Cellular Solid-State Nuclear Magnetic Resonance Spectroscopy

Cellular solid-state nuclear magnetic SEE COMMENTARY resonance spectroscopy

Marie Renaulta, Ria Tommassen-van Boxtelb, Martine P. Bosb, Jan Andries Postc, Jan Tommassenb,1, and Marc Baldusa,1

aBijvoet Center for Biomolecular Research, bDepartment of Molecular Microbiology, Institute of Biomembranes, and cDepartment of Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Edited by Robert Tycko, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board January 6, 2012 (received for review October 11, 2011)

Decrypting the structure, function, and molecular interactions of conditions (11) as a high-resolution method to investigate atomic complex molecular machines in their cellular context and at atomic structures of major cell-associated (macro)molecules. resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well- Results established imaging method that can visualize cellular entities at Sample Design for Cellular ssNMR Spectroscopy. Our goal was to the micrometer scale and can be used to obtain 3D atomic struc- establish general expression and purification procedures that lead 13 15 tures under in vitro conditions. Here, we introduce a solid-state to uniformly C, N-labeled preparations of whole cells (WC) NMR approach that provides atomic level insights into cell-asso- and cell envelopes (CE) containing an arbitrary (membrane) ciated molecular components. By combining dedicated protein pro- protein target (Fig. 1B). As our model system, we selected the duction and labeling schemes with tailored solid-state NMR pulse 150-residue integral membrane-protein PagL from Pseudomonas methods, we obtained structural information of a recombinant aeruginosa, an OM enzyme that removes a fatty acyl chain from integral membrane protein and the major endogenous molecular LPS (12). We overexpressed pagL under control of the bacter- components in a bacterial environment. Our approach permits iophage T7 promoter, which is inducible with IPTG, in a mutant studying entire cellular compartments as well as cell-associated E. coli BL21Star(DE3) strain, lacking the two major OM proteins proteins at the same time and at atomic resolution. (OMPs) OmpF and OmpA. The suppression of these major OMPs prevented to a large extent the accumulation of the unpro- cellular envelope ∣ Escherichia coli ∣ lipoprotein ∣ PagL ∣ magic angle cessed signal-peptide-bearing precursor of PagL and led to signif- spinning icant amounts of mature protein in the host membrane when mild recombinant-protein-expression conditions were used (Fig. S1). hysiological processes rely on the concerted action of mole- For optimal analysis of major cell-associated molecular compo- 15 13 Pcular entities in and across different cellular compartments. nents, E. coli cultures were switched from unlabeled to N, C- Whereas advancements in molecular imaging have provided isotope labeled growth conditions at the beginning of the expo- unprecedented insights into the macromolecular organization nential growth phase, when recombinant protein production was in the subnanometer range (1), studying atomic structure and mo- induced, leading to the incorporation of isotopes in PagL and co- tion in situ has been challenging for structural biology. NMR has expressed endogenous molecular components. WC and CE sam- provided insight into cellular processes (2–4) and can determine ples were prepared from the same exponentially growing culture. 13 15 entire 3D molecular structures inside living cells (5) provided that As a reference, (U- C, N)-labeled PagL was produced in intra- molecular entities tumble rapidly in a cellular setting. In princi- cellular inclusion bodies, purified, and reconstituted in proteoli- ple, solid-state NMR (ssNMR) spectroscopy offers a comple- posomes (PL, Fig. 1B). Before analysis, WC pellet was washed mentary spectroscopic tool to monitor molecular structure and with PBS, whereas CE and PL were resuspended in Hepes at dynamics at atomic resolution in a complex setting (see ref. 6 pH 7.0 and harvested by ultracentrifugation using identical pro- for a recent review). Indeed, ssNMR has already been used to cedures. Both in CE preparations and reconstituted in PL, PagL BIOPHYSICS AND

