Lipid Bilayer Strengthens the Cooperative Network of a Membrane-Integral Enzyme

Lipid bilayer provides a two-dimensional hydrophobic solvent milieu for membrane proteins in cells. Although the native bilayer is widely recognized as an optimal environment for folding and function of membrane proteins, the underlying physical basis remains elusive. Here, employing the intramembrane protease GlpG of Escherichia coli as a model, we elucidate how the bilayer stabilizes a membrane protein and engages the protein’s residue interaction network compared to the nonnative hydrophobic medium, micelles. We find that the bilayer enhances GlpG stability by promoting residue burial in the protein interior compared to micelles. Strikingly, while the cooperative residue interactions cluster into multiple distinct regions in micelles, the whole packed regions of the protein act as a single cooperative unit in the bilayer. Molecular dynamics (MD) simulation indicates that lipids less efficiently solvate GlpG than detergents. Thus, the bilayerinduced enhancement of stability and cooperativity likely stems from the dominant intraprotein interactions outcompeting the weak lipid solvation. Our findings reveal a foundational mechanism in the folding, function, and quality control of membrane proteins. The enhanced cooperativity benefits function facilitating propagation of local structural perturbation across the membrane. However, the same phenomenon can render the proteins’ conformational integrity vulnerable to missense mutations causing conformational diseases1,2.

The mutation-induced change in thermodynamic stability (DDG o WT-Mut ) and the activity relative to wild type in DDM micelles. The fraction of residue buried area (f ASA ) and cooperativity profile of each residue. "N" and "C" in the "Location" column denote N-and C-subdomains, respectively. In the "Cooperativity profile" column, "Local/N", "Local/C", "Moderate/N", "Moderate/C", and "Moderate/Over" denote localized in N and C subdomains, moderately localized in N and C subdomains, and moderately overpropagated, respectively. Table 3. Fitted parameters of the time-dependent contact autocorrelation data to a triple exponential decay function for the whole (a), headgroup (b), and tail (c) regions of the lipid (Lip) or detergent (Det) molecules on GlpG (Prot) and on themselves. A: % amplitude; t R : residence time; <t R >: the amplitude-weighted average residence time; Adj-R 2 : the adjusted Rsquare; DG o SolvEx : the solvation free energy of an amphiphile molecule on the protein. t R,1/e denotes the resident time at which the contact autocorrelation decays to 1/e of the initial value.     The K d,biotin of the next higher affinity variant mSA DAB -S45A was determined using mSA-W79M with known K d,biotin obtained from the preceding plot. Using the same strategy, K d,biotin 's of mSA DAB -S27A and mSA DAB -E51S were determined consecutively.

Extended Data
[mSA] (µM)    (a) Here, we compared (Top) the total incubation time of the steric trapping reaction (~72 h) to (Bottom left) the time scales of the dissociation (t off,2 ) time constants of the second mSA molecule from sterically denatured GlpG. Here, we evaluated the dissociation time constant of the mSA molecule (t off,2 ) that induces the refolding of sterically denatured GlpG. The on-rates of mSA molecules (k on = ~10 6 to 10 7 s -1 M -1 ) to transiently denatured GlpG are expected to be larger than the off-rates of the mSA-biotin complex (Srisa-Art et al. Anal Chem 2008 80, 7063-7). If the dissociation (t off,2 ) occurs in the faster time scales than the total incubation time, the observed binding isotherms for the second mSA binding (bottom right, black circles) can be regarded truly equilibrated, and thus DG o N-D of GlpG can be determined from the attenuated second mSA binding. Detailed description of each plot is shown below.
(Top) The equilibrium reaction scheme of steric trapping with the time constants of each reaction step. (Bottom left) The schematic description of measuring the time constant of a first dissociation of mSA (t off,2 ) from sterically denatured GlpG. The double-biotin variant of GlpG, 172 M 267 C -BtnPyr 2 (1 µM), is first denatured at an excess concentration of mSA DAB variants. In this initial state, denatured GlpG is strained by the steric hindrance between doubly-bound mSA DAB molecules, and pyrene fluorescence from the BtnPyr labels on GlpG are quenched by the dabcyl quencher on mSA DAB . The addition of excess free biotin (2 mM) induces the dissociation of one bound mSA DAB molecule to relieve the strain, and the pyrene fluorescence increases by dequenching. (Bottom right) An example of the binding isotherm between mSA and GlpG (denaturation, black circles) and fluorescence dequenching upon addition of free biotin (refolding, orange circles).

