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Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants.

Bottomley MJ, Cirillo A, Orsatti L, Ruggeri L, Fisher TS, Santoro JC, Cummings RT, Cubbon RM, Lo Surdo P, Calzetta A, Noto A, Baysarowich J, Mattu M, Talamo F, De Francesco R, Sparrow CP, Sitlani A, Carfí A
J Biol Chem 284 1313-23 (2009)
Related entries: 2w2m, 2w2n, 2w2o, 2w2q

Cited: 77 times
EuropePMC logo PMID: 19001363

Abstract

PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9DeltaC-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9DeltaC bound to EGF(AB)(H306Y), a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB)(H306Y)-PCSK9 interaction is pH-independent, LDLR(H306Y) binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9DeltaC containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.

Articles - 2w2p mentioned but not cited (2)

  1. Molecular modeling of the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase and predictions of structural effects of mutations associated with HIBM and sialuria. Kurochkina N, Yardeni T, Huizing M. Glycobiology 20 322-337 (2010)
  2. Computational Study of PCSK9-EGFA Complex with Effective Polarizable Bond Force Field. Chen J, Duan L, Ji C, Zhang JZH. Front Mol Biosci 4 101 (2017)


Reviews citing this publication (21)

  1. The PCSK9 decade. Lambert G, Sjouke B, Choque B, Kastelein JJ, Hovingh GK. J Lipid Res 53 2515-2524 (2012)
  2. Cholesterol, the central lipid of mammalian cells. Maxfield FR, van Meer G. Curr Opin Cell Biol 22 422-429 (2010)
  3. Molecular and cellular function of the proprotein convertase subtilisin/kexin type 9 (PCSK9). Schulz R, Schlüter KD, Laufs U. Basic Res Cardiol 110 4 (2015)
  4. The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis. Mousavi SA, Berge KE, Leren TP. J Intern Med 266 507-519 (2009)
  5. Targeting PCSK9 for hypercholesterolemia. Norata GD, Tibolla G, Catapano AL. Annu Rev Pharmacol Toxicol 54 273-293 (2014)
  6. The biology of PCSK9 from the endoplasmic reticulum to lysosomes: new and emerging therapeutics to control low-density lipoprotein cholesterol. Poirier S, Mayer G. Drug Des Devel Ther 7 1135-1148 (2013)
  7. The Multifaceted Biology of PCSK9. Seidah NG, Prat A. Endocr Rev 43 558-582 (2022)
  8. Complexity of mechanisms among human proprotein convertase subtilisin-kexin type 9 variants. Dron JS, Hegele RA. Curr Opin Lipidol 28 161-169 (2017)
  9. Proprotein convertase subtilisin/kexin type 9 inhibition. Marais DA, Blom DJ, Petrides F, Gouëffic Y, Lambert G. Curr Opin Lipidol 23 511-517 (2012)
  10. Cholesterol: the good, the bad, and the ugly - therapeutic targets for the treatment of dyslipidemia. Elshourbagy NA, Meyers HV, Abdel-Meguid SS. Med Princ Pract 23 99-111 (2014)
  11. A review of PCSK9 inhibition and its effects beyond LDL receptors. Dixon DL, Trankle C, Buckley L, Parod E, Carbone S, Van Tassell BW, Abbate A. J Clin Lipidol 10 1073-1080 (2016)
  12. Inhibition of pro-protein convertase subtilisin kexin 9 [corrected] (PCSK-9) as a treatment for hyperlipidaemia. Wierzbicki AS, Hardman TC, Viljoen A. Expert Opin Investig Drugs 21 667-676 (2012)
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  14. PCSK9 inhibition for the treatment of hypercholesterolemia: promises and emerging challenges. Norata GD, Tibolla G, Catapano AL. Vascul Pharmacol 62 103-111 (2014)
  15. Understanding PCSK9 and anti-PCSK9 therapies. McKenney JM. J Clin Lipidol 9 170-186 (2015)
  16. The promises of PCSK9 inhibition. Petrides F, Shearston K, Chatelais M, Guilbaud F, Meilhac O, Lambert G. Curr Opin Lipidol 24 307-312 (2013)
  17. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors, Reality or Dream in Managing Patients with Cardiovascular Disease. Alkhalil M. Curr Drug Metab 20 72-82 (2019)
  18. Proprotein convertase subtilisin/kexin type 9: from the discovery to the development of new therapies for cardiovascular diseases. Ferri N. Scientifica (Cairo) 2012 927352 (2012)
  19. How multi-scale structural biology elucidated context-dependent variability in ectodomain conformation along with the ligand capture and release cycle for LDLR family members. Nogi T. Biophys Rev 10 481-492 (2018)
  20. Predictive Modeling and Structure Analysis of Genetic Variants in Familial Hypercholesterolemia: Implications for Diagnosis and Protein Interaction Studies. Larrea-Sebal A, Jebari-Benslaiman S, Galicia-Garcia U, Jose-Urteaga AS, Uribe KB, Benito-Vicente A, Martín C. Curr Atheroscler Rep 25 839-859 (2023)
  21. Targeting proprotein convertase subtilisin/kexin type 9 (PCSK9): from bench to bedside. Bao X, Liang Y, Chang H, Cai T, Feng B, Gordon K, Zhu Y, Shi H, He Y, Xie L. Signal Transduct Target Ther 9 13 (2024)

