SurA is a cryptically grooved chaperone that expands unfolded outer membrane proteins

Abstract

The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the β-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network.

Chaperones Skp and SurA dynamically expand unfolded outer membrane protein X and synergistically disassemble oligomeric aggregates

“…Interestingly, at elevated temperatures, the dependency on [SurA] was enhanced and we observed a strong decrease of Ê SurA–uOmpX with increasing [SurA] at 37 °C from Ê SurA–uOmpX (1.2 μM) ≈ 0.66 to Ê SurA– uOmpX (25 μM) ≈ 0.55. This suggests that uOmpX is more expanded at elevated [SurA], which can be explained by either a different interaction mode or that uOmpX is sequestered by multiple SurAs as also suggested by previous studies 31,33 .…”

“…4b), respectively, yielding an overall stabilization of the complex of Δ G (SurA– uOmpX) = (−57 ± 1) kJ mol −1 at 37 °C, which is slightly more favorable than for Skp 3 . The change of enthalpy was two-fold lower than for Skp 3 , likely due to more transient binding as suggested in recent studies 31,33 . At the same time, the change of entropy of uOmpX upon SurA binding was three-fold lower than with Skp 3 , and close to zero, indicating only a minimal change of configurational space for the binding of SurA to uOmpX.…”

“…Using smFRET, we have characterized the structural dynamics of chaperone-free and chaperone-bound uOmpX under near-native conditions and show that both chaperones, Skp and SurA, expand uOmpX upon binding. While previous reports have observed an expansion for SurA-bound OMPs 31,38 , an expansion upon Skp binding of an OMP in its cavity has not been observed. More surprisingly, the Skp 3 -bound uOmpX state is more expanded as compared to the SurA–uOmpX state over the concentration range tested, suggesting that uOmpX interacts with Skp 3 at multiple sites by spreading across the inner surface of the chaperone cavity (Fig.…”

“…Skp depletion, on the other hand, led to an accumulation of misfolded OMPs and the activation of the cellular stress response 30 , while OMP density in the outer membrane remained the same. Interestingly, recent studies suggest substrate selectivity amongst the two chaperones 31 . Hence, it is of importance to understand the functional mechanisms underlying both Skp and SurA association with uOMPs.…”

“…Structural studies of the eight β-stranded protein outer membrane protein X (OmpX) in the presence of Skp using nuclear magnetic resonance (NMR) spectroscopy have found that unfolded OmpX (uOmpX) shows sub-millisecond backbone dynamics 32 in complex with Skp. For binding of SurA to unfolded outer membrane protein A (uOmpA), both fluid globular 32 and expanded states 31 have been proposed. Recent studies have located various interaction sites of SurA and uOmpX using cross-linking, suggesting that SurA-bound uOmpX populates multiple conformations 31,33 .…”