STRUCTURAL CHARACTERIZATION OF NOGO PROTEINS: IMPLICATIONS FOR BIOLOGICAL FUNCTIONS AND MOLECULE DESIGN OF THERAPEUTIC APPLICATION Li Minfen (B.Sc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements There are lots of people I would like to thank for a lot of reasons. Firstly, I would like to express my deep and sincere gratitude to my supervisor, Dr. Song Jianxing. I am grateful to him for his scientific emotional support. His enthusiasm and integral view on research have made a deep impression on me. His patience and consideration made me work comfortably in the lab during these years. Secondly, my thank goes to all my labmates for their valuable advice and friendly help , including Jiahai Shi, Weizheng, Jingxian Liu, Xiaoyuan Ruan and Haina Qing. In particular, I am grateful to Dr Jingsong Fan for NMR experiment training and collecting NMR spectra on the 800 MHz and 500MHz spectrometer. In addion, I appreciate Associate Professor Xiao Zhicheng and his graduate Li Yali. They gave me a great help for the neurite outgrowth experiments. Thirdly, I also want to thank all my friends. Their valuable friendship gave me energy to overcome the difficulty and let me never feel lonely. Especially, I would like to pay tribute to the constant support of my family. Without their love over these years, nothing would have been possible. Lastly, I am grateful to National University of Singapore for providing me a research scholarship, which enabled me to complete my PhD degree without financal worry in Singapore. I TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY I II VIII LIST OF TABLES X LIST OF FIGURES XI LIST OF SYMBOLS XIII CHAPTER I. INTRODUCTION 1.1. Biological Background 1.1.1. Axonal Regeneration 1.1.2 . Mechanisms Underlying the Inhibition of Axonal Regeneration in CNS 1.1.2.1. Lack of intrinsic regenerative ability 1.1.2.2. Lack of neurotrophic molecules 1.1.2.3. Inhibitory molecules in the CNS 1.1.2.3.1. Mag 1.1.2.3.2. Omagp 1.1.2.3.3. Nogo II 1.1.2.3.4. Nogo receptor complex for Myelin-associated inhibitory molecules 10 1.1.2.3.5. Strategies to block myelin inhibitory molecules effects 13 1.1.3. Biological Function Diversity of Nogos 1.2 . 1.3. 16 1.1.3.1. Nogo in Apoptosis 16 1.1.3.2 . Nogo in ALS 17 1.1.3.3. Regulating K+ channel localization at CNS paranodes 18 1.1.3.4. Nogo in Multiple sclerosis (MS) 18 1.1.3.5. Nogo in Alzheimer’s disease 19 1.1.3.6. Vascular remodeling 20 1.1.3.7. A new physiological substrate for MAPKAP-K2 20 1.1.3.8. Endoplasmic reticulum tubule formation 21 Intrinsically Disordered Protein 23 1.2.1. Reappraisal of the Protein Structure–Function Paradigm 23 1.2.2. Characterizations of Intrinsically Disordered Proteins 24 1.2.3. Biological Function of Intrinsically Disordered Proteins 25 1.2.4. Methods to Characterize Intrinsically Disorder Protein 25 1.2.4.1. Bioinformatics analysis 25 1.2.4.2. Experimental methods 26 Protein Structure Determination by NMR 1.3.1. Unique Features of NMR 29 29 III 1.3.2. NMR Phenomenon 30 1.3.3. Chemical Shift 31 1.3.4. J Coupling 31 1.3.5. Nuclear Overhauser Effect (NOE) 34 1.3.6. NMR Relaxation Parameters 35 1.3.7. General Strategies of NMR Structure Determination 36 1.3.7.1. Protein sample preparation 36 1.3.7.2. NMR spectroscopy 38 1.3.7.3. Resonance assignment 38 1.3.7.4. Collection of conformational constraints 40 1.3.7.5. Calculation of the 3D structure 40 1.3.7.6. Evaluation of structure quality 41 1.4 Aims 42 CHAPTER II. MATERIALS AND METHODS 44 2.1. Vector Construction 45 2.2. Preparation of Competent E.coli Cells 45 2.3. Transformation of E. coli Cells 46 2.4. Protein Expression and Purification 