DOCSLIB.ORG

UNIVERSITY of CALIFORNIA, IRVINE Development And

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
UNIVERSITY of CALIFORNIA, IRVINE Development And UNIVERSITY OF CALIFORNIA, IRVINE Development and application of molecular modeling methods for characterization of drug targets in Mycobacterium tuberculosis DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmaceutical Sciences by Kalistyn H. Burley Dissertation Committee: Professor David L. Mobley, Chair Professor Celia W. Goulding Professor Thomas L. Poulos 2020 Chapter 2 © 2019 American Chemical Society Chapter 3 © 2019 American Chemical Society All other materials © 2020 Kalistyn H. Burley DEDICATION To my children, Aubrey and Baron – being your mother is, and will always be, my greatest accomplishment. To Scott, an amazing partner in life and in parenthood, who has never doubted me, and has also never let me settle. To my mom, dad, and brothers Kristopher and Jacob, who gave me the confidence to “jump off cliffs and build my wings on the way down.” - Ray Bradbury, 1986 And to my mentors and role models, particularly the women before me who have paved the road for my successes, I am forever indebted and committed to paying it forward. ii TABLE OF CONTENTS LIST OF FIGURES iv LIST OF TABLES vi ACKNOWLEDGMENTS vii VITA ix ABSTRACT OF THE DISSERTATION xii CHAPTER 1: An introduction to tuberculosis and 1 computational methods for structure-based drug design References 16 CHAPTER 2: Enhancing side chain sampling using 19 non-equilibrium candidate Monte Carlo References 59 CHAPTER 3: Structure of a Mycobacterium tuberculosis heme- 63 degrading protein, MhuD, variant in complex with its product References 92 CHAPTER 4: Insights from the structure and conformational 97 dynamics of Mycobacterium tuberculosis malic enzyme, MEZ References 134 CHAPTER 5: Summary and Conclusions - Complementing structural 139 information using computational methods for drug-design References 146 APPENDIX A: Chapter 2 Supporting Information 148 APPENDIX B: Chapter 3 Supporting Information 154 APPENDIX C: Chapter 4 Supporting Information 157 iii LIST OF FIGURES Page Figure 1.1 Percentage of new TB cases that are MDR or rifampicin 2 resistant (RR-TB) Figure 1.2 Mtb cell wall architecture 5 Figure 1.3 Mtb iron uptake 7 Figure 1.4 Computational methods for drug development 12 Figure 1.5 Approximate timescales of molecular motions 14 Figure 2.1 Cycling of atom interactions during execution of NCMC 26 side chain move Figure 2.2 Move biasing based on known side chain rotamer states 30 Figure 2.3 Workflow illustration of BLUES side chain proposals 32 Figure 2.4 Rotameric states of valine side chain 35 Figure 2.5 Umbrella sampling of valine-alanine peptide in explicit 38 solvent Figure 2.6 Valine-Alanine rotamer transition data where the 40 torsional force constant, k = 3.8 kcal/mol Figure 2.7 Valine-Alanine rotamer transition data where 42 k = 10 kcal/mol. Figure 2.8 Val111 χ1 rotamer data for BLUES simulations of 47 p-xylene bound T4 lysozyme L99A in explicit solvent Figure 2.9 Val111 χ1 transition rates in T4 lysozyme L99A bound to 47 p-xylene for BLUES and MD Figure 2.10 Backbone RMSDs for T4 lysozyme L99A simulations 50 Figure 2.11 Overlay of backbone RMSDs of T4 lysozyme L99A for 51 BLUES and MD (first 100ns) 6 Figure 2.12 Val111 χ1 state transitions per 10 FEs from simulations 53 from 3 distinct starting conformations of p-xylene bound T4 lysozyme L99A Figure 3.1 Structures of tetrapyrroles and WT-MhuD-monoheme 65 Figure 3.2 Heme and αBV affinity of MhuD and the R26S variant 73 Figure 3.3 Structure of the MhuD-R26S-αBV complex 76 Figure 3.4 Structural comparison of MhuD-heme-CN and 81 MhuD-R26S-αBV Figure 3.5 Helical stability in MD simulations of MhuD 82 Figure 3.6 Orientation of His75 in MD simulations of MhuD 83 Figure 3.7 Position of Arg79 side chain during MD simulations of 85 MhuD Figure 4.1 Malic enzyme structural classes and oligomeric states 100 Figure 4.2 Structural domains of MEZ 103 Figure 4.3 Biophysical and biochemical analyses of MEZ 109 Figure 4.4 Different modes of NAD(P)+ binding after docking with Mn2+ 112 and malate iv Figure 4.5 Divalent cation stabilizes malate in binding site 114 Figure 4.6 Average RMSF per MEZ residue 115 Figure 4.7 Conformational dynamics of MEZ active site 116 Figure 4.8 Putative NAD(P)+ binding modes in MEZ after MD 121 simulations v LIST OF TABLES Page Table 2.1 Data summary for simulations of T4 Lysozyme L99A with 53 p-xylene from