Abstract
Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high-density lipoprotein (HDL) structure and metabolism, probably mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood, as lipid-free apoA-I has been notoriously resistant to high-resolution structural study. Published models from low-resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled a team of structural biologists specializing in apolipoproteins and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small-angle X-ray scattering (SAXS), hydrogen–deuterium exchange (HDX) and crystallography data, we propose a time-averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long-sought platform for understanding and testing details of HDL biogenesis, structure and function.
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Acknowledgements
This work was supported by an American Heart Association postdoctoral fellowship grant (16POST27710016 to J.T.M.), an National Institutes of Health Heart Lung and Blood Institute funded predoctoral fellowship to M.C. (HL125204-03), R01 GM098458 to W.S.D. and T.B.T., R01 HL112276 and HL127649 to M.G.S.-T., P01 HL026335 and R01 HL116518 to D.A., P01 HL12803 to W.S.D., J.P.S. and J.W.H. The MS data was acquired in the University of Cincinnati Proteomics Laboratory under the direction of K. Greis on a mass spectrometer funded in part through an NIH S10 shared instrumentation grant (RR027015 Gries-PI).
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J.T.M. and W.S.D. conceived and designed new experiments reported in this paper. J.T.M., R.G.W., A.L.C., J.M., M.C. and H.D.S. performed experiments. J.T.M., R.G.W., M.C., T.B.T., M.K.J., H.D.S., J.P.S., M.C.P. and W.S.D. analyzed data. J.T.M., M.C., T.B.T., M.K.J., H.D.S., K.-A.R., M.N.O., M.G.S.-T., M.J.T., J.W.H., X.M., D.A., J.P.S., S.L.-K., M.C.P. and W.S.D. derived the model. J.T.M., T.B.T., K.-A.R., M.N.O., M.G.S.-T., M.J.T., J.W.H., D.A., J.P.S., S.L.-K., M.C.P. and W.S.D. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Separation and purification of lipid-free apoA-I monomer by gel filtration chromatography
ApoA-I was cross-linked and subjected to gel-filtration chromatography and fractions corresponding to the stable monomeric species were pooled. Chromatograms of apoA-I cross-linked with CBDPS and BS3 are shown in panels (a) and (c), respectively. The shaded area represents the fractions corresponding to monomeric apoA-I that were pooled for cross-linking and SAXS analysis. Corresponding SDS-PAGE analysis of lipid-free apoA-I cross-linked with CBPDS and BS3 are shown in panels (b) and (d), respectively. Molecular weight markers are shown in lane 1, cross-linked apoA-I prior to separation is shown in lane 2, and cross-linked monomeric apoA-I after separation is shown in lane 3. Gels were stained with coomassie blue.
Supplementary Figure 2 Derivation of an all atom model of full-length, lipid-free monomeric apoA-I
Panel (a) shows a single molecule from the reported crystal structure of the apoA-I1–184 dimer. Panel (b) shows the folding of helix 6 previously proposed by Mei et. al.34, and the fold of helix 6 used for the time-averaged structure (right). Panel (c) shows the final time-averaged model. Molecules are colored as previously defined by Mei et. al.34. Purple and cyan represent consensus sequence peptide A and B homology sequences, green represents exon-3-encoded region (residues 1–43) and yellow are prolines.
Supplementary Figure 3 Comparison of the newest model to previous models with respect to various pieces of experimental data
The line diagrams show the fit of the models relative to the target value (black circle) derived from current and previous data on lipid-free monomeric apoA-I. Panel (a) shows the model fits to experimental cross-links from the universal cross-linking list (Supplemental Table 4) with the target being zero violations. Panel (b) shows the model fits to experimental H-DX data with the target being zero violations. Panel (c) shows the averaged χ2 values for all models fit to the scattering profiles derived from apoA-I cross-linked with BS3 and CBDPS. The target for SAXS is the lowest χ2 value possible with lower values indicating better fits to the experimental scattering curve. Panel (d) shows the fits to overall α-helical data derived values reported across 27 studies as shown in Supplementary Table 5. Panel (e) shows the rank of the MolProbity score of all reported models of apoA-I relative to 27,675 crystal structures reported in the protein database.
Supplementary Figure 4 Effect of temperature on H-DX in lipid-free apoA-I.
The plots compare the measured H-DX kinetics of the apoA-I peptide 159-169 from a helical region at pD 7 and (A) 5°C, (B) 25°C and (C) 37°C to the rate for the peptide in a dynamically disordered state (dashed line). Comparison of the rate constants derived by fitting the dashed and solid time-courses to mono-exponential rate equations yields the protection factor (Pf) and hence the free energy (ΔG) of helix stabilization. After correcting for the effect of temperature on the intrinsic chemical HX rate, the apparent ΔG of helix stability at 5°C and 25°C is 5.3 and 3.8 kcal/mol, respectively. The helix stability is less at 37°C and H-D exchange is complete in ~3 min. [From26]
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Melchior, J., Walker, R., Cooke, A. et al. A consensus model of human apolipoprotein A-I in its monomeric and lipid-free state. Nat Struct Mol Biol 24, 1093–1099 (2017). https://doi.org/10.1038/nsmb.3501
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DOI: https://doi.org/10.1038/nsmb.3501
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