Articles citing this publication (54)

  1. PCSK9: a convertase that coordinates LDL catabolism. Horton JD, Cohen JC, Hobbs HH. J Lipid Res 50 Suppl S172-7 (2009)
  2. Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route. Poirier S, Mayer G, Poupon V, McPherson PS, Desjardins R, Ly K, Asselin MC, Day R, Duclos FJ, Witmer M, Parker R, Prat A, Seidah NG. J Biol Chem 284 28856-28864 (2009)
  3. Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH. Lo Surdo P, Bottomley MJ, Calzetta A, Settembre EC, Cirillo A, Pandit S, Ni YG, Hubbard B, Sitlani A, Carfí A. EMBO Rep 12 1300-1305 (2011)
  4. Antagonism of secreted PCSK9 increases low density lipoprotein receptor expression in HepG2 cells. McNutt MC, Kwon HJ, Chen C, Chen JR, Horton JD, Lagace TA. J Biol Chem 284 10561-10570 (2009)
  5. Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9. Chaparro-Riggers J, Liang H, DeVay RM, Bai L, Sutton JE, Chen W, Geng T, Lindquist K, Casas MG, Boustany LM, Brown CL, Chabot J, Gomes B, Garzone P, Rossi A, Strop P, Shelton D, Pons J, Rajpal A. J Biol Chem 287 11090-11097 (2012)
  6. A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo. Ni YG, Di Marco S, Condra JH, Peterson LB, Wang W, Wang F, Pandit S, Hammond HA, Rosa R, Cummings RT, Wood DD, Liu X, Bottomley MJ, Shen X, Cubbon RM, Wang SP, Johns DG, Volpari C, Hamuro L, Chin J, Huang L, Zhao JZ, Vitelli S, Haytko P, Wisniewski D, Mitnaul LJ, Sparrow CP, Hubbard B, Carfí A, Sitlani A. J Lipid Res 52 78-86 (2011)
  7. A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake. Ni YG, Condra JH, Orsatti L, Shen X, Di Marco S, Pandit S, Bottomley MJ, Ruggeri L, Cummings RT, Cubbon RM, Santoro JC, Ehrhardt A, Lewis D, Fisher TS, Ha S, Njimoluh L, Wood DD, Hammond HA, Wisniewski D, Volpari C, Noto A, Lo Surdo P, Hubbard B, Carfí A, Sitlani A. J Biol Chem 285 12882-12891 (2010)
  8. Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor. Duff CJ, Scott MJ, Kirby IT, Hutchinson SE, Martin SL, Hooper NM. Biochem J 419 577-584 (2009)
  9. Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor. Zhang Y, Eigenbrot C, Zhou L, Shia S, Li W, Quan C, Tom J, Moran P, Di Lello P, Skelton NJ, Kong-Beltran M, Peterson A, Kirchhofer D. J Biol Chem 289 942-955 (2014)
  10. PCSK9 is not involved in the degradation of LDL receptors and BACE1 in the adult mouse brain. Liu M, Wu G, Baysarowich J, Kavana M, Addona GH, Bierilo KK, Mudgett JS, Pavlovic G, Sitlani A, Renger JJ, Hubbard BK, Fisher TS, Zerbinatti CV. J Lipid Res 51 2611-2618 (2010)
  11. Pharmacologic profile of the Adnectin BMS-962476, a small protein biologic alternative to PCSK9 antibodies for low-density lipoprotein lowering. Mitchell T, Chao G, Sitkoff D, Lo F, Monshizadegan H, Meyers D, Low S, Russo K, DiBella R, Denhez F, Gao M, Myers J, Duke G, Witmer M, Miao B, Ho SP, Khan J, Parker RA. J Pharmacol Exp Ther 350 412-424 (2014)
  12. A two-step binding model of PCSK9 interaction with the low density lipoprotein receptor. Yamamoto T, Lu C, Ryan RO. J Biol Chem 286 5464-5470 (2011)
  13. The E32K variant of PCSK9 exacerbates the phenotype of familial hypercholesterolaemia by increasing PCSK9 function and concentration in the circulation. Noguchi T, Katsuda S, Kawashiri MA, Tada H, Nohara A, Inazu A, Yamagishi M, Kobayashi J, Mabuchi H. Atherosclerosis 210 166-172 (2010)
