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. 2024 Apr 11;146(16):11025–11030. doi: 10.1021/jacs.3c14358

Detergents with Scalable Properties Identify Noncanonical Lipopolysaccharide Binding to Bacterial Inner Membrane Proteins

Leonhard H Urner †,‡,*, Francesco Fiorentino §, Denis Shutin ‡, Joshua B Sauer ‡, Mark T Agasid ‡, Tarick J El-Baba ‡, Jani R Bolla ‡,∥, Phillip J Stansfeld ⊥, Carol V Robinson ‡,*
PMCID: PMC11046432  PMID: 38604609

Abstract

graphic file with name ja3c14358_0004.jpg

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic–lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes, can also affect the activity of membrane proteins that are currently not assigned to be involved in outer membrane biogenesis. Our results deliver a scalable detergent chemistry for a streamlined biophysical characterization of protein–lipid interactions, provide a rationale for the high affinity of LPS-protein binding, and identify noncanonical associations between LPS and inner membrane proteins with relevance for membrane biology and antibiotic research.

Lipids surrounding proteins in the cell membrane of Gram-negative bacteria play roles in biological functions.1−3 Their identity is often difficult to specify.4−6 The cell wall of Gram-negative bacteria contains two lipid bilayers known as outer membrane and inner membrane (Figure 1).7−9 Inner membranes contain PLs, which are important for membrane integrity and protein function.7−9 LPS is synthesized across the inner membrane and translocated to the outer membrane where it is displayed at the outer leaflet.7−10 LPS secreted by bacteria during infections can cause septic shock in patients and the lipid A core in LPS is a target for polymyxin antibiotics.2,11,12 The biological relevance of LPS binding is established for proteins involved in LPS synthesis and translocation, like MsbA (Figure 1).9,13−16 LPS binding to proteins not associated with outer membrane biogenesis and function remains unexplored and is considered as purification artifact, such as in the cases of the acriflavine resistance B (AcrB) efflux pump,17 mechanosensitive channel (MscL),18 and vitamin B12 transporter (BtuCD).19 Technologies that enable the identification of biologically relevant protein-LPS interactions are needed to advance our understanding on biological membranes and to support the development of membrane-targeting antibiotics.14,20

Figure 1.

Figure 1

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Schematic snapshot of proposed Gram-negative E. coli membrane model visualizing noncanonical LPS binding to inner membrane proteins not assigned currently with outer membrane biogenesis and whose relevance is investigated in this work.

nMS enables the observation of lipids bound directly to membrane proteins21 and helps to evaluate their regulatory roles for structure and function.1 To isolate membrane proteins, inner and outer membranes are traditionally disrupted by lysis and mixed prior to solubilization with detergents.22 The solubilized mixture contains proteins, lipids, and detergents from which target proteins are isolated by affinity purification.23−25 Detergents in combination with nMS are established tools for the purification and analysis of protein–lipid complexes.26−28 Whether copurified LPS binding to inner membrane proteins currently not assigned to outer membrane biogenesis represents a protein–lipid interaction existing in inner membranes or remains an artifact of solubilization is yet to be clarified (Figure 1). To address this question, we combine rationally designed detergents,29−31 streamlined delipidation methods,27,31 and latest nMS capabilities21,32 to advance our knowledge on the role of LPS in inner bacterial membranes.

To assess the lipid complexes that copurify with proteins and may exist in membranes, we envisioned a detergent design that allows us to control the retention of protein–lipid interactions, while simultaneously ensuring that proteins will be stable in required environments. Practically, delipidation is optimized through protein purification from comparable membrane batches and controlled by variations in detergent structure and concentration.31 Since two parameters change simultaneously, i.e., detergent structure and concentration, delipidation outcomes are commonly seen as detergent-dependent.33

To uncouple protein solubilization from delipidation, we designed a library of detergents with scalable properties consisting of OG, C8E4, and 1–5 (Figure 2a). First, to monitor the role of detergent structure in delipidation, detergents 1–5 differ in terms of structure but require similar concentrations during purification (Figure 2a) (Table S1). Second, to gradually tune the delipidating properties of detergent aggregates, the detergents C8E4, OG, and 1–5 gradually differ in terms of hydrophobicity and packing density (Figure 2a) (Tables S2 and S3).34 Both hydrophobicity and packing density are estimated by the hydrophilic–lipophilic balance (HLB)35 and packing parameter values36 which we designed to match those of protein-stabilizing detergents.29,30,37 Third, to optimize for the facile nMS analysis of protein–lipid complexes, we designed detergents to be nonionic with less than five hydroxyl groups in the head (Figure 2a).31 HLB and packing parameters of our detergents are varied such that the overall hydrophobicity and packing density of their aggregates decrease gradually in the direction from OG to 5 (Figure 2a). We hypothesize that the more densely packed, hydrophobic detergent aggregates interfere more efficiently with lipid binding than more loosely packed, less hydrophobic detergent aggregates.