(b)
The assay result. Upon addition of excess free biotin, the dissociation kinetics of mSA DAB variants bound to 172 M 267 C -BtnPyr 2 were measured at an increasing concentration of mSA DAB . The data was fitted to a single exponential function under the assumption of the pseudo-first order reaction.
(c) The pseudo-first order dissociation time constants (t off,2-apparent ) of mSA DAB variants are independent of the concentration of mSA DAB . The slower time constants of mSA DAB are in the range of 2 h to 4 h, shorter than the total incubation time (~72 h). Thus, DG o N-D of GlpG can be determined from the "equilibrated" attenuated second mSA binding ( Fig. 1e and Extended Data Fig. 9).      Fig. 1c).
(a) The strategy. Native or sterically denatured GlpG in micelles is transferred to bicelles at an increasing concentration of mSA DAB -E51S labeled with dabcyl quencher. After incubation for 72 h, the activity of GlpG was measured as a folding indicator at each [mSA DAB -E51S].

(b)
The assay results for 1 µM GlpG in 3% (w/v) DMPC/CHAPS bicelles (q = 1.5) at room temperature. Quenching of NBD fluorescence was monitored upon cleavage of the model substrate LYTM2 labeled with NBD (Extended Data Fig. 3a). As the [mSA DAB -E51S] increased (black block arrows), the activity decreased (i.e., the decrease in the initial slope), indicating that the fraction of denatured GlpG increased. For each double-biotin variant of GlpG, the degree of inactivation was similar at a given [mSA DAB -E51S] regardless of the initial state in micelles before transfer to bicelles.  (a) Schematic description of a fluorescence quenching assay to measure the transfer of native (N: the folded double-biotin variants, GlpG-BtnPyr 2 ) and sterically denatured GlpG (D×2mSA) from the micellar to the bicellar phase by direct injection. The preformed bicelles contain dabcyl (quencher)labeled lipid (the molar ratio, DMPC:dabcyl-DOPE = 199:1). Pyrene-labeled mSA (mSA-Y83C-Pyr), which is soluble in water, was used as a negative control (i.e., no incorporation). Native GlpG-BtnPyr 2 , which was first reconstituted in DMPC liposomes and then solubilized by CHAPS to form bicelles, was used as a positive control (i.e., full incorporation).  Data Fig. 1), activity loss induced by steric trapping and digestion by ProK (Extended Data Figs. 3 and 4b). The incomplete digestion in the presence of excess mSA is due to the incomplete double-biotin labeling of GlpG. The free energy level (in k B T) and the degree of compactness (the end-to-end distance between the Nand C-termini) of each state are shown relative to the native state ("N"). The conformation of each state is also shown regarding whether the tertiary interaction is maintained ("compact" vs "3 o structure disrupted"), the secondary structure is maintained ("helical" vs "unfolded"), or each structural element is buried in the membrane ("transbilayer" vs "exposed to water"). "D": denatured state; "I": intermediate state; "U": unfolded state.

