University of Bath PHD A modelling study of the ligand-gated ion-channel superfamily of receptors Cockcroft, Victor Barrie Award date: 1992 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 08. Oct. 2021 A MODELLING STUDY OF THE LIGAND-GATED ION-CHANNEL SUPERFAMILY OF RECEPTORS. submitted by Victor Barrie Cockcroft for the degree of Ph.D. at the University of Bath 1992 Copyright Attention is drawn to the fact that the copyright of this thesis rests with its author. This copy of the thesis has been supplied on the condition that anyone who con­ sults it is understood to recognize that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published with­ out the prior written consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. (V. B. Cockcroft) UMI Number: U601787 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U601787 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 UNIVERSITY OF 3 a TH wrM&mr 6 6 1 5 APR 2010 Php For Connie Cockcroft. -3- Acknowledgments David Osguthorpe and George Lunt were enlightening at every level from the practical through to the imaginative. It has been enjoyable to work for them in their laboratories. Everyone in the Molecular Graphics Unit and the Biochemistry and Chemistry Departments has been friendly, making for a pleasant work environment. Some deserve special mention. They are Alison Drasdo, Liz Sanderson and Paul Burney. Apart from sound advice throughout, I am in gratitude to Dr. Pnina Dauber- Osguthorpe for tuition in a range of modelling techniques. Her help was invaluable. I am grateful to Drs. Sue Wonnacott, Stuart Reynolds, and Prem Paul for their useful discussions, and patience. I thank Dr. Adrian Friday of the Zoology Department, Cambridge University for his friendship as well as his help in generating evolutionary trees, and Jan Peder­ son for guiding me round the pitfalls of programming in C. Finally, I am most grateful to Shell Research for funding and Drs. Mike Goosey, Mike King, and Phil Jewess, at Shell Research for their keen interest and enthusi­ asm. A MODELLING STUDY OF THE LIGAND-GATED ION-CHANNEL SUPERFAMILY OF RECEPTORS ABSTRACT In this study an attempt has been made to incorporate the findings of a large number of molecular studies into a coherent view using molecular modelling to gen­ erate testable models as a basis for experimentation. Two computer programs were developed. BIOSITE provides for the interactive, comparative analysis of aligned homologous protein sequences. SCAFFOLD is a program for scanning the known protein structural database for "non-homologous similarity" based on relative residue surface-accessibility patterns of proteins. A component of the agonist/competitive antagonist binding site of the ligand- gated ion-channel (LGIC) receptors was identified as a conserved 15 residue stretch of primary structure in the N-terminal extracellular region of subunits. This subregion termed the cys-loop was modelled as an amphiphilic p-hairpin and it is proposed that it is a major determinant of the agonist binding cleft. In the model, the positive charge of agonist binds to an invariant aspartate residue at position 11, whereas recognition of a specific neurotransmitter is partly a consequence of the residue occurring at position 6. This initial, partial binding site model was extended in the case of the nicotinic acetylcholine receptor to include residues shown by experiment to be spatially adjacent to the binding cleft. -6- A model of a whole receptor oligomer was constructed using the four-heiix bundle protein myohaemerythrin as a template for the transmembrane domain of individual subunits, and the enzyme pyrophosphatase as a possible template fold for the N-terminal extracellular domain. Evolutionary analysis was performed on the LGIC nucleic acid sequences. At the molecular level, the tree showed the specialization of the cation and anion selective ion-channels, formation of distinct receptor types, and hetero-oligomeriza­ tion of receptors. Branch points were also obtained for the segregation of muscle and neuronal tissues, and CNS and ganglionic neuronal lineages. -7- Publlcatlons of the Work: 1. Modelling of Agonist Binding to the Ligand-Gated Ion-Channel Superfam- lly of Receptors. V. B. Cockcroft, D. J. Osguthorpe, E. A. Barnard, G. G. Lunt, (1990) Proteins: Struct. Func. and Genet. 