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Structure and Dynamics of a Lipid Nanodisc by Integrating NMR, SAXS and SANS Experiments with Molecular Dynamics Simulations

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Structure and Dynamics of a Lipid Nanodisc by Integrating NMR, SAXS and SANS Experiments with Molecular Dynamics Simulations bioRxiv preprint doi: https://doi.org/10.1101/734822; this version posted February 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Structure and dynamics of a lipid nanodisc by integrating NMR, SAXS and SANS experiments with molecular dynamics simulations Tone Bengtsena, Viktor L. Holmb, Lisbeth Ravnkilde Kjølbyec,d, Søren R. Midtgaardb, Nicolai Tidemand Johansenb, Sandro Bottaroa, Birgit Schiøttc, Lise Arlethb,1, and Kresten Lindorff-Larsena,1 aStructural Biology and NMR Laboratory & Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark; bStructural Biophysics, X-ray and Neutron Science, Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark; cDepartment of Chemistry, Aarhus University, Aarhus C, Denmark; dNovo Nordisk A/S, Måløv, Denmark This manuscript was compiled on February 2, 2020 1 Nanodiscs are membrane mimetics that consist of a protein belt sur- compositions(4, 5), solid state NMR have provided insights into 20 2 rounding a lipid bilayer. Nanodiscs are of broad use for solution- lipid phase transition states and lipid order (6, 7) and small 21 3 based characterization of membrane proteins, but we lack a full un- angle X-ray scattering (SAXS) and -neutron scattering (SANS) 22 4 derstanding of their structure and dynamics. Recently, NMR/EPR ex- have provided insight into the size and low resolution shape of 23 5 periments provided a view of the average structure of the nanodisc’s nanodiscs in solution(2, 8–10). These experiments have been 24 6 protein component, while small-angle X-ray and neutron scattering complemented by molecular dynamics (MD) simulations that 25 7 have provided insight into the global structure of both protein and provided both pioneering insights into the structure (11, 12) 26 8 lipids. Here, we investigate the structure, dynamics and biophysical as well as a better understanding of the assembly process, 27 9 properties of two small nanodiscs, MSP1D1∆H5 and ∆H4H5. We lipid-protein interactions and membrane mimicking effects 28 10 combine SEC-SAXS and SEC-SANS to obtain low-resolution struc- (13–15). 29 11 tures of the nanodiscs as elliptical discs. These are in apparent Recently, the first high resolution experimental structure 30 12 contrast to the NMR/EPR structure, which showed a more circular of the MSP protein belt encircling the nanodisc was ob- 31 13 conformation. We reconcile these views using a Bayesian/Maximum tained from the small, helix-5-deleted nanodisc, MSP1D1∆H5 32 14 Entropy method to combine our molecular dynamics simulations of (henceforth ∆H5), reconstituted with DMPC lipids (∆H5- 33 15 ∆ MSP1D1 H5 with the NMR and SAXS experiments. We derive a con- DMPC)(16). The structure provided an important step to- 34 16 formational ensemble that represents the structure and dynamics of wards a better understanding of the nanodisc and was ob- 35 17 the nanodisc, and find that it displays conformational heterogeneity tained by combining nuclear magnetic resonance (NMR) spec- 36 18 with various elliptical shapes. We find substantial differences in lipid troscopy, electron paramagnetic resonance (EPR) spectroscopy 37 19 ordering in the centre and rim of the discs, and support these ob- and transmission electron microscopy (TEM)(16). While these 38 20 servations using differential scanning calorimetry of nanodiscs of experiments were performed on lipid-loaded nanodiscs, the 39 21 various sizes. We find that the order and phase transition of the study focused on the protein components, and on determin- 40 22 lipids is highly dependent on the nanodisc size. Together, our re- ing a time- and ensemble averaged structure of these, but 41 23 sults demonstrate the power of integrative modelling and paves the left open the question of the role of the lipids(7) as well as 42 24 way for future studies of larger complex systems such as membrane any structural dynamics of the overall nanodisc. Intriguingly, 43 25 proteins embedded in nanodiscs. Nanodisc | Molecular Dynamics | SAXS | SANS | NMR | DSC Significance Statement 1 anodiscs are widely used membrane models that facilitate Membrane proteins play central roles in biology, but are difficult 2 Nbiophysical studies of membrane proteins(1). They are to study because they naturally function in an extended lipid 3 derived from, and very similar to, the human ApoA1 protein membrane. Nanodiscs are membrane mimetics that consist of 4 from high density lipoproteins (HDL particles) and consists of a protein belt surrounding a lipid bilayer, thus creating a well- 5 two amphipatic membrane scaffold proteins (MSPs) that stack defined and homogeneous environment for membrane proteins. 