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A Unique Structural and Dynamic Feature of Aspartic Protease Investigating plasmepsin flexibility as a function of the flap region - a unique structural and dynamic feature of aspartic protease. Lara McGillewie 214585796 A thesis submitted to the College of Health Sciences, University of KwaZulu-Natal, Westville, in fulfillment of the requirements of the degree of Doctor of Philosophy. Supervisor Professor Mahmoud Soliman KwaZulu-Natal 2015 A Investigating plasmepsin flexibility as a function of the flap region - a unique structural and dynamic feature of aspartic protease. Lara McGillewie 214585796 2015 A thesis submitted to the School of Pharmacy and Pharmacology, Faculty of Health Science, University of KwaZulu-Natal, Westville Campus, for the Doctorate of Medical Sciences (Pharmaceutical Chemistry). This is a thesis in which the chapters are written as a set of discrete research publications, with an overall introduction and final summary. This is to certify that the contents of this thesis are the original research work of Miss Lara McGillewie. As the candidate’s supervisor, I have approved this thesis for submission. Supervisor: Signed: ________________ Name: Dr. Mahmoud E. Soliman Date: ________________________ B Abstract Malaria is one of the most deadly infectious protozoan diseases known to man. It is spread by the Plasmodium parasite through the bite of the female Anopheles mosquito. Increasing resistance to currently available antimalarial drugs is a growing concern. Plasmepsins are malarial aspartic proteases, due to their characteristic mechanism of action, the fact that they are found in all Plasmodium species and are essential to parasitic survival they represent novel targets in the design of antimalarials. A unique structural feature of aspartic proteases and plasmepsins is the flap region lying perpendicular to the catalytic aspartic acid active, partially covering the active site. The flap region plays an important structural (and kinetic) role in regulating access to the active site, thereby regulating ligand binding. The present study focused on the flap dynamics of Plm I – V, proposing and validating parameters to accurately quantify the dynamic behaviour of the flap region. The catalytic aspartic acids is highly conserved in the plasmepsin family; sequence analysis revealed that although all plasmepsins are similar in structure, they differ greatly in the residues in the flap region. The heterogeneity in this region gives each plasmepsin unique substrate specificity and response to inhibitors. The parameters proposed in the present study gives a detailed account for the twisting of the flaps which move away from the active site in the absence of an inhibitor. Upon inhibitor binding, residues in the flap region form hydrogen bonds with the inhibitor pulling it inward towards the active site rendering the enzyme inactive. The parameters proposed in the present study will be of great value in the design of novel plasmepsin inhibitors, with increased efficacy and potency. i DECLARATION 1 – PLAGIARISM I, Lara McGillewie, declare that 1. The research reported in this thesis, except where otherwise indicated, is my original work. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written, but the general information attributed to them has been referenced. b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the references section. A detail contribution to publications that form part and/or include research presented in this thesis is stated (include publications submitted, accepted, in press and published). Signed: _________________ ii DECLARATION 2 - LIST OF PUBLICATIONS 1. Wilson Karubui, Soumendranath Bhakat, Lara McGillewie and Mahmoud E Soliman (2015). Flap dynamics of plasmepsin proteases: insight into proposed parameters and molecular dynamics, Molecular Biosystems (Published). Contributions: Wilson Karubui and Soumendranath Bhakat: Authors contributed to the project by performing all literature reviews, experimental work and data analysis, interpretation of the results as well as manuscript preparation and writing. Lara McGillewie: Literature review, technical support and editing of the manuscript. Mahmoud E. Soliman: Supervisor. 