8, 386-397. 2. Modelling of Binding Sites of the Nicotinic Acetylcholine Receptor and Their Relationship to Models of the Whole Receptor. V. B. Cockcroft, G. G. Lunt, D. J. Osguthorpe, (1990) In "Protein Structure: Prediction and Engineering" Kay, J., Lunt, G. G., Osguthorpe, D. J., eds. Biochemical Society Symposium No. 57, London: Portland Press, p65-79. 3. Methyllycaconitlne: A Selective Probe for Neuronal a-Bungarotoxin Bind­ ing Sites. J. M. Ward, V. B. Cockcroft, G. G. Lunt, F. S. Smiliie, S. Wonnacott, (1990) FEBS Lett. 270, 45-48. 4. Relative Residue Surface-Accesslbillty Patterns Reveal Myoglobin and Catalase Similarity. V. B. Cockcroft, D. J. Osguthorpe, (1991) FEBS Lett. 293, 149-152. 5. BIOSITE: An Interactive Program for the Comparison of Aligned Homolo­ gous Protein Sequences. V. B. Cockcroft, J.T. Pederson, G. G. Lunt, D. J. Osguthorpe, (1992) CABIOS 8, 71-73. 6. Ligand-Gated lon-Channels: Homology and Diversity. V. B. Cockcroft, D. J. Osguthorpe, E. A. Barnard, A. F. Friday, G. G. Lunt, (1992) Molecular Neurobiol­ ogy 4, 129-169. -8- 7. Modelling the Nicotinic Acetylcholine Receptor. V. B. Cockcroft, G. G. Lunt, D. J. Osguthorpe, (1992) In "The Biology of Nicotine: Current Research Issues" Lippiello, P. M., Collins, A. C., Gray, J. A., Robinson, J. H., eds New York: Raven Press, p1 -12. Poster Communications: 1. A Model of the Interactions at the Agonist Binding Site of Ligand-Gated Receptor lon-Channels. V. B. Cockcroft, G. G. Lunt, E. A. Barnard, D. J. Osguthorpe, (1988) In the Society for Neurosci. 18th Annual Meeting, Toronto, Canada. 2. Structural Models of Cloned Ligand-Gated lon-Channels. V. B. Cockcroft, D. J. Osguthorpe, A. F. Friday, E. A. Barnard, G. G. Lunt, (1989) Dupont Special Poster Session, Society for Neuroscience 19th Annual Meeting, Phoenix, USA. -9- A CURRENT VIEW OF BIOLOGICAL SCIENCE "The new paradigm, now emerging, is that all the ’genes’ will be known, and that the starting point will be theoretical ... the reagents that the scientist uses will include a knowledge of the primary sequence of the organism, together with a list of all previous deductions from that sequence." Waiter Gilbert, 1991 -10- This poem was written by John Godfrey Saxe (1816-1887). It was six men from Indostan To learning much inclined, Who went to see the Elephant, (Though all of them were blind), That each by observation Might satisfy his mind. The First approached the Elephant, And happening to fall Against his broad and sturdy side, At once began to bawl: "God bless me I but the Elephant, Is very like a wall!" The Second feeling at the tusk, Cried, "Ho? what have we here So very round and smooth and sharp? To me ’tis mighty clear This wonder of an Elephant Is very like a spearl" The Third approached the animal, And happening to take The squirming trunk within his hands, Thus boldly up and spake: "I see" quoth he, "the Elephant Is very like a snake." The Fourth reached out an eager hand, -11 - And felt about the knee. "What most this wondrous beast Is like Is mighty plain," quoth he; "Tis clear enough the Elephant Is very like a tree." The Fifth who chanced to touch the ear, Said: "E’en the blindest man Can tell what this resembles most; Deny the fact who can, This marvel of an Elephant Is very like a fan I" The Sixth no sooner began About the beast to grope, Then, seizing on the swinging tail That fell within his scope, "I seel" quoth he, "the Elephant is very like a rope." And so these men of Indostan Disputed loud and long. Each in his own opinion Exceeding stiff and strong, Though each was partly in the right, And all were in the wrong I -12- CONTENTS: Acknowledgm ents ... 3 A b s tra c t..