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Native state organization of outer membrane porins unraveled by HDx-MS Danilo Donnarumma, Claudio Maestri, Pietro Ivan Giammarinaro, Luigi Capriotti, Erika Bartolini, Daniele Veggi, Roberto Petracca, Maria Scarselli, and Nathalie Norais J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00830 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018
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Journal of Proteome Research
Native state organization of outer membrane porins unraveled by HDx-MS Danilo Donnarumma1‡, Claudio Maestri1‡†, Pietro I. Giammarinaro1§, Luigi Capriotti1, Erika Bartolini1, Daniele Veggi1, Roberto Petracca1, Maria Scarselli1, Nathalie Norais1* 1
GSK, Via Fiorentina 1, 53100 Siena, Italy
Key Words: Membrane proteins, OmpF, HDx, Outer membrane vesicles, Structural mass spectrometry
ABSTRACT: Hydrogen Deuterium exchange (HDx) associated with Mass Spectrometry (MS) is emerging as a powerful tool to provide conformational information about membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer Membrane Vesicles (OMVs) naturally released by E. coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis that avoids hindrance from the lipid contents was set-up. The extent of deuterium incorporation is in good agreement with the X-ray diffraction data of OmpF, since the buried β-barrels incorporated low amount of deuterium while the internal loop L3 as well as the external loops incorporated a higher amount of deuterium. Moreover, the kinetics of incorporation clearly highlight that peptides well segregate in two distinct groups based exclusively on a trimeric organization of OmpF in the membrane: peptides presenting fast kinetics of labeling are facing the complex surrounding environment, while those presenting slow kinetics are located in the buried core of the trimer. The data show that HDx-MS applied to a complex biological system is able to reveal solvent accessibility and spatial arrangement of an integral outer membrane protein complex.
INTRODUCTION. The study of the integral membrane proteins has always been one of the biggest challenges in structural biology. They are still sparsely represented in the protein structure databank, reflecting that the lack of adequate techniques has significantly hindered interrogation of membrane protein topologies. E. coli OmpF, a porin allowing the passive diffusion of polar solutes such as nutrients and waste products, not bigger than 600 Da, across the outer membrane1 was the first integral membrane protein structure obtained with a resolution lower than 4 Å. E. coli OmpF was also one of the first membrane proteins for which high-resolution crystallographic structure was determined more than ten years later.2 This was possible since the protein is highly abundant3 and forms extremely stable trimers.4 Crystallographic analysis of detergent-extracted OmpF revealed that the protein is organized in a homotrimeric structure. Each monomer presents 16 β-strands passing through the membrane bilayer connected by eight short turns (T1-T8) on the periplasmic face and eight loops (L1-L8) on the extracellular side, except for the loop L3 which folds back in the barrel forming the eyelet and reducing the size of the pore. The majority of the OmpF structure is in β conformation (59%) with only two well-defined α helices, one in loop L3 and the other one in loop L5. OmpF is still widely studied, to elucidate its function,5-8 and to clarify if the trimeric structure reflects the native organization of the protein in the membrane. Indeed, a dimeric folding, probably corresponding to an assembly intermediate, has been observed in both in vitro and in vivo experiments.9-12 X-ray diffraction and solution NMR structural studies of membrane proteins require their solubilisation in