  14. Isolation and characterization of the circulating truncated form of PCSK9. Han B, Eacho PI, Knierman MD, Troutt JS, Konrad RJ, Yu X, Schroeder KM. J Lipid Res 55 1505-1514 (2014)
  15. Effects of the prosegment and pH on the activity of PCSK9: evidence for additional processing events. Benjannet S, Saavedra YG, Hamelin J, Asselin MC, Essalmani R, Pasquato A, Lemaire P, Duke G, Miao B, Duclos F, Parker R, Mayer G, Seidah NG. J Biol Chem 285 40965-40978 (2010)
  16. Interaction between the ligand-binding domain of the LDL receptor and the C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDL receptor during endosomal acidification. Tveten K, Holla ØL, Cameron J, Strøm TB, Berge KE, Laerdahl JK, Leren TP. Hum Mol Genet 21 1402-1409 (2012)
  17. Comment Beyond LDL cholesterol, a new role for PCSK9. Akram ON, Bernier A, Petrides F, Wong G, Lambert G. Arterioscler Thromb Vasc Biol 30 1279-1281 (2010)
  18. New hypotheses about the structure-function of proprotein convertase subtilisin/kexin type 9: analysis of the epidermal growth factor-like repeat A docking site using WaterMap. Pearlstein RA, Hu QY, Zhou J, Yowe D, Levell J, Dale B, Kaushik VK, Daniels D, Hanrahan S, Sherman W, Abel R. Proteins 78 2571-2586 (2010)
  19. Spontaneous severe hypercholesterolemia and atherosclerosis lesions in rabbits with deficiency of low-density lipoprotein receptor (LDLR) on exon 7. Lu R, Yuan T, Wang Y, Zhang T, Yuan Y, Wu D, Zhou M, He Z, Lu Y, Chen Y, Fan J, Liang J, Cheng Y. EBioMedicine 36 29-38 (2018)
  20. Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding. Gu HM, Adijiang A, Mah M, Zhang DW. J Lipid Res 54 3345-3357 (2013)
  21. Development of vaccine for dyslipidemia targeted to a proprotein convertase subtilisin/kexin type 9 (PCSK9) epitope in mice. Kawakami R, Nozato Y, Nakagami H, Ikeda Y, Shimamura M, Yoshida S, Sun J, Kawano T, Takami Y, Noma T, Rakugi H, Minamino T, Morishita R. PLoS One 13 e0191895 (2018)
  22. An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo. Schiele F, Park J, Redemann N, Luippold G, Nar H. J Mol Biol 426 843-852 (2014)
  23. PCSK9 acts as a chaperone for the LDL receptor in the endoplasmic reticulum. Strøm TB, Tveten K, Leren TP. Biochem J 457 99-105 (2014)
  24. Pseurotin A as a novel suppressor of hormone dependent breast cancer progression and recurrence by inhibiting PCSK9 secretion and interaction with LDL receptor. Abdelwahed KS, Siddique AB, Mohyeldin MM, Qusa MH, Goda AA, Singh SS, Ayoub NM, King JA, Jois SD, El Sayed KA. Pharmacol Res 158 104847 (2020)
  25. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain. Poirier S, Hamouda HA, Villeneuve L, Demers A, Mayer G. PLoS One 11 e0157230 (2016)
  26. Treatment-Induced Viral Cure of Hepatitis C Virus-Infected Patients Involves a Dynamic Interplay among three Important Molecular Players in Lipid Homeostasis: Circulating microRNA (miR)-24, miR-223, and Proprotein Convertase Subtilisin/Kexin Type 9. Hyrina A, Olmstead AD, Steven P, Krajden M, Tam E, Jean F. EBioMedicine 23 68-78 (2017)