Figure 2.

Figure 2

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Delipidation outcomes depend on both membrane proteins and detergents. (a) Schematic showing structures of detergents and bar chart showing changes in packing parameter and HLB that translate into gradual changes in hydrophobicity, packing density, and delipidating properties detergent aggregates. (b) Overview of inner membrane proteins that are subject of this study and steps involved in relative quantification of delipidation outcomes. (c) Diagrams showing relative intensities of apo state, protein-PL complexes, and protein-LPS complexes obtained from nMS analysis of proteins that were delipidated with lower detergent concentrations during SEC (2× cac) or higher detergent concentrations during IMAC (50× cac).

To define the utility of our detergent design, six inner membrane proteins were expressed in Gram-negative E. coli, three of which bind PLs (TSPO,21 AmtB,1 AqpZ1) and the other three for which binding to PLs and LPS has been detected before by nMS (MscL,38 AcrB,17 BtuCD) (Figure 2b).19 Briefly, proteins were purified with lipids by extraction, affinity purification, and size-exclusion chromatography (SEC) using mildly delipidating n-dodecyl-ß-d-maltoside (DDM) (Figure 2b).22 Protein were then delipidated by detergent exchange into OG, C8E4, 1–5 at two times of their critical aggregation concentration (cac) using SEC (Figure 2b).38 Comparing the relative intensities of apo states and lipid-bound states of PL-binding protein by nMS (Figure 2c) indicates that PL-delipidation is sensitive to changes in hydrophobicity and packing density of micelles (Figure 2c) (Figures S1 and S2). Decreasing the hydrophobicity and packing density of detergent micelles enhances the retention of protein-PL interactions during purification.

To evaluate whether altering detergent properties biases relative abundances of copurifying PL species, relative intensities of PL species that bind to proteins were compared by native top-down MS.21 Similar relative abundances were observed among all three PL classes that copurify with bacterial inner membrane proteins, such as PE, PG, and CDL (Figures S3 and S4). All PL classes are equally delipidated by altering the hydrophobicity and shape of the detergents.

When extending our method to inner membrane proteins for which LPS-binding has been detected before by nMS, we found that relative amounts of apo form and LPS-complexes did not change with the hydrophobicity and shape of detergents (Figure 2c) (Figure S5). While PL-binding can be gradually controlled at low detergent concentrations (2× cac) using SEC, LPS-binding proteins need to be immobilized on affinity resin and washed with larger column volumes (CVs) of buffer with high detergent concentrations (50× cac; Figure 2c) (Figures S5 and S6). Combining our scalable detergent design with comparable purification conditions and nMS allows to differentiate the degree to which proteins copurify with PLs and/or LPS and to identify conditions needed to control delipidation and nMS analysis (Figure 2c).

LPS plays protective and structural roles in the outer membrane and is important for the function of inner membrane proteins involved in LPS translocation.10 Previous studies on MscL, AcrB, and BtuCD illustrated LPS copurification without ascribing a role with LPS (Figure 2c) (Figure S5).17−19 To investigate whether LPS complexes are artifactually produced by membrane mixing or reflect membrane environments, we incubated fully delipidated AmtB and AcrB with PLs and different LPS forms and compared their relative affinities by nMS using an established procedure.39 Regardless of the protein, higher relative affinities were observed in the cases of CDL and LPS (Figure S7). This suggests that both proteins preferentially bind CDL and LPS over PE and PG under the assay conditions. In contrast, we do see that LPS copurification is protein dependent (Figure 2c), which leads us to conflicting explanations. Proteins either have different selectivity for PLs and LPS and get lipidated accordingly during membrane solubilization with detergents or protein–lipid complexes obtained upon detergent purifications are biased by the lipid environment surrounding proteins in membranes (Figure 1). Our methodology does not allow us to rule out that AcrB never encounters LPS in membranes until cells are lysed and dissolved with detergent at which point it binds.

To investigate the relevance of tackling this question in years to come, we analyzed the effect that LPS binding can have on the activity of proteins not assigned with outer membrane biogenesis. We extended our method to BtuCD, a drug target that mediates the membrane transport of vital molecules.40 We were unable to fully deplete LPS during purification (Figure S5). To understand the origin of strong LPS binding, we monitored the relative abundance of lipid contacts among the surface of BtuCD in a simulated inner membrane bilayer containing PLs and LPS using molecular dynamics (MD). Relative abundance of lipid contacts with the soluble domain BtuD increased with the size of the glycan head in LPS (Figure S8). While protein-PL interactions were mainly limited to the membrane domain of BtuC, LPS contains glycans that reach out into the aqueous phase surrounding membranes and can interact with the soluble domain of BtuCD (Figure 3a). To validate whether the glycan units are important for binding, we isolated BtuCD under conditions comparable to those of an LPS-deficient E. coli strain. The LPS synthesis in this expression strain is halted at the level of lipid IVA—a LPS precursor that has no additional glycan units attached to its membrane-embedded lipid IVA core (Figure S9). BtuCD isolated from LPS-lacking E. coli could be quantitatively delipidated. This result implies that the glycan profile of LPS is important for binding (Figure S9).