46 2.5. Preparation of Isotope Labeled Proteins 47 2.6. Protein Analysis by SDS-PAGE 47 2.7. Determination of Protein Concentration by Spectroscopy 48 IV 2.8. Circular Dichrosim (CD) spectroscopy 48 2.9. Isothermal Calorimetry (ITC) 48 2.10. Fluorescence 49 2.11. NMR Sample Preparation and Experiments 49 2.12. NMR Structure Determination 50 2.13. HSQC Characterization of the Binding between the SH3 Domains with Nogo-A Fragments 51 CHAPTER III. RESULTS AND DISCUSSIONS 53 3.1. Structural Characterization of Inhibitory Domains of Nogo 54 3.1.1. Expression and structural characterization of NogoA-(567-748) and Nogo-66 55 3.1.2. CD and NMR characterization of Nogo-40 60 3.1.3. NMR structure determination of Nogo-40 66 3.1.4. Discussion 73 3.2. Solution Structure of Nogo-60 and Design of Structured and Active Nogo-54 76 3.2.1. Preliminary structural characterization 77 3.2.2. Structure and 15N backbone dynamics of Nogo-60 78 3.2.3. Design of structured and active Nogo-54 85 3.2.4. Discussion 91 V 3.3. Structural Characterization of Nogo-B and Systematic Dissection of the Am-Nogo-A 3.4. 92 3.3.1. Bioinformatics analysis 93 3.3.2. CD and NMR characterization of N- and C-termini of Nogo-B 97 3.3.3. CD and NMR characterization of the dissected Nogo-A fragments 100 3.3.4. Discussion 107 Identification of the Specific Molecular Interaction between the Nogo and Nck2 Proteins 110 3.4.1. NMR and ITC characterization of the binding between the hNck2 SH3 domains and Nogo-A fragments 111 3.4.2. Identification of the Binding residues involved in the interaction between the third hNck2 SH3 Domain and Nogo-A Peptide 116 3.4.3. Discussion 118 3.5. CD and NMR Studies of Protein Fragments Solubilized in Salt-Free Water 122 3.5.1. Nogo-66 fragments 123 3.5.2. Extracellular receptor (NgR) domains 126 3.5.3. Other proteins 127 3.5.4. Discussion 129 3.6. Structural Characterization of NgBR 134 3.6.1. Cloning and expression of NgBR and its dissected domains 136 3.6.2. Bioinformatics characterization 138 3.6.3. Structural characterization of the NgBR ectodomain 138 VI 3.6.4. Structural characterization of NgBR and its cytoplasmic domain 141 3.6.5. Disscussion 144 CHAPTER IV. CONCLUTION AND FURTHER WORK 147 REFERENCES 151 Appendix I Analysis of globularity and disordered regions of Nogo proteins 164 Appendix II alignment of human RTNs 169 Appendix III Publications 173 VII SUMMARY Nogo-A, the largest isoform of RTN4/Nogo, was originally identified as an inhibitor of neurite outgrowth of the central nervous system (CNS) and has received intensive studies. In addition, more and more novel functions have been recently found to be associated with Nogo molecules, including vascular remodeling, apoptosis, interaction with β-amyloid protein converting enzyme, formation/maintenance of the tubular network of the endoplasmic reticulum (ER), and so on. In order to understand the structure-function relationship of Nogo molecules, we initiated a systematical investigation of structural properties of Nogo molecules by a combined use of bioinformatics analysis, and biochemical and biophysical approaches. Firstly, two identified inhibitory domains (Ngogo-A_567-748 and Nogo-66) had been studies. CD and NMR characterization indicated that Ngogo-A_567-748 was partially structured while Nogo-66 was highly insoluble. Therefore, we targeted at buffer-soluble Nogo-40, which is a truncated form of Nogo-66 and was demonstrated as an NgR antagonist. Nogo-40 was flexible in the phosphate buffer while in presence of TFE Nogo-40 revealed two well-defined helices linked by an unstructured loop. The surprising discovery that