(c) Correlation between the efficiency of double biotinylation (Extended
The stability of GlpG that we determined directly under native condition (DG o N-D = -12k B T, "This study") in bicelles is much larger than that from the single-molecule magnetic tweezer study in the same neutral bicelles "Min et al."). 54 In the latter, DG o N-D was obtained by extrapolating the unfolding and refolding rates measured in the two distinct force ranges (12-30 pN and 2-7 pN, respectively) to zero force. At the higher force, GlpG unfolds via a single cooperative step or multiple steps with one or two intermediates to the fully stretched coil. 54 At the lower force, the conformation of the starting unfolded state prior to refolding is not defined ("?"). A simulation study ("Lu et al. AWSEM") predicts that the unfolded state at low force is I 1 (TM1-TM4 folded). 55 Notably, our DG o N-D is similar to the free energy difference between the native state and I 2 (TM1-TM2 folded) from the same simulation (-10k B T) 55 as well as that between the native state and I 1 (TM1-TM2 folded) from the more recent tweezer study in the negatively charged bicelles (-13k B T) 56 at low force ("Choi et al."). We note that the end-to-end distances for "N" and "D" states in "This study" were taken from our previous work (Gaffney et al.) measured in the negatively charged bicelles (DMPC/DMPG/CHAPS) using DEER.
Previously, we have shown that while both N-(TM1-TM3) and C-(TM4-TM6) subdomains in sterically denatured GlpG expand relative to those in the native state in bicelles, N-subdomain denatures close to the collapse limit with C-subdomain close to the full-expansion limit, resembling the conformation expected from I 2 by "Lu et al." and from I 1 by "Choi et al." 42 Thus, we reason that the stability discrepancy likely stem from the different conformation of the denatured state in the steric trapping ("This study") and magnetic tweezer ("Min et al.") studies.

Transbilayer
Transbilayer ↔ water exposed  TM1TM2TM3   TM4TM5TM6   TM1TM2TM3TM4  TM5TM6   TM1TM2   TM3TM4TM5TM6   TM1TM2TM3   TM4TM5TM6   TM1TM2TM3TM4   TM5TM6   TM1TM2   TM3TM4TM5TM6   TM1TM2TM3   TM4TM5TM6   TM1TM2TM3TM4  TM5TM6   TM1TM2   TM3TM4TM5TM6   TM1TM2TM3   TM4TM5TM6   TM1TM2TM3 Fig. 11│ Binding isotherms between the double-biotin variant of GlpG (95 N 172 M -BtnPyr 2 or 172 M 267 C -BtnPyr 2 ) and monovalent streptavidin (mSA) to determine the thermodynamic stability of GlpG in DMPC/CHAPS bicelles using steric trapping. Binding was measured by quenching of pyrene fluorescence from the BtnPyr labels on GlpG, which was induced by the dabcyl quencher conjugated to mSA (mSA DAB ). The first mSA binds either biotin label with an intrinsic binding affinity (black dashed lines). Binding of the second mSA is attenuated depending on the stability of GlpG (DG o N-D ), which was obtained by fitting the attenuated second binding phase to Methods Eq.'s 1-2. In each plot, the fluorescence intensity was normalized to the intensity change of the second binding phase and the WT data is shown.  The residues that participate in the packing of N-(blue, top), C-(orange, bottom) subdomain, and subdomain interface (green, middle) in the structure of GlpG (PDB code: 3B45). The interior of Nsubdomain and the subdomain interface are mainly formed by the extensive knob-into-hole type vdW packing of the large (Thr, Val, Leu, Met, Phe and Trp) and small (Gly, Ala and Ser) nonpolar residues. C-subdomain is primarily mediated by the face-to-face backbone contact between TM4 and TM6 through the Gly-zipper motifs (GlyxxxGlyxxxGly: x is any residue and Gly can be replaced by Ala or Ser). The catalytic dyad Ser201-His254 (black, bottom) is mounted on the helix-helix interface formed by TM4 and TM6.

