Protein prenylation plays a critical role in regulating the cellular localization of small GTPases and is essential for multiple myeloma (MM) pathology. Geranylgeranyl diphosphate synthase (GGPPS), producing a key prenylation moiety, exists in a dimeric or hexameric form, depending on the species. However, the functional significance of this oligomerization remains unclear. Using crystallography, mass spectrometry, and fluorescence spectroscopy, we show that the GGPPSR235C mutant-found in the widely studied MM cell line RPMI-8226-exhibits weakened inter-dimer interactions, reduced hexamer stability, and increased apparent substrate affinity and product release kinetics. These effects are even more pronounced in a dimeric mutant, GGPPSY246D, demonstrating that interdimer interactions within the hexamer help stabilize a lid region over the active site, thereby stabilizing product binding in an inhibitory conformation. Together, these findings reveal that hexamerization regulates GGPPS activity through product inhibition and underscore the importance of cell line selection and characterization in drug discovery efforts.

BLAVATNIK CENTER for Drug Discovery Tel Aviv University Israel

Department of Biochemistry Faculty of Science Charles University Prague 2 Czech Republic

Department of Physiology and Pharmacology Gray Faculty of Medical and Health Sciences Tel Aviv University Israel

Division BioCeV Institute of Microbiology of the Czech Academy of Sciences Vestec Czech Republic

Sagol School of Neuroscience Tel Aviv University Israel

Tel Aviv Sourasky Medical Center Israel

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Waller DD, Park J & Tsantrizos YS (2019) Inhibition of farnesyl pyrophosphate (FPP) and/or geranylgeranyl pyrophosphate (GGPP) biosynthesis and its implication in the treatment of cancers. Crit Rev Biochem Mol Biol 54, 41–60. PubMed

Xu N, Shen N, Wang XX, Jiang S, Xue B & Li CJ (2015) Protein prenylation and human diseases: a balance of protein farnesylation and geranylgeranylation. Sci China Life Sci 58, 328–335. PubMed

Palsuledesai CC & Distefano MD (2015) Protein prenylation: enzymes, therapeutics, and biotechnology applications. ACS Chem Biol 10, 51–62. PubMed PMC

Takai Y, Sasaki T & Matozaki T (2001) Small GTP‐binding proteins. Physiol Rev 81, 153–208. PubMed

Kavanagh KL, Dunford JE, Bunkoczi G, Russell RGG & Oppermann U (2006) The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding. J Biol Chem 281, 22004–22012. PubMed

Coxon FP, Helfrich MH, Van't Hof R, Sebti S, Ralston SH, Hamilton A & Rogers MJ (2000) Protein geranylgeranylation is required for osteoclast formation, function, and survival: inhibition by bisphosphonates and GGTI‐298. J Bone Miner Res 15, 1467–1476. PubMed

Guerra‐rodr M & Ferna L (2021) The mevalonate pathway, a metabolic target in cancer therapy. Front Oncol 11, 1–21. PubMed PMC

Park J, Matralis AN, Berghuis AM & Tsantrizos YS (2014) Human isoprenoid synthase enzymes as therapeutic targets. Front Chem 2, 1–21. PubMed PMC

Lu YP, Liu HG & Liang PH (2009) Different reaction mechanisms for cis‐ and trans‐prenyltransferases. Biochem Biophys Res Commun 379, 351–355. PubMed

Li Z‐H, Cintrón R, Koon NA & Moreno SNJ (2012) The N‐terminus and the chain‐length determination domain play a role in the length of the isoprenoid product of the bifunctional toxoplasma gondii farnesyl diphosphate synthase. Biochemistry 51, 7533–7540. PubMed PMC

Chang HY, Cheng TH & Wang AHJ (2021) Structure, catalysis, and inhibition mechanism of prenyltransferase. IUBMB Life 73, 40–63. PubMed PMC

Pham AC, Holstein SA & Borgstahl GEO (2024) Structural insight into geranylgeranyl diphosphate synthase (GGDPS) for cancer therapy. Mol Cancer Ther 23, 14–23. PubMed PMC