  27. Immunization against proprotein convertase subtilisin-like/kexin type 9 lowers plasma LDL-cholesterol levels in mice. Fattori E, Cappelletti M, Lo Surdo P, Calzetta A, Bendtsen C, Ni YG, Pandit S, Sitlani A, Mesiti G, Carfí A, Monaci P. J Lipid Res 53 1654-1661 (2012)
  28. Discovery of a cryptic peptide-binding site on PCSK9 and design of antagonists. Zhang Y, Ultsch M, Skelton NJ, Burdick DJ, Beresini MH, Li W, Kong-Beltran M, Peterson A, Quinn J, Chiu C, Wu Y, Shia S, Moran P, Di Lello P, Eigenbrot C, Kirchhofer D. Nat Struct Mol Biol 24 848-856 (2017)
  29. Internalized PCSK9 dissociates from recycling LDL receptors in PCSK9-resistant SV-589 fibroblasts. Nguyen MA, Kosenko T, Lagace TA. J Lipid Res 55 266-275 (2014)
  30. Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9. Le QT, Blanchet M, Seidah NG, Labonté P. J Biol Chem 290 23385-23400 (2015)
  31. The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation: A NOVEL MECHANISM CAUSING FAMILIAL HYPERCHOLESTEROLEMIA. Susan-Resiga D, Girard E, Kiss RS, Essalmani R, Hamelin J, Asselin MC, Awan Z, Butkinaree C, Fleury A, Soldera A, Dory YL, Baass A, Seidah NG. J Biol Chem 292 1573-1590 (2017)
  32. Calcium-independent inhibition of PCSK9 by affinity-improved variants of the LDL receptor EGF(A) domain. Zhang Y, Zhou L, Kong-Beltran M, Li W, Moran P, Wang J, Quan C, Tom J, Kolumam G, Elliott JM, Skelton NJ, Peterson AS, Kirchhofer D. J Mol Biol 422 685-696 (2012)
  33. Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody. Schroeder KM, Beyer TP, Hansen RJ, Han B, Pickard RT, Wroblewski VJ, Kowala MC, Eacho PI. J Lipid Res 56 2124-2132 (2015)
  34. Structure and protective efficacy of the Staphylococcus aureus autocleaving protease EpiP. Kuhn ML, Prachi P, Minasov G, Shuvalova L, Ruan J, Dubrovska I, Winsor J, Giraldi M, Biagini M, Liberatori S, Savino S, Bagnoli F, Anderson WF, Grandi G. FASEB J 28 1780-1793 (2014)
  35. Inhibition of PCSK9D374Y/LDLR Protein-Protein Interaction by Computationally Designed T9 Lupin Peptide. Lammi C, Sgrignani J, Roda G, Arnoldi A, Grazioso G. ACS Med Chem Lett 10 425-430 (2019)
  36. APP, APLP2 and LRP1 interact with PCSK9 but are not required for PCSK9-mediated degradation of the LDLR in vivo. Fu T, Guan Y, Xu J, Wang Y. Biochim Biophys Acta Mol Cell Biol Lipids 1862 883-889 (2017)
  37. In silico Screening of Chemical Libraries to Develop Inhibitors That Hamper the Interaction of PCSK9 with the LDL Receptor. Min DK, Lee HS, Lee N, Lee CJ, Song HJ, Yang GE, Yoon D, Park SW. Yonsei Med J 56 1251-1257 (2015)
  38. Affinity and kinetics of proprotein convertase subtilisin/kexin type 9 binding to low-density lipoprotein receptors on HepG2 cells. Mousavi SA, Berge KE, Berg T, Leren TP. FEBS J 278 2938-2950 (2011)
  39. Exemplification of the challenges associated with utilising fluorescence intensity based assays in discovery. Gul S, Gribbon P. Expert Opin Drug Discov 5 681-690 (2010)
  40. Identification of amino acid residues in the ligand binding repeats of LDL receptor important for PCSK9 binding. Deng SJ, Alabi A, Gu HM, Adijiang A, Qin S, Zhang DW. J Lipid Res 60 516-527 (2019)