study individual molecular components in the context of natural exhibited similar heat modifiability, a property typical of the well- COMPUTATIONAL BIOLOGY bilayers (7, 8), bacterial cell walls (9), and cellular organelles (10). folded protein (12, 13) (Fig. 1C). Toverify that PagL was correctly Here, we introduce a general approach to investigate structure folded in vivo and in vitro, we monitored its LPS 3-O-deacylase and dynamics of an arbitrary molecular target and its potential activity in CE and PL preparations as described previously (12, molecular partners in a cellular setting. Our studies focuses on 13). In both cases, LPS was converted into a form with higher the Gram-negative bacterial cell that is characterized by a mole- electrophoretic mobility (Fig. 1D), in agreement with the ex- cularly complex but architecturally unique envelope, consisting of pected hydrolysis of the primary acyl chain at position 3 of lipid two lipid bilayers, the inner and outer membrane (IM, OM), se- A. Taken together, our data (heat modifiability and activity as- parated by the periplasm containing the peptidoglycan (PG) layer says) indicate that cellular and PL preparations contained well- (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical folded and functional PagL. proteins, whereas the OM is asymmetrical and consists of phos- pholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrel- NMR Spectra of E. coli Whole Cells and Cell Envelopes Versus Proteo- fold integral membrane proteins. LPS forms the outermost layer liposomes. To characterize rigid—presumably membrane-asso- of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell Author contributions: M.R., J.T., and M.B. designed research; M.R., R.T.-v.B. and M.P.B. its shape and rigidity. performed research; J.-A.P. contributed new reagents/analytic tools; M.R., J.T., and M.B. 13 15 Using uniformly C, N-labeled cellular preparations of analyzed data; and M.R., J.T., and M.B. wrote the paper. Escherichia coli, we characterized the structure and dynamics The authors declare no conflict of interest. of a recombinant integral membrane protein (PagL) and other This article is a PNAS Direct Submission. R.T. is a guest editor invited by the Editorial Board. major endogenous molecular components of the cell envelope See Commentary on page 4715. including lipids, the peptidoglycan, and the lipoprotein Lpp (also 1To whom correspondence may be addressed. E-mail: [email protected] or known as Braun’s lipoprotein). These studies highlight the [email protected]. influence of the surrounding compartment on molecular struc- This article contains supporting information online at www.pnas.org/lookup/suppl/ ture and establish ssNMR under magic angle spinning (MAS) doi:10.1073/pnas.1116478109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1116478109 PNAS ∣ March 27, 2012 ∣ vol. 109 ∣ no. 13 ∣ 4863–4868 Downloaded by guest on September 30, 2021 ciated—molecular components in WC and CE, we performed a set of 2D 13C-13C correlation experiments employing dipolar- based magnetization transfer steps. Overall, both preparations yielded NMR spectra of astonishing quality considering sample complexity and noncrystallinity (Fig. 2 A and B), with well-dis- persed cross-peaks characteristic for protein and lipid signals. As anticipated, we observed an improvement in both sensitivity and spectral resolution for the CE preparation (Fig. S2A), potentially due to the single contribution of CE-associated components. These results were corroborated by SDS/PAGE analysis (Fig. 1C), which showed a significant decrease of the amount of proteins after removal of the protoplasm by cell lysis and ultracentrifugation. Over time, WC and CE preparations did not reveal any marked spectroscopic changes at −2 °C, and the cell morphology and the structural integrity of the CE were preserved after extended NMR studies (Fig. S3). When comparing WC and CE spectra with the reference PL spectrum recorded under similar measurement conditions (Fig. 2C), we found that a large set of intraresidue correlations from PagL, notably cross-peaks of Ala, Thr, and Ser residues in β-sheet protein segments (see below), are well preserved in WC, CE, and PL spectra.