Kazandjian D (2016) Multiple myeloma epidemiology and survival: a unique malignancy. Semin Oncol 43, 676–681. PubMed PMC

Palumbo A & Anderson K (2011) Multiple myeloma. N Engl J Med 364, 1046–1060. PubMed

Wennerberg K, Rossman KL & Der CJ (2005) The ras superfamily at a glance. J Cell Sci 118, 843–846. PubMed

Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10, 513–525. PubMed

Hutagalung AH & Novick PJ (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91, 119–149. PubMed PMC

Haney SL, Chhonker YS, Varney ML, Talmon G, Murry DJ & Holstein SA (2018) Preclinical investigation of a potent geranylgeranyl diphosphate synthase inhibitor. Investig New Drugs 36, 810–818. PubMed PMC

Lacbay CM, Waller DD, Park J, Palou MG, Vincent F, Huang XF, Ta V, Berghuis AM, Sebag M & Tsantrizos YS (2018) Unraveling the prenylation‐cancer paradox in multiple myeloma with novel geranylgeranyl pyrophosphate synthase (GGPPS) inhibitors. J Med Chem 61, 6904–6917. PubMed

Holstein SA, Tong H & Hohl RJ (2010) Differential activities of thalidomide and isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells. Leuk Res 34, 344–351. PubMed PMC

Holstein SA & Hohl RJ (2011) Isoprenoid biosynthetic pathway inhibition disrupts monoclonal protein secretion and induces the unfolded protein response pathway in multiple myeloma cells. Leuk Res 35, 551–559. PubMed PMC

Haney SL, Varney ML, Williams JT, Smith LM, Talmon G & Holstein SA (2022) Geranylgeranyl diphosphate synthase inhibitor and proteasome inhibitor combination therapy in multiple myeloma. Exp Hematol Oncol 11, 5. PubMed PMC

Bamford S, Dawson E, Forbes S, Clements J, Pettett R, Dogan A, Flanagan A, Teague J, Futreal PA, Stratton MR PubMed PMC

Luo J, Solimini NL & Elledge SJ (2009) Principles of cancer therapy: oncogene and non‐oncogene addiction. Cell 136, 823–837. PubMed PMC

Hanahan D & Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144, 646–674. PubMed

Abaan OD, Polley EC, Davis SR, Zhu YJ, Bilke S, Walker RL, Pineda M, Gindin Y, Jiang Y, Reinhold WC PubMed PMC

Lisnyansky M, Yariv E, Segal O, Marom M, Loewenstein A, Ben‐Tal N, Giladi M & Haitin Y (2019) Metal coordination is crucial for geranylgeranyl diphosphate synthase‐bisphosphonate interactions: a crystallographic and computational analysis. Mol Pharmacol 96, 580–588. PubMed

Aparicio A, Gardner A, Tu Y, Savage A, Berenson J & Lichtenstein A (1998) PubMed

Krissinel E & Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774–797. PubMed

Wallrapp FH, Pan J‐J, Ramamoorthy G, Almonacid DE, Hillerich BS, Seidel R, Patskovsky Y, Babbitt PC, Almo SC, Jacobson MP PubMed PMC

Lisnyansky M, Kapelushnik N, Ben‐Bassat A, Marom M, Loewenstein A, Khananshvili D, Giladi M & Haitin Y (2018) Reduced activity of geranylgeranyl diphosphate synthase mutant is involved in bisphosphonate‐induced atypical fractures. Mol Pharmacol 94, 1391–1400. PubMed

Giladi M, Bar‐El ML, Vaňková P, Ferofontov A, Melvin E, Alkaderi S, Kavan D, Redko B, Haimov E, Wiener R PubMed PMC

Teng KH, Chen APC, Kuo CJ, Li YC, Liu HG, Chen CT & Liang PH (2011) Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time. Anal Biochem 417, 136–141. PubMed

Teng KH, Hsu ET, Chang YH, Lin SW & Liang PH (2016) Fluorescent farnesyl diphosphate analogue: a probe to validate trans‐Prenyltransferase inhibitors. Biochemistry 55, 4366–4374. PubMed

