STRUCTURAL BASIS FOR MOLECULAR RECOGNITION OF FOLIC ACID BY FOLATE RECEPTORS CHEN CHEN B.Sc (Hons.), Nanyang Technological University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously 陈晨 Chen Chen i ACKNOWLEDGEMENT Acknowledgement I would like to express my heartfelt gratitude to all of them who have helped me during the course of my Ph.D First and foremost, I wish to thank my main supervisor, Professor Yong Eu-Leong for providing me the opportunity to start this wonderful research experience I appreciate his scientific advice in conceptualizing the project and valuable training for my presentation skills Besides, I am deeply grateful for his continuous support and guidance during my 2-year overseas attachment I would also like to acknowledge the Department of Obstetrics and Gynaecology for supporting my studies in NUS I would like to thank my TAC members Dr Song Hai-wei and Dr Li Jun for their incessant monitoring of my research progress and keeping me on track I appreciate their precious advice in my Ph.D qualifying examination and thesis I would also like to thank Dr Inthrani Raja Indran for taking the time to comment on this thesis I am very grateful for Dr Eric Xu and Dr Karsten Melcher, who supervised me in Van Andel Institute of Research, US Their close guidance not only equipped me with technical skills in protein crystallography, but also inspired my enthusiasm in research I wish to thank Dr Jiyuan Ke, who worked closely with me in my Ph.D project I have learnt a lot from his scientific advice and professional experience I am thankful for all the members in Eric's lab, who helped me immensely in my research as well as daily life, and made my overseas attachment enjoyable and memorable I would like to acknowledge and thank NGS for providing me the 4-year PhD scholarship and the opportunity to participate the "2+2" collaborative program ii ACKOWLEDGEMENT In addition, I would like to thank Van Andel Institute of Research for hosting my overseas attachment Last but not least, I would like to express my deepest gratitude to my family and friends Without their unfailing encouragement and support, I would not finish this challenging journey iii TABLE OF CONTENT TABLE OF CONTENTS Declaration .i Acknowledgements ii Table of contents iv Summary .vii List of tables ix List of figures x List of abbreviations xii Chapter 1: Literature review 1.1 Folate 1.1.1 Introduction of folate 1.1.2 Folate metabolism .4 1.1.3 Folate transport system 1.2 Folate deficiency 1.2.1 Neural tube defects 1.2.2 Vascular diseases .12 1.2.3 Cancer 12 1.3 Antifolate 14 1.4 Folate receptors 19 1.4.1 Expression, localization and function of folate receptors 19 1.4.2 Physiological roles of folate receptors .20 1.4.3 Implication of folate receptors in human diseases 21 1.5 Current drugs targeting folate receptors 24 1.5.1 FRα-antibody .24 1.5.2 Novel antifolates .25 1.5.3 Folate conjugates .25 1.6 Aims and significance of the study 29 iv TABLE OF CONTENT Chapter 2: Materials and methods .31 2.1 Plasmid construction 32 2.2 Cell culture .33 2.3 Stable cell selection 34 2.4 Protein expression and purification 35 2.4.1 Small scale purification of recombinant proteins 35 2.4.2 Large scale purification of recombinant proteins 36 2.5 Protein crystallization and data collection 38 2.6 Structure determination 40 2.7 Mutagenesis 41 2.8 Western blot .41 2.9 Radioligand binding assay .42 Chapter 3: Results 43 3.1 Bacterial expression of FRs 44 3.1.1 Screening of bacterial expression tags for FRα 44 3.1.2 Screening of FRs from different species 45 3.1.3 Purification and in vitro refolding of MBP-FRα fusion protein 48 3.2 Mammalian expression of FRs 50 3.2.1 Transient expression of MBP-FRα-MBP fusion protein 50 3.2.2 Stable clone expression of FRα-Fc fusion protein 52 3.2.3 Crystallization of wild type FRα protein 54 3.3 Deglycosylation of FRs 56 3.3.1 Point mutation of glycosylation sites 56 3.3.2 Endo F3 treatment of FRα 58 3.3.3 Combined treatment of kifunensine and Endo H 60 3.4 Structure determination 63 3.4.1 Molecular replacement 63 3.4.2 Isomorphous replacement 64 v TABLE OF CONTENT 3.4.3 Single anomalous diffraction 65 3.5 Overall structure of FRα and ligand binding pocket 67 3.6 Mutagenesis and ligand binding assay .72 Chapter 4: Discussion 76 4.1 Structure comparison between FRα and chicken RfBP 77 4.2 Structure of FRβ and pH-dependent ligand release mechanism 80 4.3 Structure-based rational drug design 86 4.3.1 DHFR structure and drug discovery 88 4.3.2 GARFT structure and AG2034 discovery 92 4.3.3 TS structure and Nolatrexed discovery .96 4.4 Development of Novel FR-targeted antifolate .101 4.4.1 Current efforts in developing FR-targeted antifolates .102 4.4.2 Structural insights of antifolate binding by FRs 105 4.5 Conclusions and perspectives 109 References 110 Publications 122 vi SUMMARY SUMMARY Folates also known as vitamin B9 are essential substrates for de novo nucleic acid synthesis and for many biological methylation processes As a result, folate deficiency is associated with numerous diseases such as fetal neural tube defects, cardiovascular diseases, and cancers High affinity uptake of folates requires folate receptors (FRα, β, γ), which are cell surface glycoproteins mediating folate intake by endocytosis FRα is the most prevalent isoform of FRs, and its expression is restricted to apical surface of epithelial cells in choroid plexus, proximal kidney tubules and placenta In contrast to limited normal tissue distribution, FRα is overexpressed in a spectrum of tumors, such as ovarian cancer, endometrial cancer and breast cancer As such, FRα has become the molecular target for the development of many cancer therapeutics Despite intense research on the folate structureactivity relationship, the molecular basis for the high affinity recognition of folates by FRα remains elusive due to the technical difficulties in expression, purification, and crystallization of FRα for structural studies Here, we developed