Conformational Analysis of the PagL Protein in the E. coli Cell Envel- ope. To examine in further detail the conformation of PagL in CE, we performed 2D 15N-13C correlation experiments (14) in which signals arising from nonproteinaceous molecular components are largely reduced. Comparison with the reference PL spectrum (Fig. 3A, red) revealed astonishing similarities. With average 13C and 15N line widths of 0.6–0.8 and 1.5–1.6 ppm, respectively, Fig. 1. Cellular ssNMR spectroscopy: overall strategy and sample prepara- ssNMR spectra of the CE preparation exhibited comparable, if tion, including inner and outer membrane proteins (IMP, OMP). (A) Schematic not superior spectral resolution (Fig. S2B). Because of the favor- structure of the E. coli K-12 cell envelope. (B) Overall scheme for the prepara- able spectroscopic dispersion among Thr, Ser, and Gly residues in tion of WC and CE from strain CE1535 carrying plasmid pPagLð Þ, and of pur- Pa standard 2D CC/NC correlation experiments, we could subse- ified PagL protein from strain BL21Star(DE3) carrying plasmid pPagLðPaÞ(-) reconstituted in PL. (C) Coomassie-stained SDS/PAGE analysis of WC, CE, quently perform a residue-specific analysis, which consisted of and protoplasm (P) fractions obtained from exponentially growing E. coli three stages. First we determined sequential resonance assign- CE1535 cells containing plasmid pPagLðPaÞ and comparison with the reference ments in PagL-PL preparations in spectroscopically favorable PL sample. Molecular-mass markers (MM) are indicated next to the gels. Sam- regions. Second, comparison to the same spectral regions in da- ples were denatured by boiling in SDS (d) or left on ice (n) before electrophor- tasets obtained for CE served to obtain tentative assignments for esis. F and U denote the positions of folded and heat-denatured forms of the same PagL residues in the CE case. These were finally cross- PagL, respectively. (D) In vitro and in vivo LPS deacylase activity of PagL. validated using additional 2D and 3D datasets. With this strategy, (Upper) Purified Neisseria meningitidis LPS was incubated in a detergent-con- 15 13 taining buffer with (lane 3) or without (lane 1) PagL-containing PL and ana- we obtained N and C resonance assignments for 13 PagL lyzed by Tricine SDS/PAGE and staining with silver. Membranes from N. residues located in different topological regions of the protein meningitidis harboring functional PagL were coanalyzed for reference (lane using CE and PL preparations (Fig. S4 and Table S1). An analysis 13 2). (Lower) Silver-stained Tricine SDS/PAGE analysis of CE isolated from plas- based on C secondary chemical shifts of PagL in CE and in PL midless CE1535 cells (lane 1) or noninduced (lane 2) and IPTG-induced (lane 3) was consistent with the presence of Ser33, Thr34, and Thr91 in CE1535 cells carrying the PagL-encoding plasmid. unstructured protein regions and Ala9, Thr10, Thr16, Thr32,

Fig. 2. NMR spectra of whole cells, cell envelopes, and proteoliposomes. 13C-13C correlation spectra of fully hydrated IPTG-induced WC (A), CE isolated from IPTG-induced WC (B), and (U-13C, 15N)-labeled PagL-containing PL (C) recorded using respectively 224, 336, and 192 scans and processed identically using a sine bell function (SSB of 3.5) and linear prediction in the indirect dimension. Contour plots were adjusted to the same noise level. Characteristic cross-peaks of Ala, Ser, and Thr residues located within PagL β-sheet protein segments and of endogenous E. coli lipids (Lip) are indicated in red and green, respectively.

4864 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1116478109 Renault et al. Downloaded by guest on September 30, 2021 Arg36-Thr38, and Thr46 situated in β-strands (Fig. S5A). These and 0.4 ppm, respectively (Fig. 3C, arrows). In addition, signifi-

