promotes a ‘lateral open’ BAM state, exposing the β1 strand of BamA. BamA β1 then templates β-sheet formation in the nascent OMP, possibly via β-hairpin units. Folding is completed by concerted OMP insertion and tertiary structure formation, releasing the BamA barrel and allowing BAM to return to the ground state. In all models BamA is involved in destabilising the membrane to aid insertion and folding (not shown). The lipoproteins BamB–E and the chaperone SurA have been omitted for clarity. Note that there is currently little direct experimental evidence to favour one model over another (see main text for more details)

Fig. 6

Cryo-EM structure of the BAM complex. Solution structure of the BAM complex viewed from a the membrane plane, and b the periplasm. Image created with PyMOL (5LJO [79]). Individual BAM subunits are labelled in different colours, as indicated