buffer-insoluble protein domains could be easily solubilized in salt-free water allowed us to go back to determine the structure of Nogo60 in water. Nogo-60 adopted a structure with the N- and C-terminal helices connected by a long middle helix and it appeared that the packing between the C-helix and the 20-residue middle helix triggered the formation of the stable Nogo-60 structure. Based on Nogo-60 structure, a structured and buffer-soluble Nogo-54 was successfully designed, which may hold promising potential to be used as a novel NgR VIII antagonist to enhance CNS axonal regeneration. We further extended the structural study to the full-length of NogoA/B. Except for the helical loop of Nogo-66 and Cterminal transmembrane domains, NogoA/B were predominantly unstructured in solution. We speculated that being intrinsically unstructured may allow Nogo molecules to serve as double-faceted functional players, with one set of functions involved in cellular signaling processes essential for CNS neuronal regeneration, vascular remodeling, apoptosis and so forth; and with another in generating/maintaining membrane-related structures. Interestingly, proline rich N-terminus of Nogo-A/B possesses many short binding motifs. With the assistance of NMR spectroscopy and ITC, for the first time we identified a tight and specific binding between Nogo-A (171-181) and the third Nck2 SH3 domain with a Kd of 2.8 µM , which provides a novel clue for the investigation of the Nogo functions and its cross-talks with other Nck2-coordinated signaling networks such as Eph-ephrinB signaling. In addition, we characterized the structural properties of 297-residue Nogo B receptor (NgBR). Our results indicate that the NgBR ectodomain is intrinsically unstructured without both secondary and tertiary structures while the cytoplasmic domain is only partially folded with secondary structures but without a tight tertiary packing. 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Interactions of macromolecules with salt ions: An electrostatic theory for the Hofmeister effect. Proteins. 61: 69–78 164 Appendix I Analysis of globularity and disordered regions of Nogo proteins Reticulon 1A reticulon 1C 165 Reticulon 2A reticulon 2B 166 Reticulon 2C reticulon 3A 167 reticulon 3B reticulon 3C 168 reticulon 3D 169 Appendix II alignment of human RTNs cladogram Clustal W (1.83) RTNs sequence alignment reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C -----MAAPGDPQDELLPLAGPGSQWLRHRGEGENEAVTPKGATPAPQAGEPSPGLGARA ------------------------------------------------------------MEDLDQSPLVSSSDSPPRPQPAFKYQFVREPEDEEEEEEEEEEDEDEDLEELEVLERKP -MEDLDQSPLVSSSDSPPRPQPAFKYQFVREPEDEEEEEEEEEEDEDEDLEELEVLERKP -----------------------------------------------------------------------------------------------------------MAEPSAATQSHS MGQVLPVFAHCKEAPSTASSTPDSTEGGNDDSDFRELHTAREFSEEDEEETTSQDWGTPR MGQVLPVFAHCKEAPSTASSTPDSTEGGNDDSDFRELHTAREFSEEDEEETTSQDWGTPR ------------------------------------------------------------ 55 reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C REAASREAGSGPARQSPVAMETASTGVAGVSSAMDHTFSTTSKDGEGSCYTSLISDICYP -----------------------------------------------------------AAGLSAAPVPTAPAAGAPLMDFGNDFVPPAPRGPLPAAPPVAPERQPSWDPSPVSSTVPA AAGLSAAPVPTAPAAGAPLMDFGNDFVPPAPRGPLPAAPPVAPERQPSWDPSPVSSTVPA -----------------------------------------------------------ISSSSFGAEPSAPGGGG------------------------SPGACPALGTKSCSSSC-ELTFSYIAFDGVVGSGGRRDSTARRPRPQGRSVSEPRDQHPQPSLGDSLESIPSLSQSPE ELTFSYIAFDGVVGSGGRRDSTARRPRPQGRSVSEPRDQHPQPSLGDSLESIPSLSQSPE ------------------------------------------------------------ reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C