correlations as well as the comparison to the CC correlation pat- cant side-chain chemical-shift changes were observed for residues SEE COMMENTARY tern predicted on the basis of the X-ray structure suggested that within transmembrane segments (Ala9, Thr10, Leu37), the first the backbone fold of PagL seen in crystals is largely conserved in periplasmic turn (Ser33), and the third extracellular loop the cellular envelope as well as in proteoliposomes (Fig. S5B). In (Thr91). For several assigned correlations, we also observed a 15 13 addition to using N and C chemical shifts, we investigated sizable attenuation in NMR signal intensity (ranging between which protein regions were sensitive to the cellular environment 30% and 50%), notably for backbone resonances involving Ala9, by analyzing cross-peak amplitudes as spectral parameters Thr91, and Thr32 (Fig. S6A). Even in the absence of residue- (Fig. 3B). Detailed ssNMR signal sets extracted from 2D correla- specific assignments, we could analyze other amino acid types, tion spectra of CE isolated from induced cells (black) or nonin- including Ser, Ala, Gly, Val, Lys, Tyr, and Trp, based on their 15 13 duced cells (green) and PL (red) at selected PagL N and C characteristic intraresidue correlation pattern and peak positions. resonance frequencies are shown in Fig. 3B, Left. The bar dia- Besides subtle backbone chemical-shift changes, we found signif- gram (Fig. 3B, Right) displays chemical-shift differences in back- icant alterations in side-chain resonances of Val, Lys, and Tyr bone 15N,13Cα and side-chainCβ resonances between CE and PL preparations. Overall, most differences in the backbone chemical shifts were small. Only for Ala9, Thr16, Thr32, and Arg36 we observed 15Nor13Cα chemical-shift deviations of around 0.6 BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Fig. 3. Conformational analysis of PagL. (A) Overlay of 2D NCA correlation spectra of CE isolated from IPTG-induced WC (black) and PL (red), recorded Fig. 4. Identification and characterization of the lipoprotein Lpp. (A) Two- with identical acquisition and processing parameters—i.e., with a sine bell dimensional (13C-13C) and (B) 2D NCA correlation spectra obtained on the CE function (SSB ¼ 4.5) and using linear prediction in indirect dimension. (B) isolated from noninduced WCs overlaid with backbone Cα-Cβ and N-Cα (red Comparison of PagL in CE and PL environments. (Left) Selected spectral re- crosses) intraresidue correlations predicted from the crystal structure of Lpp gions from 2D homonuclear and heteronuclear spectra of CE isolated from (Protein Data Bank ID code 1EQ7) using SPARTA+ (33). For other carbon posi- IPTG-induced WC (black) showing isolated PagL resonances, and overlaid tions (black crosses), average 13C chemical-shift values given in the Biological with spectra obtained on CE isolated from noninduced WC (green) and Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/ref_info/statsel PL (red). Assignments are indicated where available. (Right) Backbone N, .htm) were used. Characteristic correlations are labeled and color coded: or- α β C , and C chemical-shift changes observed for PagL embedded in E. coli ange for correlations absent in the experimental data and green for correla- CE and in PL. Horizontal lines indicate the threshold for significant chemi- tions that are in agreement with chemical-shift predictions. (Inset, Upper Δðδ − δ Þ¼ pcal-shiftffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi changes. The threshold was set to 2 times CE PL Right) Spectral region characteristic of Gly residues. (Inset, Lower Left)Anover- ½Δðδ Þ2 þ½Δðδ Þ2 α CE PL . Residues with a chemical-shift deviation larger than lay of 2D NCA spectra for α-helical Thr, Val, and Ile N-C correlations in CE iso- the threshold (+ 2 SD) are labeled. (C) Summary of PagL ssNMR spectral lated from non- (black) and IPTG-induced (blue) cells. (C) Summary of the changes between CE and PL preparations plotted onto the topological repre- structural analysis of Lpp based on ssNMR spectra obtained on CE isolated from sentation of PagL according to the crystal structure (β-sheet protein segments noninduced cells. Residues for which backbone correlations significantly devi- are represented by open rectangles and transmembrane segments TM1–8 ate or not from predictions are labeled and highlighted in orange or green, are labeled). Arrows point to residues that experienced significant backbone respectively. (D) Two-dimensional NCA correlation spectrum including side- (solid lines) and side-chain (dashed lines) chemical-shift changes, whereas chain amide 15Nζ resonances of Lys revealing distinct and characteristic NC cor- orange filled bars indicate major alterations in signal intensities (>50%) be- relation patterns for the free (Upper) and the bound (Lower) forms of Lpp. tween CE and PL. Meso-diaminopimelic acid, m-DAP.