Konermann L, Pan J & Liu Y‐H (2011) Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem Soc Rev 40, 1224–1234. PubMed

Hanahan D & Weinberg RA (2000) The hallmarks of cancer. Cell 100, 57–70. PubMed

Sahai E & Marshall CJ (2002) RHO–GTPases and cancer. Nat Rev Cancer 2, 133–142. PubMed

Downward J (2003) Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 3, 11–22. PubMed

Mullen PJ, Yu R, Longo J, Archer MC & Penn LZ (2016) The interplay between cell signalling and the mevalonate pathway in cancer. Nat Rev Cancer 16, 718–731. PubMed

Gillet J‐P, Calcagno AM, Varma S, Marino M, Green LJ, Vora MI, Patel C, Orina JN, Eliseeva TA, Singal V PubMed PMC

Notarnicola M, Messa C, Cavallini A, Bifulco M, Tecce MF, Eletto D, Di Leo A, Montemurro S, Laezza C & Caruso MG (2004) Higher farnesyl diphosphate synthase activity in human colorectal cancer inhibition of cellular apoptosis. Oncology 67, 351–358. PubMed

Marianayagam NJ, Sunde M & Matthews JM (2004) The power of two: protein dimerization in biology. Trends Biochem Sci 29, 618–625. PubMed

Nishi H, Hashimoto K, Madej T & Panchenko AR (2013) Chapter one–evolutionary, physicochemical, and functional mechanisms of protein Homooligomerization. In Progress in Molecular Biology and Translational Science (Giraldo J & Ciruela F, eds), pp. 3–24. Academic Press, PubMed PMC

Gerhart JC & Pardee AB (1962) The enzymology of control by feedback inhibition. J Biol Chem 237, 891–896. PubMed

Miziorko HM (2011) Enzymes of the mevalonate pathway of isoprenoid biosynthesis. Arch Biochem Biophys 505, 131–143. PubMed PMC

Park J, Zielinski M, Magder A, Tsantrizos YS & Berghuis AM (2017) Human farnesyl pyrophosphate synthase is allosterically inhibited by its own product. Nat Commun 8, 14132. PubMed PMC

Liang PH, Ko TP & Wang AHJ (2002) Structure, mechanism and function of prenyltransferases. Eur J Biochem 269, 3339–3354. PubMed

Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66, 125–132. PubMed PMC

Strong M, Sawaya MR, Wang S, Phillips M, Cascio D & Eisenberg D (2006) Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from mycobacterium tuberculosis. Proc Natl Acad Sci USA 103, 8060–8065. PubMed PMC

McCoy AJ, Grosse‐Kunstleve RW, Adams PD, Winn MD, Storoni LC & Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40, 658–674. PubMed PMC

Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ , Grosse‐Kunstleve RW PubMed PMC

Emsley P & Cowtan K (2004) Coot: model‐building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132. PubMed

Marty MT, Baldwin AJ, Marklund EG, Hochberg GKA, Benesch JLP & Robinson CV (2015) Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal Chem 87, 4370–4376. PubMed PMC

Manori B, Vaknin A, Vaňková P, Nitzan A, Zaidel‐Bar R, Man P, Giladi M & Haitin Y (2024) Chloride intracellular channel (CLIC) proteins function as fusogens. Nat Commun 15, 2085. PubMed PMC

Yang M, Hoeppner M, Rey M, Kadek A, Man P & Schriemer DC (2015) Recombinant Nepenthesin II for hydrogen/deuterium exchange mass spectrometry. Anal Chem 87, 6681–6687. PubMed

Trcka F, Durech M, Vankova P, Chmelik J, Martinkova V, Hausner J, Kadek A, Marcoux J, Klumpler T, Vojtesek B PubMed PMC

Kavan D & Man P (2011) MSTools ‐ web based application for visualization and presentation of HXMS data. Int J Mass Spectrom 302, 53–58.

Niesen FH, Berglund H & Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2, 2212–2221. PubMed