IN SILICO STUDY OF LEGUME AND LEGUME-TYPE LECTINS ___________________________ A Dissertation Presented to The Faculty of the Graduate School At the University of Missouri-Columbia _______________________________________________________ In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy _____________________________________________________ By CUONG THE NGUYEN Dr. Gary Stacey, PhD Dissertation Supervisor NOVEMBER 2016 The undersigned, appointed by the Dean of the Graduate School, have examined the Dissertation entitled: IN SILICO STUDY OF LEGUME AND LEGUME-TYPE LECTINS Presented by CUONG THE NGUYEN A candidate for the degree of Doctor of Philosophy of Plant Breeding, Genetics, and Genomics And hereby certify that, in their opinion, it is worthy of acceptance. ______________________________________________________________ Professor Gary Stacey ______________________________________________________________ Professor Dong Xu ______________________________________________________________ Professor Melissa Goellner Mitchum ______________________________________________________________ Professor James Schoelz ACKNOWLEDGEMENTS My special thanks to my advisor, Dr. Gary Stacey, for giving me the privilege and a great opportunity to work in his lab under his whole-hearted guidance during my Ph.D. program. My sincere thanks to the Advisory Committee members, Dr. Dong Xu, Dr. Melissa G. Mitchum, and Dr. James Schoelz, who gave me valuable, constructive advice and directions for this project. My appreciation also goes to Dr. Jianlin Cheng, who provided me opportunity to work in his lab in the first two years. I would like to extend my deepest gratitude to Dr. Jerry Nelson, who first introduced the University of Missouri (MU) to me and then helped me to settle in Columbia during my first days in the United States. Thanks to his dedicated help and strong support, my time as a graduate student at the University has been a very memorable and productive experience. I would like to give my thanks to Dr. Kiwamu Tanaka, Dr. Bing Stacey, other members and colleagues in the laboratories of Dr. Gary Stacey, Dr. Dong Xu, and Dr. Jianlin Cheng for their time and help on my projects; and for the joyful and happy moments they have shared with me. I am grateful to all of my friends; especially those in Columbia, Missouri and in my hometown in Cantho, Vietnam for their continuous support. i I must express my heartfelt gratitude to my parents, parents-in-law, my wife, and two wonderful daughters who are always with me in hard times and during happy moments. Without their dedicated love, encouragement, patience and constant support, I would not be able to achieve one of my major goals in life today. I would like to thank the Vietnamese International Education Development, the US Department of Energy, the US National Institute of Health, and the Systems and Synthetic Agrobiotech Center of Korea for their funding support. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ________________________________________________________ i TABLE OF CONTENTS _________________________________________________________ iii LIST OF TABLES _____________________________________________________________ vi LIST OF FIGURES ___________________________________________________________ vii ABSTRACT _________________________________________________________________ ix Chapter 1 - GENERAL INTRODUCTION _______________________________________ 1 I. Research rationale: ________________________________________________________ 1 II. Literature review: _________________________________________________________ 3 1. Lectin super family: _______________________________________________________________ 3 2. L-type lectin proteins (LLPs): _______________________________________________________ 7 3. L-type lectin receptor-like kinases (LLRKs): ___________________________________________ 14 4. Animal legume-like lectins: _______________________________________________________ 18 5. Genome-scale analysis, expansion mechanisms and molecular evolution of LLDPs ___________ 20 III. Outline of the dissertation ________________________________________________ 23 Chapter 2 - ORIGIN, DISTRIBUTION, DIVERSIFICATION AND EVOLUTION OF PROTEINS CONTAINING THE L-TYPE LECTIN DOMAIN __________________________________ 25 Abstract __________________________________________________________________ 25 I. Introduction _____________________________________________________________ 26 II. Materials and methods ___________________________________________________ 28 1. Sequence databases used for identification of LLDPs ___________________________________ 28 2. Computational tools used for identification of LLDPs ___________________________________ 29 3. Computational approaches to identify members of the LLDP family _______________________ 30 4. Folding recognition and template search ____________________________________________ 32 5. Analysis of PFAM domains in association with L-type lectin domain of LLDPs _______________ 32 6. Linear structural analysis of plant LLDP genes _________________________________________ 33 7. LLDP phylogenetic tree reconstruction ______________________________________________ 33 8. Analysis of expansion mechanisms of LLDPs in soybean genome _________________________ 35 10. Analysis of LLDP gene expression patterns in soybean _________________________________ 37 11. Gene ontology (GO) term enrichment analysis for soybean LLDPs _______________________ 37 III. Results ________________________________________________________________ 38 1. Global searching for LLDPs ________________________________________________________ 38 2. Distribution of LLDPs in bacteria ___________________________________________________ 39 3. Distribution of the LLDP family in eukaryotes _________________________________________ 41 4. Genome-wide distribution of LLDPs in the plant kingdom _______________________________ 43 5. Fold topology of bacterial and lower eukaryote LLDs ___________________________________ 47 iii 6. Domain architectures of the LLDP family_____________________________________________ 48 7. Exon and intron structure of LLDP family members ____________________________________ 51 8. Phylogenetic analysis of the LLDP family _____________________________________________ 55 9. Duplication and expansion of the LLDP gene family in the soybean genome ________________ 61 10. Distribution of soybean LLDP genes on the chromosomes ______________________________ 64 12. The nature of selection pressure acting on soybean LLDP coding genes ___________________ 66 13. Expression profiles of soybean LLDP genes in different tissues and development stages _____ 68 14. Gene ontology (GO) enrichment of soybean LLDPs and functional implication _____________ 73 IV. Discussion _____________________________________________________________ 75 1. Occurrence and origin of LLDPs ____________________________________________________ 76 2. Domain diversification of LLDPs across the kingdoms __________________________________ 78 3. Gene exon and intron structure and divergence of plant LLDPs __________________________ 80 4. Expansion of the plant and soybean LLDP family ______________________________________ 82 5. Purifying selection of the soybean LLDP family ________________________________________ 84 6. Molecular evolution of the LLDPs __________________________________________________ 85 7. Involvement of LLDP genes in soybean growth and development _________________________ 88 8. Functional implication of the LLDP family members ____________________________________ 88 V. Concluding remarks ______________________________________________________ 90 VI. Supplementary figures ___________________________________________________ 91 VII. Supplementary tables ___________________________________________________ 96 Chapter 3 - COMPUTATIONAL ANALYSIS OF THE LIGAND BINDING SITE OF THE EXTRACELLULAR ATP RECEPTOR, DORN1 __________________________________ 104 Abstract _________________________________________________________________ 104 I. Introduction ____________________________________________________________ 105 II. Materials and Methods __________________________________________________ 108 1. In silico design for three dimensional (3D) modeling, binding site prediction and ligand docking of eATP to the DORN1 L-type lectin domain ___________________________________________ 108 2. Folding recognition and template search ___________________________________________ 109 3. Alignment, homology modeling and quality assessment of DORN1 models ________________ 110 4. Molecular dynamic simulation ____________________________________________________ 111 5. Identification of ligand binding sites _______________________________________________ 112 6. In vitro ATP binding assay for site-directed mutagenesis _______________________________ 113 7. Template redocking experiments _________________________________________________ 116 8. Target docking experiments ______________________________________________________ 116 III. Results _______________________________________________________________ 117 1. Fold topology, template search and sequence-structure alignment ______________________ 117 2. Homology model of the DORN1 lectin domain _______________________________________ 121 3. Prediction of ATP binding sites on the DORN1 lectin domain ___________________________ 123 4. Site-directed mutagenesis studies for the ATP binding site _____________________________ 124 5. High-resolution, template and target docking _______________________________________