Renault et al. PNAS ∣ March 27, 2012 ∣ vol. 109 ∣ no. 13 ∣ 4865 Downloaded by guest on September 30, 2021 residues, whereas Thr residues are not affected (Fig. 3 B, Left and tral resolution are well preserved in the presence of recombinant C, orange bars; see also Fig. S6B). Taken together, these findings protein PagL in the OM (Fig. 4B, Lower Left Inset, blue spec- suggest an overall conservation of the backbone structure of trum). In contrast, we observed a systematic deviation between PagL in the cellular envelope. Larger changes observed for some our data and predicted (Fig. 4 A and B, orange) 13C and 15N re- backbone residues and, in particular, side-chain conformations sonances for N- and C-terminal residues (Ser3, Asn4, Met52, (Fig. 3C) may reflect structural and/or dynamical alterations that Thr54), which exceeded the standard deviation. We thus specu- are potentially related to the presence of endogenous membrane- late that potential conformational and/or dynamical changes oc- associated molecular components in the CE environment. cur between crystalline Lpp and native Lpp around these residues. In vivo, Lpp has characteristic covalent modifications Characterization of the Endogenous E. coli Lipoprotein Lpp. With up at its N and C termini (19). About one-third of Lpp molecules to about 7.2 × 105 copies per cell, the lipoprotein Lpp, or Braun’s is covalently bound to the PG layer. Such a modification involves lipoprotein (15), belongs to the most abundant CE proteins in the formation of a peptide bond between the free ϵ-amino group exponentially growing E. coli cells. Lpp is found in both “free” of the C-terminal Lys of Lpp and the free carboxylate group of and “bound” forms, the latter being covalently attached to the meso-diaminopimelic acid residues in PG (Fig. 4C). We thus re- PG network (16, 17). In solution, the 56-residue polypeptide moi- corded a 2D (15N- 13Ca) correlation spectrum using a larger spec- ety, called Lpp-56, associates to form a hydrophilic homotrimer tral window that includes the side-chain region (Fig. 4D). We composed of a three-stranded coiled-coil domain and two helix- identified two intense cross-peaks in the amide region at the ex- capping motifs (18), but a model for a lipophilic superhelical as- pected Cϵ carbon frequency of free Lys. These signals were cor- sembly containing six subunits has also been proposed (16). We related to a set of additional signals at 32, 27, and 29.5 ppm, in first monitored the presence of free Lpp by SDS/PAGE analysis good agreement with averaged 13C chemical shifts of Lys Cβ,Cγ, of CE preparations followed by immunoblotting (Fig. S7A). We and Cδ side-chain resonances and strongly suggesting that a next performed a series of 2D 15N-13C and 13C-13C correlation bound Lpp contribution is detected in the CE spectrum. experiments on CE isolated from noninduced WC and compared results with predictions based on the available high-resolution 3D Resonance Assignment of Mobile LPS and PG. We could readily char- structure of Lpp-56. We found good agreement between our data acterize highly flexible PG, located in the periplasm, and LPS in and predicted intraresidue backbone C–C (Fig. 4A) as well as the OM in 2D (1H,13C) as well as (13C-13C) through-bond corre- N–Cα (Fig. 4B) correlation patterns, suggesting the predomi- lation spectra obtained on CE preparations. LPS consists of a nance of well-folded Lpp in the CE preparations. These results 1,4′-bisphosphorylated β-1,6-linked glucosamine disaccharide were further supported by weak signal intensities in spectral re- (α- and β-GlcN), substituted with fatty acid chains at positions gions characteristic for glycine (Fig. 4B, Upper Right Inset), which 2, 3, 2′, and 3′ and with an oligosaccharide core at position 6′. is missing in the amino acid composition of Lpp. In addition, iso- PG is a giant heteropolymer made of linear glycan strands of lated backbone and side-chain resonances corresponding to Ala, alternating β-1,4-linked N-acetylglucosamine (GlcNAc) and N- Val, and Ile residues were readily observed around peak positions acetylmuramic acid (NAM) residues, which are cross-linked by predicted from the crystal structure (18) (Fig. 4 A and B, red short peptides. First, we recorded a 2D (1H-13C) insensitive crosses). In the X-ray structure, these residues pack against each nuclei enhanced by polarization transfer (INEPT) spectrum to other to form the hydrophobic interface between the three he- examine the sugar heterogeneity/composition in our sample. lices (Fig. 4C, green). Interestingly, both peak positions and spec- Intense and very well-resolved cross-peaks were found at peak