detergents since lipid bilayers are incompatible with the threedimensional crystallization and isotropic motion. To overcome this limitation, in the last years solid state NMR has become the most used methodology to determine the structure of membrane proteins in a wide variety of lipid bilayers.13, 14 Hydrogen Deuterium exchange (HDx) associated with Mass Spectrometry (MS) is also emerging as an alternative tool to provide conformational information and dynamics of membrane proteins. Despite the low resolution output, HDx-MS has the advantage of requiring smaller amounts of material, it is not limited by the protein size, and it has the ability to distinguish proteins from lipids.15 Exchange has been performed in in vitro systems mimicking the membrane environment, such as detergent micelles, liposomes,16 nanodiscs17 or Langmuir monolayers.18 Unfortunately, like for X-ray diffraction and NMR, these conditions might not represent the native environment since neither membrane lipid composition nore protein complexity is represented. This is especially true for the outer membrane of Gram-negative bacteria, which is asymmetric, with lipopolysaccharides (LPS) (or lipooligosaccharides, LOS) forming its outer leaflet and phospholipids forming its inner leaflet.3 Influence of different LPS environments on the structure and dynamics of OmpF, accessibility of monoclonal antibodies, and ion permeability and selectivity have been reported.19 In this study, we took advantage of the Outer Membrane Vesicles (OMVs) naturally released by E. coli to carry out conformational and dynamic analysis of native OmpF through HDx-MS. OMVs are heterogeneously sized closed spheroid particles (10–300 nm in diameter) released by all
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Gram-negative bacteria and generated through the “blebbing out” of the bacterial outer membrane.20 The OMV blebbing was originally described as an essential step for rapid adaptation to environmental changes, but a multitude of other functions has now been attributed to them, including delivery of toxins and virulence factors to host cells, inter-species and intra-species cell-to-cell cross-talk, biofilm formation, genetic transformation and defense against innate and adaptive host immune responses.20 The OMVs have the advantage of carrying membrane proteins in their native conformation and natural environment since no chemical or mechanical treatment are applied for their production.21 Moreover, the use of OMVs considerably reduces the sample complexity compared to intact bacteria, since proteomics analysis identified around 100 proteins, mainly from outer membrane and periplasmic compartments.21,22 Specific engineered strains, usually with deletions of proteins involved in membrane structure, such as the Tol-pal system,23 release large amount of OMVs. Recently, an E. coli ∆-OmpA mutant has been proven to overproduce OMVs.24 We used these OMVs to develop a new protocol compatible with HDx-MS analysis avoiding hindrance from the lipid content and monitor incorporation of deuterium on more than 90% of the OmpF, to gain insights on the solvent accessibility and spatial arrangement of the porin in its native state.
EXPERIMENTAL SECTION. Preparation of the OMVs. E. coli BL 21 ∆ompA24 was plated on a Luria-Bertani cell-culture dish and grown overnight. Bacteria were recovered and suspended in 25 mL of Luria-Bertani broth at 0.5 OD (Optical Density) and grown for 5 hours, shaking at 150 RPM at 37°C. The pre-inoculum was diluted in 200 mL of LuriaBertani broth at 0.5 OD and grown overnight at 150 RPM shaking and 37°C. T. Bacteria were then collected by 15 min centrifugation at 3,200 x g and the recovered culture medium was filtered through a 0.22 µm pore size filter (Millipore, Bedford, MA, USA) and protease inhibitors (SIGMAFAST, Sigma Aldrich, Merck KGaA, Darmstadt, Germany) were added to the suspension. The filtrates were subjected to ultracentrifugation (200,000 x g, for 180 min) and the pellets containing the OMVs were then suspended either with PBS or 5 mM KCl, and stored at -20°C. Deuteration and sample preparation for mass spectrometry analysis. The deuterium labeling was initiated by diluting 10 µL of E. coli BL21 OMV with 44 µL of deuterated PBS buffer (pH 7.4), or D2O (99.9% atom D (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) used without additional purification), reaching a deuterium excess of 81.5%. The labeling was performed at room temperature (RT), and at the end of each selected time point, 6 µL of cold TCA (Trichloroacetic Acid, Sigma Aldrich) 60% (w/v) were added and the sample was immediately centrifuged at 16,000 x g at 4 °C for 4 minutes. The supernatant was removed and the protein pellet was washed with 60 µL of cold acetone (-20 °C) and immediately centrifuged at 16,000 x g at 4 °C for 4 min. The supernatant was removed and the protein pellet was re-suspended with 60 µL of an ice-cold 200 mM sodium phosphate 2 M guanidinium chloride pH 2.4 buffer, frozen in liquid nitrogen and stored at −80°C for