a mammalian expression system which yielded correctly folded cysteine-rich FRα in sufficient amount for crystallization By combining the treatments of glycosylation inhibitor and enzymatic deglycosylation, we obtained homogenous protein which produced crystals diffracting to 2.8Å We solved the crystal structure of FRα-folic acid complex which provided the molecular basis for the high affinity binding Folate receptor is a globular protein which consists of six helices, two pairs of β-sheets and many loop regions which are stabilized by eight disulfide bonds vii SUMMARY FRα has a deep binding pocket with one end open, which accommodates folic acid with its pteroate moiety buried inside and glutamate group sticking outside The overall structure assumes a hand-like structure The binding pocket is almost perpendicular to the plane formed by helix α1, α2 and α3, which is the palm of the hand Whereas the N-terminal loop, loops between α1-α2, β1-β2 and α3-α4 are the fingers which grab the folic acid in middle The crystal structure also revealed the detailed folic acid binding mechanism of FRα First, the overall shape and charge distribution of FRα ligand binding pocket is complementary to folic acid The basic pteroate head of folic acid is buried within the positively charged interior of the pocket, whilst the acidic tail of folic acid is stabilized by the negatively charged exterior Second, the parallel side chains of Y85 and W171 stacking the pterin ring in between, together with D81, which forms a pair of strong hydrogen bonds with N1 and N2 of pterin, anchor folic acid inside the binding pocket Third, there is an extensive network of hydrogen bonds and hydrophobic interactions lining the binding pocket To validate these structural observations, we examined the ligand-binding affinities of FRα mutants that have alanine mutations in the key folate-contacting residues Results indicate that the extensive interaction network makes FRα–folic acid binding remarkably resistant to single point mutations In summary, the FRα–folic acid complex structure illustrates how the receptor assumes a deep folate-binding pocket that is formed by conserved residues across all receptor subtypes and provides detailed insights into how folic acid interacts with its receptors Together, these observations lay a foundation for future FR-targeted anticancer drug development viii LIST OF TABLES LIST OF TABLES Table 1: SNPs in folate-metabolism genes associated with NTDs 11 Table 2: Summary of antifolates 18 Table 3: Sequence identity between FRs from different species 46 Table 4: Statistics of data collection for deglycosylated FRα 63 Table 5: X-ray diffraction data and refinement statistics .66 ix DISCUSSION here Thus, a bulkier ligand which utilizes more space in the binding cleft can be synthesized and analyzed for increasing affinity Armed with the structural knowledge of FRs and folate metabolic enzymes (DHFR, GARFT and TS), we are now at the frontline of the development of novel FR-targeted antifolates, which is specific to tumor overexpressing FRs and cytotoxic by inhibiting nucleotides synthesis Structural studies of RFC and PCFT will be the way forward to provide critical information on how the transport of drugs to normal tissue via these two transporters can be minimized to eliminate toxicity 108 DISCUSSION 4.5 Conclusions and perspectives Folates are one-carbon donors that are required for the synthesis of nucleic acids Rapidly dividing cells, such as cancer cells, are therefore much more dependent on folates than quiescent body cells FRs are cell surface glycoproteins that mediate high affinity folate uptake through endocytosis FRs are significantly expressed only in few cells, in particular cells important for embryonic development (e.g placenta) and folate reabsorption (kidney), to allow selective folate uptake under folate-limiting conditions A large proportion of tumors therefore hijack FR and express it at very high levels to meet their folate demand High level expression of folate receptor is so cancerspecific that it is hotly pursued as an anticancer drug target In this study, we have overcome numerous technical barriers in the purification and crystallization of cysteine-rich glycoproteins and successfully solved the long awaited crystal structure of FRα bound to folic acid This structure provided a detailed map of the extensive interaction network between folic acid and FRs, and rationalizes previous structure-activity relationship studies of several folate derivatives, including antifolates and folate conjugates The 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al (2013) Structural basis for RNA recognition by a dimeric PPR-protein complex Nature structural & molecular biology 20, 1377-1382 Ke, J., Harikumar, K.G., Erice, C., Chen, C., Gu, X., Wang, L., Parker, N., Cheng, Z., Xu, W., Williams, B.O., et al (2013) Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex Genes & development 27, 2305-2319 Tiong, C.T., Chen, C., Zhang, S.J., Li, J., Soshilov, A., Denison, M.S., Lee, L.S., Tam, V.H., Wong, S.P., Xu, H.E., et al (2012) A novel prenylflavone restricts breast cancer cell growth through AhRmediated destabilization of ERalpha protein Carcinogenesis 33, 10891097 Notes and author contributions: *These authors contribute equally to this work The scope of this thesis is based on publication 122 ... form of folate in circulation is 5-methyl tetrahydrofolate (5-methyl THF) [7] a) b) Figure Chemical structure of folic acid and folate derivatives a) Constitution of folic acid b) Tetrahydrofolate... crystal structure of FRα -folic acid complex which provided the molecular basis for the high affinity binding Folate receptor is a globular protein which consists of six helices, two pairs of β-sheets... complementary to folic acid The basic pteroate head of folic acid is buried within the positively charged interior of the pocket, whilst the acidic tail of folic acid is stabilized by the negatively