  41. A New Strategy for Rapidly Screening Natural Inhibitors Targeting the PCSK9/LDLR Interaction In Vitro. Li L, Shen C, Huang YX, Li YN, Liu XF, Liu XM, Liu JH. Molecules 23 E2397 (2018)
  42. Short- and long-term effects of xuezhikang, an extract of cholestin, on serum proprotein convertase subtilisin/kexin type 9 levels. Jia YJ, Zhang Y, Liu J, Guo YL, Xu RX, Li JJ. Chin J Integr Med 22 96-100 (2016)
  43. In Silico Insights into Protein-protein Interaction Disruptive Mutations in the PCSK9-LDLR complex. Martin WR, Lightstone FC, Cheng F. Int J Mol Sci 21 E1550 (2020)
  44. Mutational Spectrum of LDLR and PCSK9 Genes Identified in Iranian Patients With Premature Coronary Artery Disease and Familial Hypercholesterolemia. Moradi A, Maleki M, Ghaemmaghami Z, Khajali Z, Noohi F, Moghadam MH, Kalyinia S, Mowla SJ, Seidah NG, Malakootian M. Front Genet 12 625959 (2021)
  45. Studies of the autoinhibitory segment comprising residues 31-60 of the prodomain of PCSK9: Possible implications for the mechanism underlying gain-of-function mutations. Wierød L, Cameron J, Strøm TB, Leren TP. Mol Genet Metab Rep 9 86-93 (2016)
  46. The Cholesterol-Lowering Effect of Capsella Bursa-Pastoris Is Mediated via SREBP2 and HNF-1α-Regulated PCSK9 Inhibition in Obese Mice and HepG2 Cells. Hwang JT, Choi E, Choi HK, Park JH, Chung MY. Foods 10 408 (2021)
  47. Characterisation of de novo mutations in the C-terminal domain of proprotein convertase subtilisin/kexin type 9. Geschwindner S, Andersson GM, Beisel HG, Breuer S, Hallberg C, Kihlberg BM, Lindqvist AM, O'Mahony G, Plowright AT, Raubacher F, Knecht W. Protein Eng Des Sel 28 117-125 (2015)
  48. Structures of LRP2 reveal a molecular machine for endocytosis. Beenken A, Cerutti G, Brasch J, Guo Y, Sheng Z, Erdjument-Bromage H, Aziz Z, Robbins-Juarez SY, Chavez EY, Ahlsen G, Katsamba PS, Neubert TA, Fitzpatrick AWP, Barasch J, Shapiro L. Cell 186 821-836.e13 (2023)
  49. Letter Antihyperlipidemic therapies targeting PCSK9: Novel therapeutic agents for lowering low-density lipoprotein cholesterol. Liu MH. Int J Cardiol 195 212-214 (2015)
  50. Construction of point mutation rabbits using CRISPR/Cas9. Yan K, Zhang T, Zha Y, Liang J, Cheng Y. Zhejiang Da Xue Xue Bao Yi Xue Ban 50 229-238 (2021)
  51. Fragment-based design of small molecule PCSK9 inhibitors using simulated annealing of chemical potential simulations. Guarnieri F, Kulp JL, Kulp JL, Cloudsdale IS. PLoS One 14 e0225780 (2019)
  52. Functional Characterization of p.(Arg160Gln) PCSK9 Variant Accidentally Found in a Hypercholesterolemic Subject. Larrea-Sebal A, Trenti C, Jebari-Benslaiman S, Bertolini S, Calandra S, Negri EA, Bonelli E, Benito-Vicente A, Uraga-Gracianteparaluceta L, Martín C, Fasano T. Int J Mol Sci 24 3330 (2023)
  53. In Vitro Assays for the Discovery of PCSK9 Autoprocessing Inhibitors. Salowe SP, Zhang L, Zokian HJ, Gesell JJ, Zink DL, Wiltsie J, Ai X, Kavana M, Pinto S. J Biomol Screen 21 1034-1041 (2016)
  54. Pathogenic gain-of-function mutations in the prodomain and C-terminal domain of PCSK9 inhibit LDL binding. Sarkar SK, Matyas A, Asikhia I, Hu Z, Golder M, Beehler K, Kosenko T, Lagace TA. Front Physiol 13 960272 (2022)


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