alternate starting conformations. Table 3.1 Heme and αBV binding affinities (Kd) for MhuD 72 Table 3.2 Statistics for X-ray diffraction data collection and atomic 75 refinement for the MhuD-R26S-αBV complex. Table 4.1. Data collection and refinement statistics for MEZ crystal 106 structure. vi ACKNOWLEDGMENTS In my time as a graduate student, I have been fortunate to have had the guidance and mentorship of two outstanding research advisors: Dr. Celia Goulding and Dr. David Mobley. They are each highly revered in their respective fields and are deeply committed to their responsibility to nurture and train future scientists. Thank you, Celia, for applying steady pressure to move me forward in my research, persistently advocating on my behalf, and for helping me navigate the highs, lows, and idiosyncrasies of life as a graduate student. Thank you to David, for always making yourself available, encouraging and providing opportunities to engage in the wider computational chemistry community, looking out for my general welfare and for trusting in my ability to work independently. Thank you both for unequivocally supporting and respecting my decision to start and grow my family while in graduate school. To that end, I would also like to acknowledge and thank my husband Scott - I could not have done this without you. Thank you for being an amazing father, partner, and coach. Finally, a special thank you to Mary and Henry Bugielski - for providing outstanding care and a warm and nurturing environment for our children, each and every weekday. Chapter 2 is a minimally modified reprint of the published work: Burley KH, Gill SC, Lim NM, Mobley DL. Enhancing Side chain sampling using non-equilibrium Monte Carlo. J Chem Theory Comput. 2019 Jan 24;15(3):1848-1862. I would like to acknowledge financial support from the National Science Foundation (CHE 1352608) and the National Institutes of Health (1R01GM108889-01 and T32GM108561), and the Vertex Fellowship as well as computing support from the UCI GreenPlanet cluster, supported in part by NSF Grant CHE- 0840513 and the infrastructure support from the Triton Shared Computing Cluster (TSCC) at the San Diego Supercomputing Center (SDSC). I particularly thank my co-authors, Sam Gill for developing and pioneering the BLUES method and Nathan Lim for helping transform BLUES into a developer-friendly and user-friendly software package. I would also like to recognize Sukanya Sasmal (UCI) for help with docking, Gaetano Calabró (UCI) for basic MD training when joining the lab, and Victoria Lim (UCI) for help with umbrella sampling. Chapter 3 is a minimally modified reprint of the published work: Chao A, Burley KH, Sieminski PJ, de Miranda R, Chen X, Mobley DL, Goulding CW. Structure of Mycobacterium tuberculosis heme-degrading protein, MhuD, variant in complex with its product. Biochemistry. 2019 Oct 22;58(46):4610-20. This work and its authors were financially supported by the National Institutes of Health (NIH) (P01-AI095208, T32GM108561), the National Science Foundation (DGE-1321846). The authors are grateful to Tom Poulos for discussions and critical reading of the manuscript, as well as Dr. Fengyun Ni, Baylor College of Medicine for advice on the more difficult aspects of structure refinement. The authors also acknowledge the Advanced Light Source at Berkeley National Laboratories (ALS) and the Stanford Synchrotron Radiation Lightsource (SSRL) for their invaluable help in data collection, and The Triton Shared Computing Cluster (TSCC) at the San Diego Supercomputing Center (SSDC) for their computing support. Regarding my co- authors, I especially thank Alex Chao for solving the structure, Xiaorui Chen for her vii contributions in data processing and refinement, as well as Paul Sieminski and Rodger de Miranda for their biochemical characterization of the MhuD variant with its product. Chapter 4 is part of a manuscript in preparation. I would like to acknowledge Bonnie Cuthbert for her tenacity and expertise in refining the crystal structure of MEZ, her analysis of the structure, as well as her moral support and friendship, Ervin Irimpan for his assistance in preparing protein and setting up crystal trays, Ilona Foik for the well-kept records of her prior crystallization attempts and purification methods, and Robert Quechol for his work in performing critical biochemical assays. I also appreciate financial support from the National Institutes of Health (NIH) (P01-AI095208, T32GM108561) and