Bicelles
Extended Data Fig. 14│ The features of cooperativity profiles in lipid bilayers still preserve those in micelles, but to a less extent.
(c) Cooperativity profiles in bilayers on the basis of smaller cut-off values, ⎼RT, ⎼1/2RT, +1/2RT, and +RT (i.e., the 1/2RT scale). Overall, except for several residues which display distinctively different profiles (Phe135, Phe136, Ala203, Ala206, Leu225, Gln226, and Arg214), the reconstructed profiles using the 1/2RT scale in bilayers has an overall similarity to those using the RT scale in micelles. The preserved features include: the cooperative packing core (formed by TM1, TM2 and TM3), the cooperative cluster in the active site (Ser201, His150 and Asn154), the localized cluster in L1, and the overpropagated cluster at the TM4-TM6 interface.  (a) The effect of the location of the biotin pair (172/267 C -BtnPyr 2 vs 172/267 C -BtnPyr 2 ) on GlpG activity for the TM substrate LYTM2 in micelles and bilayers. In both environments, the slopes (the activities of 172/267 C -BtnPyr 2 vs 172/267 C -BtnPyr 2 ) are close to the unity, indicating that the location of the biotin pair does not affect the mutational impacts on activity.

(b)
The effect of the hydrophobic environment (micelles vs bicelles) on GlpG activity for the TM substrate LYTM2. All activity values correspond to the fractional substrate turnover rate (min -1 ) out of the initial substrate concentration (10 µM) in DDM micelles (5 mM) or DMPC:CHAPS (2% w/v, q = 1.5) bicelles as measured by NBD fluorescence (Extended Data Figs. 3a-b). Errors denote SEM (n = 3). For each mutation, the relative activities measured in the backgrounds of 95 N 172 M -BtnPyr 2 and 172 M 267 C -BtnPyr 2 (Extended Data Tables 1 and 2) were averaged and color-coded in the heat map on the structure (PDB code: 3B45). Overall, the inactivating mutations are distributed at the TM4/TM6 interface harboring the catalytic dyad (Ser201-His254 marked with a rectangular box), in the L1 loop (Arg136 and Trp137, Wang & Ha 2007J Mol Biol 374, 1104, and the substrate binding site at the TM2/TM5 interface. The activity is slightly more tolerant to mutation in bicelles.

Figure 16
DDM150 DDM120 (a) Two micellar systems in this study, one with 120 DDM molecules (DDM120) and the other with 150 molecules (DDM150). The shapes of both micellar systems were oblate with the axial dimension (2xc), which remained constant at c = ~22.5 Å.
The shape of micelles was assessed for DDM120 and DDM150 without protein to understand overall packing. The structure of a DDM micelle was approximated by a spheroid as below: , where x, y, and z are the cartesian coordinates in the 3D-space. The semi-axes a and c are aligned along each symmetry axis, each indicating the equatorial radius in the xy-plane and the distance from the spheroid center to the pole along the symmetry axis of z, where a>c forms an oblate spheroid, while a<c a prolate. All coordinates of DDM 2O4 atoms in a micelle were utilized to describe the spheroidal shape of the micelle, which then were subjected to a parametric fitting for obtaining the semi-axes and . We found that both DDM120 and DDM150 create oblate spheroidal shapes (i.e., a>c), from which we estimated the effective surface area by using A oblate = 2pa 2 + pc 2 /e •ln[(1 + e)/(1 -e)] (the eccentricity, e is defined by e = [1 -c 2 /a 2 ] 1/2 ) (Kruger, DM & Kamerlin, SCL 2017 ACS Omega 2, 4524-4530).
(b) As the number of DDM molecules increases from 120 to 150, the equatorial dimension accordingly increases from a = 29.5 Å to 32.9 Å,. The area per DDM at the micellar surface is larger in DDM120 providing room for each DDM molecule to relax fast in the micelles relative to that in DDM150 (Fig. 4b).
The experimental values obtained from small-angle X-ray scattering (SAXS) are also shown for comparison (Oliver RC, Lipfert J, Fox DA, Lo RH, Doniach S, et al. 2013 PLoS ONE 8: e62488). N A,DDM : the aggregation number of DDM. a b