Structures of Unfolded Outer Membrane Proteins in Complex with Chaperones

Abstract

The viability of Gram-negative bacteria is ensured by the presence of outer membrane proteins (OMPs) acting as channels, porins, lipases and receptors. They are considered as interesting drug targets and thus obtaining a molecular perspective about various stages of their biogenesis is of great interest. However, the biogenesis of OMPs is biologically puzzling because it is not driven by ATP. We lack a detailed understanding of the various steps involved in this process. Unlike other proteins, OMPs adopt an active conformation only on reaching the outer membrane and thus have to be transferred from the cytoplasm through the periplasm in a folding competent state. Chaperones like Seventeen kilodalton protein (Skp) and Survival Factor A (SurA) protect these nascent hydrophobic proteins against aggregation. Yet, it remains elusive which conformations and interaction dynamics occur in the chaperone and unstructured OMP interplay. Using single molecule Förster Resonance Energy Transfer (smFRET), we investigate the structure of unfolded OMPs in chaperone-bound and unbound states. We find that both OMPs adopt a rather compact conformation in a low denaturant buffer. Remarkably, when bound with periplasmic chaperones, we observe that this compact state is dissolved by the chaperones differently for both OMPs. The small 8 beta-stranded OmpX shows heterogeneous conformations with sub-millisecond dynamics, while the 12 beta-stranded Outer membrane phospholipase A (OmpLA) appears to be pulled and pushed upon by the chaperones in expanded conformations. In both the cases, the chaperone-OMP interactions were remarkably long-lasting for hours, even below the dissociation constant. Our findings hint towards mechanisms by which OMPs are arranged in a folding competent state before reaching the outer membrane and shed light on the elusive mechanism of chaperone-OMP interaction in periplasm.