PQEDSTYFTGILQKENGHVTISESPEELGTPGPSLPDVPGIESRGLFSSDSGIEMTPAES -----------------------------------------------------------PSPLS-------AAAVSPSKLPEDDEPPARPPPPPPASVSPQAEPVWTPPAPAPAAPPST PSPLS-------AAAVSPSKLPEDDEPPARPPPPPPASVSPQAEPVWTPPAPAPAAPPST ----------------------------------------------------------------------------------------------------------------------PGRRG-------DPDTAPPSERPLEDLRLRLDHLGWVARGTGSGEDSSTSSSTPLEDEEP PGRRG-------DPDTAPPSERPLEDLRLRLDHLGWVARGTGSGEDSSTSSSTPLEDEEP -----------------------------------------------------------: :: reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C TEVNKILADPLDQMK-------------------------------------------------------------------------------------------------------PAAPKRRGS------SGSVDETLFALPAASEPVIRSSAENMDLKEQPGNTISAGQEDFPS PAAPKRRGS------SGSV--------------------------------------------------------------------------------------------------------------------------------------------------------------QEPNRLETG--------------------------------------------------QEPNRLETG-------------------------------------------------------------------------------------------------------------: : reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C ---------------------------------------AEAYKYIDITRPEEVKHQEQH 211 -----------------------------------------------------------VLLETAASLPSLSPLSAASFKEHEYLGNLSTVLPTEGTLQENVSEASKEVSEKAKTLLID 286 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- reticulon1A HPELEDKDLDFKNKDTDISIKPEGVREPDKPAPVEGKIIKDHLLEESTFAPYIDDLSEEQ 271 59 59 12 60 60 115 119 119 46 120 120 175 172 172 173 173 190 226 185 182 182 170 reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C -----------------------------------------------------------RDLTEFSELEYSEMGSSFSVSPKAESAVIVANPREEIIVKNKDEEEKLVSNNI--LHNQQ 344 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EAGEELDLRLRLAQPSSPEVLTPQLSPGSGTPQAGTPSPSRSRDSNSGPEEPLLEEEEK 241 -EAGEELDLRLRLAQPSSPEVLTPQLSPGSGTPQAGTPSPSRSRDSNSGPEEPLLEEEEK 241 -----------------------------------------------------------: reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C RRAPQITTPVKITLTEIEPSVETTTQEKTPEKQDICLKPSPDTVPTVTVSEPEDDSPGSI -----------------------------------------------------------ELPTALTKLVKEDEVVSSEKAKDSFNEKRVAVEAPMREEYADFKPFERVWEVKDSKEDSD ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------QWGPLEREPVRGQCLDSTDQLEFTVEPRLLGTAMEWLKTSLLLAVYKTVPILELSPPLWT QWGPLEREPVRGQCLDSTDQLEFTVEPRLL----------------------------------------------------------------------------------------: : reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C TPPSSGTEPSAAESQGKGSISEDELITAIKEAKGLS------------------------ 367 -----------------------------------------------------------MLAAGGKIESNLESKVDKKCFADSLEQTNHEKDSESSNDDTSFPSTPEGIKDRSGAYITC 464 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AIGWVQRGPTPPTPVLRVLLKWAKSPRSSGVPSLSL------------------------ 337 ----------------------------------------------------------------------------------------------------------------------- reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C ----------------------------------------------------------------------------------------------------------------------APFNPAATESIATNIFPLLGDPTSENKTDEKKIEEKKAQIVTEKNTSTKTSNPFLVAAQD 524 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C ----------------------------------------------------------------------------------------------------------------------SETDYVTTDNLTKVTEEVVANMPEGLTPDLVQEACESELNEVTGTKIAYETKMDLVQTSE 584 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C ----------------YETAENPRPVGQLADRPEVKARSGPPTIPSPLDHEASSAESGDS 411 -----------------------------------------------------------VMQESLYPAAQLCPSFEESEATPSPVLPDIVMEAPLNSAVPSAGASVIQPSSSPLEASSV 644 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: : reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C EIELVSEDPMAAEDALPSGYVSFGHVGGPPPSPASPSIQYSILREEREAELD-------- 463 -----------------------------------------------------------NYESIKHEPENPPPYEEAMSVSLKKVSGIKEEIKEPENINAALQETEAPYISIACDLIKE 704 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: : : : : : 331 404 301 271 171 reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C --------------SELIIESCDASSASEESPKREQDSPPMKPSALDAIREETGVRAEER 509 ------------------------MQATADSTK------------MDCVWSN-------- 16 TKLSAEPAPDFSDYSEMAKVEQPVPDHSELVEDSSPDSEPVDLFSDDSIPDVPQKQDETV 764 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: : : reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C APSRRGLAEPGSFLDYPSTEPQPGPELPPGDGALEPETPMLPRKPEEDS----------- 558 -----------------------------------------------------------MLVKESLTETSFESMIEYENKEKLSALPPEGGKPYLESFKLSLDNTKDTLLPDEVSTLSK 824 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: : : reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C ----------------------------------------------------------------------------------------------------------------------KEKIPLQMEELSTAVYSNDDLFISKEAQIRETETFSDSSPIEIIDEFPTLISSKTDSFSK 884 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C ----------------------------------------------------------------------------------------------------------------------LAREYTDLEVSHKSEIANAPDGAGSLPCTELPHDLSLKNIQPKVEEKISFSDDFSKNGSA 944 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C ------------------------------SSNQSPAATKGPGPLGPGAPPPLLFLNKQK --------------------------------------------------------WKSQ TSKVLLLPPDVSALATQAEIESIVKPKVLVKEAEKKLPSDTEKEDRSPSAIFSAELSKTS ------------------------------------------------------------------------------------------------------------MDGQKKNWKDK -----------------------------------------------------------A -----------------------------------------------------GADMGSK -------------------------------------------------------------------------------------------------------------------MGSK 588 20 1004 reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C AIDLLYWRDIKQTGIVFGSFLLLLFSLTQFSVVSVVAYLALAALSATISFRIYKSVLQAV AIDLLYWRDIKQTGIVFGSFLLLLFSLTQFSVVSVVAYLALAALSATISFRIYKSVLQAV VVDLLYWRDIKKTGVVFGASLFLLLSLTVFSIVSVTAYIALALLSVTISFRIYKGVIQAI VVDLLYWRDIKKTGVVFGASLFLLLSLTVFSIVSVTAYIALALLSVTISFRIYKGVIQAI VVDLLYWRDIKKTGVVFGASLFLLLSLTVFSIVSVTAYIALALLSVTISFRIYKGVIQAI VHDLIFWRDVKKTGFVFGTTLIMLLSLAAFSVISVVSYLILALLSVTISFRIYKSVIQAV VADLLYWKDTRTSGVVFTGLMVSLLCLLHFSIVSVAAHLALLLLCGTISLRVYRKVLQAV VADLLYWKDTRTSGVVFTGLMVSLLCLLHFSIVSVAAHLALLLLCGTISLRVYRKVLQAV VADLLYWKDTRTSGVVFTGLMVSLLCLLHFSIVSVAAHLALLLLCGTISLRVYRKVLQAV . **::*:* : :*.** :. *:.* **::**.::: * *. ***:*:*: *:**: 648 80 1064 245 71 107 404 331 64 reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C QKTDEGHPFKAYLELEITLSQEQIQKYTDCLQFYVNSTLKELRRLFLVQDLVDSLKFAVL QKTDEGHPFKAYLELEITLSQEQIQKYTDCLQFYVNSTLKELRRLFLVQDLVDSLKFAVL QKSDEGHPFRAYLESEVAISEELVQKYSNSALGHVNCTIKELRRLFLVDDLVDSLKFAVL QKSDEGHPFRAYLESEVAISEELVQKYSNSALGHVNCTIKELRRLFLVDDLVDSLKFAVL QKSDEGHPFRAYLESEVAISEELVQKYSNSALGHVNCTIKELRRLFLVDDLVDSLKFAVL QKSEEGHPFKAYLDVDITLSSEAFHNYMNAAMVHINRALKLIIRLFLVEDLVDSLKLAVF HRGDGANPFQAYLDVDLTLTREQTERLSHQITSRVVSAATQLRHFFLVEDLVDSLKLALL HRGDGANPFQAYLDVDLTLTREQTERLSHQITSRVVSAATQLRHFFLVEDLVDSLKLALL HRGDGANPFQAYLDVDLTLTREQTERLSHQITSRVVSAATQLRHFFLVEDLVDSLKLALL :: : .:**:***: ::::: * . : : . : ::***:*******:*:: 708 140 1124 305 131 167 464 391 124 11 47 344 172 reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C