2. Lara McGillewie and Mahmoud E Soliman (2015). Flap flexibility amongst plasmepsins I, II, III, IV, and V: Sequence, structural, and molecular dynamics analyses, Proteins (Published). Contributions: Lara McGillewie: Author contributed to the project by performing all literature reviews, experimental work, and data analysis, interpretation of the results as well as manuscript preparation and writing. Mahmoud E. Soliman: Supervisor. iii 3. Lara McGillewie and Mahmoud E Soliman (2015). The binding landscape of plasmepsin V and the implications on flap dynamics. (Submitted). Contributions: Lara McGillewie: Author contributed to the project by performing all literature reviews, experimental work, and data analysis, interpretation of the results as well as manuscript preparation and writing. Mahmoud E. Soliman: Supervisor. 4. Lara McGillewie and Mahmoud E Soliman (2015). Flap dynamics as unique conformational features among aspartic proteases. (Submitted). Contributions: Lara McGillewie: Author contributed to the project by performing all literature reviews, experimental work, and data analysis, interpretation of the results as well as manuscript preparation and writing. Mahmoud E. Soliman: Supervisor. iv RESEARCH OUTPUTS A. PUBLICATIONS i. Karubui W, Bhakat S, McGillewie L and Soliman M.E (2015). Flap dynamics of plasmepsin proteases: insight into proposed parameters and molecular dynamics. Molecular Biosystems (Published). ii. McGillewie L and Soliman M.E (2015). Flap flexibility amongst plasmepsins I, II, III, IV, and V: Sequence, structural, and molecular dynamics analyses. Proteins (Published). iii. McGillewie L and Soliman M.E (2015). The binding landscape of plasmepsin V and the implications on flap dynamics (Submitted). iv. McGillewie L and Soliman M.E (2015). Flap dynamics as unique conformational features among aspartic proteases. (Submitted). B. CONFERENCES i. Oral presentation: ‘Flap flexibility amongst plasmepsins I, II, III, IV, and V: Sequence, structural, and molecular dynamics analyses.’ UKZN College of Health Sciences Symposium September 2015 (awarded 3rd prize). v ACKNOWLEDGEMENTS First and foremost, I would like to express my sincerest gratitude and appreciation to my supervisor, Prof Soliman, whose guidance, expertise and understanding has mentored me throughout my thesis. His motivation, support and encouragement has made all this possible, thank you Prof. I would also like to thank the members and friends in the Molecular Modelling and Drug Design research group for all the laughs, all the support and encouragement. To the Centre of High Performance Computing in Cape Town, as well as the San Diego Computer Centre for computational resources. And to the School of Health Sciences, UKZN Westville Campus. To the National Research Foundation (NRF) who funded me through my thesis. And lastly to my family, to my mom and dad, Helena and David, you have made this dream a reality. Your unwavering love and support knows no limits or boundaries. Thank you for being the most incredible parents, I am eternally grateful. To my brother, Dane, for always believing in me no matter the odds. And last but not least to my Daniel, thank you for walking this journey with me, for always encouraging and supporting me. Love you all more than words could ever express. vi TABLE OF FIGURES Chapter 1 Figure 1. Structures of the antimalarial drugs used to treat the asexual blood stage of the Plasmodium lifecycle. ------------------------------------------------------------------------------------------------------------------- 3 Figure 2. Current antimalarials and their intracellular targets and pathways they disrupt, and potential antimalarial targets . ----------------------------------------------------------------------------------------------------- 4 Chapter 2 Figure 1. World map representative of the regions with ongoing malarial transmission in 2013 and the severity of each. --------------------------------------------------------------------------------------------------------- 12 Figure 2. Malaria parasite in both the mosquito vector (sexual phase) and mammalian (human) host (asexual) 25. --------------------------------------------------------------------------------------------------------------- 15 Figure 3. Hemoglobin degradation in the host RBC through numerous parasitic proteases37. ---------- 16 Figure 4. The general acid-base mechanism of proteolytic hydrolysis of aspartic proteases via the formation of the tetrahedral intermediate 45. ----------------------------------------------------------------------- 17 Chapter 3 Figure 1. A two-dimensional model of the potential energy surface (PES) 4. ------------------------------ 27 Chapter 4 Figure 1. An illustration of the different
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