Fig. 5. Resonance assignment of flexible LPS and PG using through-bond ssNMR correlation spectroscopy. (A) Expansion of the 2D (1H-13C) insensitive nuclei enhanced by polarization transfer (INEPT) correlation spectrum obtained on CE isolated from IPTG-induced WC, showing dispersion and resolution of individual α- and β-anomeric resonances of LPS and PG sugar moieties. Splitting due to the C1-C2 scalar coupling is visible for all anomeric resonances and varied between 47 and 53 Hz. Color coding: β-GlcN, dark blue; α-GlcN, light blue; PG GlcNAc, orange; NAM, red; LPS core, black. (B) Sections from the 2D (13C-13C) INEPT-total through-bond correlation spectroscopy showing characteristic correlations between anomeric C1 and one-bond nitrogen-substituted C2 carbons (Upper) or one-to-three bonded non nitrogen-substituted C2-C4 carbons (Lower) of LPS and PG sugar moieties. The CC and HC correlations that belong to the same spin system are connected by dashed lines. (C) Residue-specific ssNMR assignment of LPS and PG glucosamine units based on 13C-13C connectivities within the sugar rings and their substituents (see also Fig. S7A). Assigned resonances from LPS and PG glucosamine units are highlighted by filled circles onto chemical structures with the same color coding as in A.

4866 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1116478109 Renault et al. Downloaded by guest on September 30, 2021 positions corresponding to anomeric H1/C1 atoms from carbohy- 13

drate units (Fig. 5A). The C dispersion between 90 and 104 ppm SEE COMMENTARY was consistent with the presence of nine carbohydrate species, in α- and β-configuration. Glucosamine units, constituting exclu- sively the PG backbone (GlcNAc and NAM) and the lipid A moiety of the LPS (β-D-GlcN and α-D-GlcN) can be distinguished from sugars of the LPS core (labeled A–E) based on a characteristic one-bond CC connectivity between anomeric carbon and nitrogen-substituted C2 in a 2D (13C,13C) correlation spectrum (Fig. 5B). Next, we identified carbohydrate units by tracing CC and HC connectivities within the sugar rings and their substituents (20, 21) (Fig. 5C and Fig. S7B). Using this strategy, we obtained de novo ssNMR assignments of LPS and PG glucosamine units and some of their substituents (Table S1). Solid-state NMR chemical shifts were compatible with the presence of polysubsti- tuted glucosamine units within the lipid A of LPS and backbone PG moieties, as previously deduced from the analysis of purified molecules (21–23), even when part of cell envelope preparations. However, carbohydrates from the core of the LPS could not be identified unambiguously due to the large overlap of 13C and 1H resonances and the inherent structural heterogeneity of the LPS core region (Fig. S7C). Discussion Our results demonstrate that cellular ssNMR spectroscopy can be used to probe atomic details of integral membrane proteins and endogenous membrane-associated molecular components in a bacterial cellular setting. We showed that 15N-edited datasets as well as the combined use of through-space and through-bond ssNMR experiments reduces spectral complexity and facilitates the discrimination between proteinaceous and nonproteinaceous molecular components at different levels of molecular mobility. By analyzing (U-13C,15N)-labeled E. coli cell envelopes, we found that LPS and PG moieties can exhibit a remarkable degree Fig. 6. Atomic-level insights of E. coli CE-associated macromolecules revealed of molecular motion (Fig. 6, red), while the protein components by cellular ssNMR spectroscopy. Schematic representation of the CE from investigated were well folded in the cellular membrane context E. coli showing rigid (blue) and flexible (red) (non)proteinaceous molecular (Fig. 6, blue). In the case of Lpp, our results strongly support components characterized by through-bond and through-space ssNMR experi- the anchoring of the protein to the underlying PG layer by virtue ments, respectively. The topological representations of PagL and Lpp as seen in the available 3D models of isolated molecules are indicated. For PagL, residues of the side chain of the C-terminal lysine. Although our data are located in β-strand and random-coil protein segments are represented by consistent with the overall 3D model for the isolated Lpp protein squares and circles, respectively. Residues that were not included in the analysis moiety, substantial conformational changes are predicted for re- are colored in gray. Amino acids are given in single-letter notation. Spectro- sidues located at the N- and C-terminal extremities of the protein, scopic changes of large (chemical-shift deviation >0.4 ppm and/or signal inten- which are covalently substituted in vivo. Further cellular ssNMR sity variations >50%) and small magnitude that are potentially related to studies may reveal the conformation of the free form of Lpp that association between membrane protein and OM (or PG) are indicated for both BIOPHYSICS AND