MWLLTYVGALFNGLTLLLMAVVSMFTLPVVYVKHQAQIDQYLGLVRTHINAVVAKIQAKI MWLLTYVGALFNGLTLLLMAVVSMFTLPVVYVKHQAQIDQYLGLVRTHINAVVAKIQAKI MWVFTYVGALFNGLTLLILALISLFSVPVIYERHQAQIDHYLGLANKNVKDAMAKIQAKI MWVFTYVGALFNGLTLLILALISLFSVPVIYERHQAQIDHYLGLANKNVKDAMAKIQAKI MWVFTYVGALFNGLTLLILALISLFSVPVIYERHQAQIDHYLGLANKNVKDAMAKIQAKI MWLMTYVGAVFNGITLLILAELLIFSVPIVYEKYKTQIDHYVGIARDQTKSIVEKIQAKL FYILTFVGAIFNGLTLLILGVIGLFTIPLLYRQHQAQIDQYVGLVTNQLSHIKAKIRAKI FYILTFVGAIFNGLTLLILGVIGLFTIPLLYRQHQAQIDQYVGLVTNQLSHIKAKIRAKI FYILTFVGAIFNGLTLLILGVIGLFTIPLLYRQHQAQIDQYVGLVTNQLSHIKAKIRAKI ::::*:***:***:***::. : :*::*::* ::::***:*:*:. : . **:**: reticulon1A reticulon1C reticulon4A reticulon4B reticulon4C reticulon3a reticulon2A reticulon2B reticulon2C PG-------------AKRHAE PG-------------AKRHAE PG-------------LKRKAE PG-------------LKRKAE PG-------------LKRKAE PGI------------AKKKAE PGTGALASAAAAVSGSKAKAE PGTGALASAAAAVSGSKAKAE PGTGALASAAAAVSGSKAKAE 768 200 1184 365 191 227 524 451 184 776 208 1192 373 199 236 545 472 205 173 PUBLICATIONS 1. Li, M., Shi, J., Wei, Z., Teng, F.Y., Tang, B.L., and Song, J. 2004. Structural characterization of the human Nogo-A functional domains: solution structure of Nogo40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration. Eur. J. Biochem. 271: 3512–3522. 2. Li, M., J. Liu, and J. Song. 2006. Nogo goes in the pure water: solution structure of Nogo-60 and design of the structured and buffer-soluble Nogo-54 for enhancing CNS regeneration Protein Sci. 15(8):1835-41. 3. Liu J*, Li M*, Ran X, Fan JS, Song J. 2006. Structural insight into the binding diversity between the human Nck2 SH3 domains and proline-rich proteins. Biochemistry. 45(23):7171-84 * co-first authored 4. Li M, Liu J, Ran X, Fang M, Shi J, Qin H, Goh JM, Song J.2006 Rescuing Abandoned Proteins with Water: CD and NMR Studies of Protein Fragments Solubilized in Salt-Free Water. Biophys J. 91(11):4201-9. 5. Li M, Song J, 2006 The N- and C-termini of the Human Nogo Molecules are Intrinsically Unstructured: Bioinformatics, CD, NMR Characterization and its Functional Implications. Proteins: structure, function and bioinformatics. (Accepted) 6. Li M, Song J 2007, Nogo-B Receptor Possesses an Intrinsically-unstructured Ectodomain and a Partially-folded Cytoplasmic Domain. Biochemical and Biophysical Research Communications 2007 Aug 17;360(1):128-34. 7. Li M, Song J 2007, Structural and function perspectives of RTN4/Nogo proteins: implications for their functions and molecule design of therapeutic application. CURRENT PROTEIN AND PEPTIDE SCIENCE, (invited review, under preparation) 174 [...]... 3.1.5 NMR characterization of Nogo- 24 64 Figure 3.1.6 CD and NMR characterization of Nogo- 40 65 Figure 3.1.7 NMR spectral assignment of Nogo- 40 68 Figure 3.1.8 secondary structures of Nogo- 24 and Nogo- 40 69 Figure 3.1.9 Solution structure of Nogo- 40 72 Figure 3.2.1 CD and NMR characterization of Nogo peptides 79 Figure 3.2.2 The NOE patterns critical for defining the secondary structure of Nogo- 60 in... 3.3.4 CD and NMR characterization of the Nogo- B N-terminus 99 Figure 3.3.5 CD and NMR characterization of the Nogo- 33 101 Figure 3.3.6 CD and NMR characterization of the first group of the dissected Nogo- A Fragments 103 Figure 3.3.7 CD and NMR characterization of the second group of the dissected Nogo- A fragments 104 Figure 3.3.8 CD and NMR characterization of the mixture of nine dissected Nogo- A fragments... structure of Nogo- 60 84 Figure 3.2.4 15N NMR backbone dynamics 86 Figure 3.2.5 Solution structure of Nogo- 54 87 XI Figure 3.2.6 Neurite outgrowth assay of Nogo- 54 90 Figure 3.3.1 The domain organization and