6 and encircle a small patch of lipids in a membrane bilayer to Unfortunately, we lack a detailed structural and biophysical 7 form a discoidal assembly. The popularity of nanodiscs arises description of the protein and lipid components, hampering 8 from their ability to mimic a membrane while at the same interpretation of experiments and design of improved nanodisc 9 time ensuring a small system of homogeneous composition, systems. We combined molecular simulations with small-angle 10 the size of which can be controlled and can give diameters in X-ray and neutron scattering experiments and previously mea- 11 a range from about 7 to 13 nm(2, 3). sured nuclear magnetic resonance experiments to study the 12 Despite the importance of nanodiscs in structural biology structure and dynamics of the proteins and lipids in a nano- 13 research and the medical importance of HDL particles, we still disc. Our results highlight spatial heterogeneity of lipid and 14 lack detailed structural models of these protein-lipid particles. protein components, providing new insights into the behaviour 15 The nanodisc has so far failed to crystallize, so in order to study of nanodiscs. 16 its structure and dynamics a range of biophysical methods have 17 been used to provide information about specific characteristics. 18 For example, mass spectrometry experiments have provided 1To whom correspondence should be addressed. 19 insight into lipid-water interactions and heterogeneous lipid E-mail: [email protected] (L.A.) and [email protected] (K.L.-L.) | February 2, 2020 | vol. XXX | no. XX | 1–11 bioRxiv preprint doi: https://doi.org/10.1101/734822; this version posted February 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 44 the resulting structure of the belt proteins corresponded to 45 that of an almost circularly-shaped disc, while our previous 46 SAXS/SANS investigations are clearly consistent with discs 47 with an on-average elliptical cross-section(8, 10). 48 Here we continue towards the goal of understanding the 49 structure and dynamics of the nanodisc and its membrane 50 mimicking capabilities. We performed SAXS and SANS ex- 51 periments on the ∆H5-DMPC variant, and integrated these 52 with MD simulations and previously published NMR data(16) 53 through an integrative Bayesian/maximum entropy (BME) 54 approach (17–21). We thereby obtain a model of the con- 55 formational ensemble of the ∆H5-DMPC nanodisc that is 56 consistent with the structural information obtained from each 57 method, as well as our molecular simulations, and successfully 58 explains differences in previous structural interpretations. In 59 addition, we study the membrane mimicking capabilities by 60 both simulation studies of lipid order parameters using the 61 reweighted simulation ensemble and by Differential Scanning 62 Calorimetry (DSC) measurements of the melting transition 63 of DMPC in differently sized nanodiscs. The results provide 64 information about the internal dynamics of the encircled lipid 65 bilayer. This study shows how multiple methods and their 66 integration helps simultaneously understand the structure and 67 the dynamics of a complex protein-lipid system, and paves the 68 way for future studies of complex biophysical systems such as Fig. 1. SEC-SAXS and SEC-SANS analysis of nanodiscs. A) SEC-SAXS (dark 69 membrane proteins embedded in nanodisc systems. purple) and SEC-SANS (light purple) for ∆H5-DMPC nanodiscs at 10 ◦C. The continuous curve show the model fit corresponding to the geometric nanodisc model shown in E. B) SEC-SAXS (dark orange) and SEC-SANS (light orange) data for the 70 Results and Discussion ∆H4H5-DMPC nanodiscs at 10 ◦C. C,D) Corresponding pair-distance distribution functions. E, F) Fitted geometrical models for the respective nanodiscs (drawn to 71 Structural investigations of ∆H5-DMPC and ∆H4H5-DMPC scale relative to one another). 72 nanodiscs by SAXS and SANS. We determined optimal re- 73 constitution ratios between the DMPC lipids and the ∆H5 74 and ∆H4H5 protein belts to form lipid-saturated nanodiscs We analyzed the SEC-SAXS and -SANS data simultane- 104 75 based on a size-exclusion chromatography (SEC) analysis (Fig. ously by global fitting of a previously developed molecular- 105 76 S1 and Methods). In line with previous studies(3), we found constrained geometrical model for the nanodiscs (8, 22) using 106 77 that reconstitution ratios of 1:33 for ∆H4H5:DMPC and 1:50 the WillItFit Software developed in our laboratory (23). The 107 78 for ∆H5:DMPC were optimal in order to form single and rela- nanodisc model (see further details in methods section) de- 108 79 tively narrow symmetric peaks. We then performed combined scribes the interior of the nanodisc as a stack of flat, elliptically- 109 80 SEC-SAXS and SEC-SANS experiments to determine the size shaped bilayer discs to account for the hydrophobic bilayer that 110 81 and shape of DMPC loaded ∆H5 and ∆H4H5 nanodiscs (Fig.
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