has been postulated to cross the OM (17). In the case of PagL, for proteins in orange and blue, respectively. COMPUTATIONAL BIOLOGY which high-resolution ssNMR spectra were obtained for all studied preparations, we demonstrated that the global fold of the WC preparations can be readily combined with state-of-the-art protein as seen in the crystal structure was largely preserved in ssNMR signal enhancement methods operating at low tempera- both PL and CE environments, including the presence of β-sheet ture, thereby reducing acquisition times significantly (25). Such structure in the first periplasmic turn, which involved Thr32. On technologies are likely to reduce the expression levels needed the other hand, several protein backbone resonances related to to perform cellular ssNMR experiments. In the present study, residues located in the first periplasmic turn, the transmembrane the expression levels of PagL were comparable to those of the segments 1–3, and in the third extracellular loop were sensitive to endogenous protein Lpp (Fig. S2B). Dedicated preparation the molecular environment. Alterations in ssNMR signal inten- methods (see, e.g., ref. 6) including the single-protein production sity were detected predominantly for aromatic, hydrophobic, and method (26) are available to further reduce unwanted back- charged residues. Interestingly, these PagL residues are located in ground contributions. Cellular ssNMR spectroscopy as described protein segments potentially exposed to other major membrane- herein opens opportunities for the structural investigation of associated cellular components, including LPS and the PG (Fig. 6, large and/or membrane-associated macromolecules. Already, the orange). Similar to the case of Lpp, further ssNMR studies will cell envelope of Gram-negative bacteria, including the IM, the help to establish the structural details of the molecular interac- periplasm, and the OM, epitomizes a model organelle involved tion of PagL and its substrate LPS, to dissect active and latent in a large number of functions critical for cellular physiology. PagL conformations that depend on its molecular environment With recent advances in using insect and mammalian cells for (24), and to establish the physiological relevance of PagL dimers producing recombinant eukaryotic proteins, cellular ssNMR as (13) in a native-like environment. shown here could also be applicable to recombinant eukaryotic With increasing levels of molecular complexity, spectroscopic proteins in specific compartments—i.e., after isolation of the cel- sensitivity becomes a critical factor. Nevertheless, one- and two- lular membrane or intracellular organelles. Using cellular ssNMR, dimensional ssNMR spectroscopy of whole cell preparations the structural investigation of fundamental processes, such as such as shown in this study is possible (Fig. 2A). In addition, ligand/drug binding or membrane-protein folding mediated by