Figure 3.

Figure 3

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LPS glycans modulate the binding and activity of BtuCD. (a) Protein structures obtained from MD simulations visualizing amino acid surface patches on the BtuCD dimer surface involved in binding to “core” LPS. (b) Bar chart showing the ATPase activity of BtuCD in the presence of different lipids. The molecular structure of KLA is shown, which is a truncated version of “core” LPS that has two additional glycan units (n = 2) attached to lipid A. The glycan profile of KLA (n = 2) and “core” LPS (2 < n ≤ 10) can interact with the soluble domain of BtuCD. ATPase activity increases with the number of glycan units attached to lipid A. Data were obtained from three independent biological repeats (n = 3) and are plotted with standard deviation (±SD).

Moreover, MD simulations suggested that the glycan profile of LPS interacts with the nucleotide binding pocket located at the soluble domain BtuD (Figure S10). To evaluate whether this interaction is relevant for activity, we monitored the ATPase activity of BtuCD in the presence and absence of different LPS forms. The ATPase activity increased with the number of glycan units (n) attached to the membrane-embedded lipid core of LPS (Figure 3b). Taken together, the glycan profile of LPS can be important for the binding and activity of inner membrane proteins. Since BtuCD is not involved in LPS translocation, our data suggest that LPS plays roles beyond the outer membrane structure and function.

Current protein purification protocols inevitably involve the mixing of inner and outer membrane components, which complicates our understanding and therefore assessment of critically important membrane lipids in the structural and functional regulation of inner membrane proteins in Gram-negative bacteria. Here we introduced a rationally designed detergent chemistry and demonstrated its utility by developing a delipidation method in combination with MD simulations and nMS. Our approach enables to qualitatively differentiate the degree to which membrane proteins copurify with PLs and/or LPS. Furthermore, our approach enables a gradual control of PL- and LPS-delipidation and can be used to decipher how the structure of lipids affects their interactions with membrane proteins. A limitation of our approach is that it cannot be used to clarify whether protein–lipid interactions obtained upon purification reflect true membrane environments. However, harnessing our improved methodological capabilities, we uncovered a previously underappreciated association of LPS, a predominantly thought of outer membrane component involved in biogenesis and structural integrity, with BtuCD, an inner membrane type II ABC importer, which suggests an importance of LPS and its precursors in modulating functional properties beyond outer membranes. Using MD simulations and ATPase activity assays, we illustrate that hydrophilic interactions with the glycan headgroups of LPS markedly affect binding to BtuCD and turnover rates. Our data spotlight a new membrane protein–lipid interaction that may be a suitable target for antibiotic discovery.14,20

Acknowledgments

The authors acknowledge R. Haag, K. Pagel, K. Goltsche, C. Fasting, V. Wycisk, A. Hardy, J. Gault, A. Dolan, V. L. Cullen, and I. Liko for continuous support.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c14358.

  • Data supporting the findings presented in this manuscript, including details on detergent design, protein delipidation, nMS analysis, MD simulations, ATPase activity measurements (PDF)

Author Contributions

# F.F. and D.S. contributed equally to this work. All authors have given approval to the final version of the manuscript.

The Ministry of Culture and Science of the German State of North Rhine-Westphalia (NRW return program) and the European Research Council (ERC Advanced Grant No. 695511, ENABLE) are gratefully acknowledged for financial support. This project made use of time on ARCHER and JADE granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://hecbiosim.ac.uk), supported by EPSRC (grant no. EP/R029407/1). The University of Warwick Scientific Computing Research Technology Platform is gratefully acknowledged for computational access. J.B.S. was supported by the Oxford interdisciplinary DTP and the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/M011224/1). J.R.B. acknowledges the support of the Royal Society through the University Research Fellowship grant (URF\R1\211567). F.F. acknowledges Sapienza University of Rome funding “Progetti per Avvio alla Ricerca – Tipo 2” (AR22218162B6F5D1) and “INCENTIVE MS” (GA12117 A8711CC9F).

The authors declare no competing financial interest.

Supplementary Material

ja3c14358_si_001.pdf (1.2MB, pdf)

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Supplementary Materials

ja3c14358_si_001.pdf (1.2MB, pdf)

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