Abstract
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol (LDL-c) levels by promoting the degradation of liver LDL receptors (LDLRs). Antibodies that inhibit PCSK9 binding to the EGF(A) domain of the LDLR are effective in lowering LDL-c. However, the discovery of small-molecule therapeutics is hampered by difficulty in targeting the relatively flat EGF(A)-binding site on PCSK9. Here we demonstrate that it is possible to target this site, based on the finding that the PCSK9 P′ helix displays conformational flexibility. As a consequence, the vacated N-terminal groove of PCSK9, which is adjacent to the EGF(A)-binding site, is in fact accessible to small peptides. In phage-display experiments, the EGF(A)-mimicking peptide Pep2-8 was used as an anchor peptide for the attachment of an extension peptide library directed toward the groove site. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which encroach on the EGF(A)-binding site and inhibit LDLR binding.
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Acknowledgements
We would like to thank M. Dillon and S.H. Kim for sequencing Ab7G7, P. Luan for preparing the Fab7G7, P. Liu for N-terminal sequencing, T. Lipari for identifying F.XIa and B. Lazarus for his ingenious suggestions regarding phage-display strategies. Synchrotrons at the ALS and the APS are supported by the Director, Office of Science, Office of Basic Energy Sciences (BES) of the US Department of Energy (DOE) under contracts DE-AC02-05CH11231 and DE-AC02-06CH11357, respectively. The Berkeley Center for Structural Biology is supported in part by the NIH, NIGMS, and HHMI. Supporting institutions of the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at APS may be found at http://www.ser-cat.org/members.html. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS (including grant no. P41GM103393).
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Contributions
Y.Z. identified peptide ligands by phage display, performed phage ELISA and wrote the manuscript. M.U. and S.S. crystallized proteins and solved structures. C.E. coordinated crystallography and solved structures and wrote the manuscript. N.J.S. designed peptides for SAR studies and the antagonist phage library and wrote the manuscript. D.J.B. designed and performed peptide SAR studies and synthesized peptides. M.H.B. developed and conducted TR-FRET assays. A.P. developed experimental strategies for antibody generation and groove validation studies and wrote the manuscript. W.L. developed and conducted PCSK9 P′-helix cleavage assays, protein ELISA and affinity measurements of PCSK9 variants. M.K.-B. performed HepG2 assays, J.Q. conducted SPR studies measuring peptide binding to PCSK9 variants, C.C. and Y.W. generated Ab33 and Ab20 and determined binding affinities by SPR, and P.M. identified inhibitory antibodies and designed PCSK9 open-groove variants and purified protein reagents. P.D.L. conducted NMR studies, and D.K. designed and coordinated the overall study and wrote the manuscript. All authors analyzed and interpreted experimental data.
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Integrated supplementary information
Supplementary Figure 1 Electron density 2mFo-DFc maps contoured at 1 times rmsd shown for a subset of atoms.
(a) Part of CDR-H2 of Fab33. (b) Fusion2 peptide. (c) Pep1. (d) Pep3. (e) WNLV(hR)IGLLR. (f) MESFPGWNLV(hR)IGLLR.
Supplementary Figure 2 Surface plasmon resonance measurements of PCSK9 variants binding to immobilized LDLR-Fc.
Shown are sensorgrams (different color for each concentration) and fitted curves (black) of (a) PCSK9 (KD 103 ± 3.4 nM), (b) PCSK9-AAA (KD 138 ± 1.3 nM) and (c) PCSK9ΔP' (KD 541 ± 8.2 nM). The KD values, calculated from the determined kon and koff values, were the average ± s.d. of three independent experiments.
Supplementary Figure 3 Binding of phage displaying Pep2-8 (TVFTSWEEYLDWVGSG) or Pep2-8V2A (TAFTSWEEYLDWVGSG) to PCSK9.
The single mutation of valine2 to an alanine reduced binding affinity to PCSK9. The reduced but still detectable binding of Pep2-8V2A to PCSK9 was suitable for its use as an anchor peptide to C-terminally attach an extension peptide library via the GSG linker.
Supplementary Figure 4 Groove-binding peptides approach Pep2-8 and EGF(A) in a similar fashion as the native P'-helix.
(a) The Pro4 residue of Pep1 is in the same location as Pro155 of the native P'-helix and contacts the side chains of residues Trp6 and Tyr9 of Pep2-8. Analogous contacts are observed for Pep3 (not shown). (b) The structurally analogous PCSK9 P'-helix Pro155 contacts EGF(A) Leu298 in the PCSK9/EGF(A) complex (PDB 2W2M). (c) Cyclic peptides Pep1 (green-yellow) and Pep3 (brown) are closely similar in sequence and structure when bound to PCSK9 and display a residue (Arg2, Phe2) close to the volume occupied by Ser153 in the native P’-helix (Ser153-Ile154-Pro155 in white).
Supplementary Figure 5 Sequential and medium-range NOEs observed for two groove-binding peptides.
(a) MESFPGWNLV(hR)IGLLR peptide in H2O/CD3CN (70:30), at 284K. (b) WNLV(hR)IGLLR peptide in H2O/CD3CN (70:30), at 284K. The thickness of the bars is proportional to the intensities of the NOE signals. The detection of weak, medium-range NOEs indicate a helical propensity for residues W7-L14 of the MESFPGWNLV(hR)IGLLR peptide and for residues W1-L8 of the WNLV(hR)IGLLR peptide.
Supplementary Figure 6 Structure of MESFPGWNLV(hR)IGLLR bound to PCSK9ΔP’ explains antagonism.
Hydrophobic contacts involving side chains from new residues M1 and F4 and residues W7, V10 and hR11 help determine the conformation of the N-terminal extension. Carbon (and sulfur) atoms from these side chains are shown with dotted surfaces.
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Supplementary Figures 1–6 and Supplementary Tables 1–11. (PDF 1635 kb)
Supplementary Data Set 1
Uncropped blot for Figure 2a–d and Figure 4b. (PDF 3594 kb)
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Zhang, Y., Ultsch, M., Skelton, N. et al. Discovery of a cryptic peptide-binding site on PCSK9 and design of antagonists. Nat Struct Mol Biol 24, 848–856 (2017). https://doi.org/10.1038/nsmb.3453
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DOI: https://doi.org/10.1038/nsmb.3453
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