Renault et al. PNAS ∣ March 27, 2012 ∣ vol. 109 ∣ no. 13 ∣ 4867 Downloaded by guest on September 30, 2021 complex proteinaceous machineries, should be possible, thereby centrifugation (90;000 × g for 45 min at 4 °C). Freshly prepared and fully bridging the gap between structural and cellular biology. hydrated WC, CE, and PL samples were transferred into 3.2-mm MAS rotors by low-speed centrifugation, packed with bottom and top spacers, and sub- Materials and Methods sequently analyzed by ssNMR spectroscopy. In the PagL-PL case, we estimate Expression Vectors and Bacterial Strains. The pET11a-derived plasmids 8 mg of protein present in the NMR rotor.

pPagLðPaÞ and pPagLðPaÞ(-) encoding P. aeruginosa PagL with and without signal sequence, respectively, have been described (12). Mutant derivatives of E. NMR Spectroscopy. The ssNMR analysis of cellular and proteoliposome pre- coli BL21Star(DE3) lacking the OmpF and/or OmpA proteins were isolated by parations was based on a set of multidimensional homonuclear and hetero- selection for resistance to the OmpF- and OmpA-specific bacteriophages TuIa nuclear ssNMR experiments employing dipolar- or scalar-based magneti- (27) and K3 (28), respectively. Mutants lacking OmpF, OmpA, or both were zation transfer steps as detailed in SI Materials and Methods. Solid-state designated CE1536, CE1537, and CE1535, respectively. NMR experiments were performed at a regulated sample temperature of −2 °C on a narrow-bore Bruker Avance 700 spectrometer equipped with a Sample Preparation. WC and CE NMR samples were prepared from 50 and 3.2-mm triple-resonance (1H,13C,15N) Efree MAS probe. 13C and 1H resonances 200 mL of the cultures, respectively, following the procedure described in were calibrated using adamantane as an external reference. The upfield 13C SI Materials and Methods. For CE NMR samples, cells were disrupted in pre- resonance and isotropic 1H resonance of adamantane were set to 31.47 and sence of EDTA-free protease inhibitor cocktail (Sigma-Aldrich) using a pre- 1.7 ppm, respectively, to allow for a direct comparison of the solid-state che- cooled French pressure cell (8,000 psi). Unbroken cells were removed by 15 multiple centrifugation steps (1;000 × g, 10 min, 4 °C) until a pellet was mical shifts to solution-state NMR data. Accordingly, N resonances were ca- no longer detectable. The CE fraction was isolated by ultracentrifugation librated using the tripeptide AGG (31) as an external reference. A summary of of the cell lysate for 8 min at 150;000 × g and at 4 °C. The correct localization acquisition and process parameters is given in Table S2. NMR spectra were and membrane insertion of PagL was confirmed on Coomassie-stained SDS/ processed using Topspin 3.0 (Bruker Biospin) and analyzed with Sparky (32). PAGE gels after extracting the CE with 1% N-lauroylsarcosine or 6 M urea as described previously (29, 30). To obtain the reference PL ssNMR spectra, PagL ACKNOWLEDGMENTS. Technical assistance by Deepak Nand, Christian Ader, was expressed as intracellular inclusion bodies in E. coli BL21Star(DE3) harbor- and Hans Meeldijk is gratefully acknowledged. This work was supported ing the plasmid pPagLðPaÞ(-), then purified, and reconstituted in dimyristoyl- by Nederlandse organisatie voor wetenschappelijk onderzoek (700.26.121, phosphatidylcholine vesicles at a final protein-to-lipid molar ratio of 1∶50 as 700.10.433, and 815.02.012) and received funding from the European Com- described in SI Materials and Methods. Before packing, the CE and PL pellets munity’s Seventh Framework Program (FP7/2007-2013) under Grant Agree- were resuspended in 500 μL of 10 mM Hepes (pH 7.0) and harvested by ultra- ment 211800.

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