HYPER-PLASTIC STRUCTURAL EVOLUTION OF THE PEX DOMAIN - A MODEL OF EVOLUTIONARY EXAPTATION AND NEOFUNCTIONALIZATION AT THE MOLECULAR LEVEL A DISSERTATION IN Molecular Biology and Biochemistry and Cell Biology and Biophysics Presented to the Faculty of the University of Missouri-Kansas City in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY by LEE LIKINS B.S., University of West Florida, Pensacola, FL 1985 M.A., University of Kansas, Lawrence, KS 1996 Kansas City, Missouri 2017 © 2017 LEE LIKINS ALL RIGHTS RESERVED HYPER-PLASTIC STRUCTURAL EVOLUTION OF THE PEX DOMAIN - A MODEL OF EVOLUTIONARY EXAPTATION AND NEOFUNCTIONALIZATION AT THE MOLECULAR LEVEL Lee Likins, Candidate for the Doctor of Philosophy Degree University of Missouri – Kansas City, 2017 ABSTRACT With the advent of sophisticated genetic, biophysical and in silico technology an enormous amount of information is being generated regarding the structural, biochemical and physiological aspects of proteins. Tertiary protein structural domains are assumed to be features of proteins whose bio-historical relationships can be traceable over relevant evolutionary space. The phylogenetics of protein domains, including their genesis, duplication, combination, selectively derived loss and potential horizontal capture to derive novel functional rearrangements, are of great interest to molecular evolutionists. Evolutionary and bioinformatic analyses of a considerable collection of variable protein primary, secondary, tertiary, quaternary and biochemical structures has established the principle that, from a functional perspective, many, if not most, proteins are evolutionarily dependent on the functional capacity of readily defined and identifiable, globular components defined as "domains". Therefore, it is reasonable to infer that specific secondary and tertiary folds, or arrangements, represent functional phenotypic characters that can be analyzed to provide insights into the evolutionary history of any given protein domain. The evolution of proteins, at the domain level, has a significant impact iii on the overall functionality of metabolic pathways in general. Therefore, insights into the evolutionary trajectories of protein domains have the potential to inform the understanding of every aspect in which any given protein has a role of functional significance including, but certainly not limited to basic metabolic equilibrium, potential physical compromise at the phenotypic level, and deeper insights into corruptions that often lead to metabolic dysfunction and potential progression to full blown disease states. The prime focus of this study is to investigate the evolutionary relationships of proteins, across all Kingdoms of life, that contain within their tertiary phenotypic structure the 4-bladed β-propeller domain (the "Hemopexin" or "PEX" domain), towards illuminating the biophysical and biochemical significance of this specific domain's impact on protein functionality. While the phylogenetic relationships of entire proteins that have PEX domains is relatively straight forward, the mutational accumulation in gene sequences that lead to the tertiary structure of the PEX domain itself seems to have partially, if not entirely, obscured the evolutionary history at the domain level. The phylogenetic analyses presented here allow for several novel conclusions. First, this research demonstrates that a derived primary amino acid sequence in mammalian Hemopexin proteins (the JEN-14 epitope) represents a functional synapomorphy at the molecular level. Secondly, that there is substantial evidence for horizontal gene transfer of PEX domain proteins into specific Fungi. Additionally, there are no proteins containing a PEX domain in Kingdom Archaea. Lastly, it argues that the PEX domain itself represents an evolutionary “spandrel” (sensu Gould and Lewontin) with specifically derived functions existing around a core, preserved structural architecture. iv The faculty listed below, appointed by the Dean of the School of Graduate Studies, have examined a dissertation titled "Hyper-Plastic Structural Evolution Of The PEX Domain - A Model Of Evolutionary Exaptation And Neofunctionalization At The Molecular Level," presented by Lee Likins candidate for the Doctor of Philosophy degree, and hereby certify that in their opinion it is worthy of acceptance. Supervisory Committee Gerald J. Wyckoff, Ph.D. (Committee Chair) Department of Molecular Biology and Biochemistry Alex Idnurm, Ph.D., Department of Cell Biology and Biophysics Ann Smith, Ph.D., Department of Molecular Biology and Biochemistry Jakob Waterborg, Ph.D., Department of Cell Biology and Biophysics Xiaolan Yao, Ph.D., Department of Molecular Biology and Biochemistry v CONTENTS ABSTRACT ................................................................................................................... iii LIST OF ILLUSTRATIONS .......................................................................................... ix LIST OF TABLES ........................................................................................................ xii ACKNOWLEDGMENTS ............................................................................................ xiv DEDICATION ............................................................................................................. xvi Chapter 1. STRUCTURAL ANALYSES OF THE HEMOPEXIN PROTEIN AS A MEANS TO INFER THE EVOLUTION OF FUNCTIONALITY BETWEEN THE PEX DOMAIN CONTAINING PROTEINS a). Introduction......................................................................................................... 1 b). Methods .............................................................................................................. 7 c). Results ................................................................................................................ 8 2. DISCOVERY AND CHARACTERIZATION OF THE JEN-14 EPITOPE AS A MOLECULAR SYNAPOMORPHY IN HEMOPEXIN a). Introduction ....................................................................................................... 14 b). Methods ............................................................................................................ 15 c). Results .............................................................................................................. 16 d). Discussion and Conclusions .............................................................................. 18 3. PHYLOGENOMIC ANALYSES PROVIDE INSIGHTS INTO PATTERNS OF FUNCTIONAL DIVERSITY BETWEEN PEX DOMAIN CONTAINING PROTEINS a). Introduction ....................................................................................................... 22 b). Methods ............................................................................................................ 32 c). Results .............................................................................................................. 33 4. DISCUSSION AND CONCLUSIONS OF RESULTS FROM CHAPTERS 1-4 a). Discussion ......................................................................................................... 39 b). Conclusions ...................................................................................................... 48 vi 5. DETERMINATION OF THE TERTIARY STRUCTURE OF THE PEX DOMAIN IN THE HUMAN PROTEOGLYCAN-4 (LUBRICIN) PROTEIN a). Introduction ....................................................................................................... 54 b). Methods and Results ......................................................................................... 59 6. EVOLUTIONARY ANALYSES OF TARGET GENES IDENTIFIED AS POTENTIAL GENETIC MARKERS OF COMPLICATIONS ASSOCIATED WITH DIET-INDUCED OBESITY a). Introduction ....................................................................................................... 92 b). Methods and Results ......................................................................................... 99 c). Discussion ....................................................................................................... 108 Appendix A. SUPPLEMENTAL INFORMATION ON 4-BLADED β-PROPELLER DOMAIN CONTAINING PROTEINS ................................................................................. 112 B. LIST OF THE HUMAN MMPs INCLUDED IN THE PHYLOGENIES ALONG WITH A BRIEF DESCRIPTION OF KNOWN FUNCTIONALITY .................... 114 C. PRIMARY SEQUENCE IDENTITY BETWEEN VARIOUS PEX DOMAINS: HPX, MMPs PRG4, VTN ..................................................................................... 117 D. PRIMARY SEQUENCE AND HOMOLOGY MODEL OF THE PROTEIN LIMUNECTIN FROM HORSESHOE CRAB ..................................................... 118 E. PRG4 - PEX-DOMAIN DNA NUCLEOTIDE SEQUENCE (SHOWN AS CODONS) USED FOR ORDERING OF THE GBLOCK FOR CLONING, EXPRESSION AND PURIFICATION ................................................................ 119 F. pET-44a-c(+) VECTOR MAP ................................................................................. 120 G. RETURN OF RESULTS FROM MU-DNA LIMS SEQUENCING FACILITY SUBMISSION OF SAMPLE FROM COLONY 8 FROM PCR OF TRANSFORMED LIGATION PRODUCTS OF THE PRG4 PEX DOMAIN pET44 CONSTRUCT ........................................................................................... 121 H. ANALYSES FROM MASS SPECTROMETRY ON PRG4 PEX-DOMAIN .......... 122 I. SEQUENCE ALIGNMENTS FOR THE Atp1a3 PROTEIN IN PRIMATES ROOTED WITH MURINES ............................................................................... 126 vii J. ACCESSION NUMBERS