dissection of the human Nogo proteins 94 Figure 3.3.2 Analysis of globularity and disordered regions of Nogo proteins 95 Figure 3.3.3 Functional sites prediction of Nogo- B by ELM 96 Figure 3.3.4 CD and. .. Conformational properties of Nogo- 54 125 Figure3.5.3 CD and HSQC characterization of extracellular domains of transmembrane receptors 128 Figure 3.6.1 Domain organization of NgBR and its expression and purification 137 Figure 3.6.2 Bioinformatics analysis of NgBR 139 Figure 3.6.3 CD and NMR characterization of the NgBR ectodomain 140 Figure 3.6.4 CD and NMR characterization of the full-length NgBR and its cytoplamic... structures of Nogo- 40 71 Table 3.2.1 Chemical shifts of Nogo- 60 in a 90/10 % (H2O/D2O) 81 Table 3.2.2 NMR constraints and structural statistics for the 20 accepted CYANA structures of Nogo- 60 83 Table3.2.3 NMR constraints and structural statistics for the 20 accepted CYANA structures of Nogo- 54 88 Table3.4.1 Binding interaction between the human Nogo- A fragments and hNck2 SH3 domain monitored by NMR and ITC... 1.5 Strategy of structure determination by NMR 37 Figure 1.6 Outline of structure calculation by NMR 39 Figure 3.1.1 Schematic representation of the domain organization of the human Nogo- A protein 56 Figure 3.1.2 Expression and purification of Nogo- A-(567-748) and Nogo- 66 57 Figure 3.1.3 CD and NMR characterization of NogoA-(567-748) 59 Figure 3.1.4 Secondary structural prediction of Nogo- 40 by DNAMAN... pathogenesis of human disease, especially the inhibition effect in CNS neuron regeneration RTN4 /Nogo (on human chromosomal 2p14-p13) gene generate three different isoforms, termed Nogo- A, -B and –C, by alternative promoter usage and /or splicing Nogo- A, -B and –C proteins and mRNAs are expressed ubiquitously, although there is some tissue specificity For example, NogoA is mainly expressed in brain and heart; NogoB... 3.4.1 ITC characterization of the binding interaction between the third hNck2 SH3 domain and Nogo- A fragments 114 Figure 3.4.2 NMR structures of the third hNck2 SH3 domain 117 Figure 3.4.3 The interaction between the third hNck2 SH3 domain and the prolinerich motifs located at the N-terminus of Nogo- A 119 Figure 3.5.1 CD and HSQC characterization of Nogo- 66 fragments 124 Figure 3.5.2 Conformational... majority of the Nogo proteins are localized to ER, with a small amount on the cell surface (Oertle et al., 2003) The Nterminal regions of RTN4/Nogos are highly variable (Figure 1.1a) The 1192-residue Nogo- A is the largest isoform with an ~1016-residue N-terminus while the 373residue Nogo- B owns a N-terminus almost identical to the first 200 residues of NogoA The smallest variant Nogo- C consisting of 199... explanation for this divergence and this divergence has evidenced the need of further investigation about Nogos on axonal regeneration failure 8 (a) Nogo- A-specific region Nogo- 66 C RTN4-A/NogoA T M N N T M T M N T M C RTN4-B/NogoB T M T M C RTN4-C/NogoC (b) Cytopl Extracell/ER lumen Cytopl Extracell/ER lumen Figure 1.1 Three transcripts from the RTN4 and membrane topologies and subcellular localization of Nogo/ RTN4 . Structural Characterization of Inhibitory Domains of Nogo 54 3.1.1. Expression and structural characterization of NogoA-(567-748) and Nogo- 66 55 3.1.2. CD and NMR characterization of. Structural Characterization of Nogo- B and Systematic Dissection of the Am -Nogo- A 92 3.3.1. Bioinformatics analysis 93 3.3.2. CD and NMR characterization of N- and C-termini of Nogo- B 97. STRUCTURAL CHARACTERIZATION OF NOGO PROTEINS: IMPLICATIONS FOR BIOLOGICAL FUNCTIONS AND MOLECULE DESIGN OF THERAPEUTIC APPLICATION