the National Science Foundation (DGE-1321846), as well as computing support from The Triton Shared Computing Cluster (TSCC) at the San Diego Supercomputing Center (SSDC) and UCI GreenPlanet cluster, supported in part by NSF Grant CHE-0840513. viii VITA Kalistyn Hope Burley EDUCATION Ph.D. in Pharmaceutical Sciences June 2020 University of California, Irvine Irvine, CA Co-Advisors: Dr. David Mobley, Dr. Celia Goulding A.B. in Biomedical Engineering June 2009 Dartmouth College Hanover, NH PROFESSIONAL EXPERIENCE Graduate Student Researcher September 2015 – April 2020 Mobley Lab, UC Irvine, Department of Pharmaceutical Sciences Irvine, CA Graduate Student Researcher January 2016 – April 2020 Goulding Lab,
Load more

Recommended publications
  • Targeting Proteins Self-Association in Drug Design
    Targeting proteins self-association in drug design Léopold Thabault,1,2 Maxime Liberelle,1 Raphaël Frédérick1* 1 Louvain Drug Research Institute (LDRI), Université catholique de Louvain (UCLouvain), B-1200 Brussels, Belgium. 2 Pole of Pharmacology and Therapeutics, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain (UCLouvain),B-1200 Brussels, Belgium. * Corresponding Author : Prof. Raphaël Frédérick Université catholique de Louvain (UCLouvain) Louvain Drug Research Institute (LDRI) Tour Van Helmont, B1.73.10 73 avenue Mounier 1200 Brussels Belgium Phone : +32 2 764 73 41 1 Abstract Protein self-association is a universal phenomenon essential for stability and molecular recognition. Disrupting constitutive homomers constitutes an original and emerging strategy in drug design. Inhibition of homomeric proteins can be achieved through direct complex disruption, subunits intercalation or by promoting inactive oligomeric states. Targeting self- interaction grants several advantages over active site inhibition thanks to the stimulation of protein degradation, the enhancement of selectivity, a sub-stoichiometric inhibition and a by- pass of compensatory mechanisms. This new landscape in protein inhibition is driven by the development of biophysical and biochemical tools suited for the study of homomeric proteins, such as DSF, native MS, FRET spectroscopy, two-dimensional NMR and X-ray crystallography. The present review covers the different aspects of this new paradigm in drug design. Keywords Protein-protein interaction; Homo-oligomers Disruptor; Protein destabilisation; Allosteric inhibition; Self-association. Teaser Disrupting protein self-association bears great therapeutic promesses and could provide exciting alternatives to active-site inhibitors. 2 1. PPI as a well-accepted paradigm in drug design The human interactome is nowadays widely considered as one of the keys to cell mechanistic regulation.
    [Show full text]
  • Moonlighting Proteins in the Fuzzy Logic of Cellular Metabolism
    molecules Review Moonlighting Proteins in the Fuzzy Logic of Cellular Metabolism Haipeng Liu 1 and Constance J. Jeffery 2,* 1 Center for Biomolecular Sciences, College of Pharmacy, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60607, USA; [email protected] 2 Department of Biological Sciences, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, IL 60607, USA * Correspondence: cjeff[email protected]; Tel.: +1-312-996-3168 Academic Editor: Pier Luigi Gentili Received: 30 May 2020; Accepted: 23 July 2020; Published: 29 July 2020 Abstract: The numerous interconnected biochemical pathways that make up the metabolism of a living cell comprise a fuzzy logic system because of its high level of complexity and our inability to fully understand, predict, and model the many activities, how they interact, and their regulation. Each cell contains thousands of proteins with changing levels of expression, levels of activity, and patterns of interactions. Adding more layers of complexity is the number of proteins that have multiple functions. Moonlighting proteins include a wide variety of proteins where two or more functions are performed by one polypeptide chain. In this article, we discuss examples of proteins with variable functions that contribute to the fuzziness of cellular metabolism. Keywords: moonlighting proteins; fuzzy logic; intrinsically disordered proteins; metamorphic proteins; morpheeins 1. Introduction Fuzzy logic systems include variables that can be any real number between 0 and 1 instead of being limited to the Boolean logic variables of only 0 and 1. This enables expression of complexity, uncertainty, and imprecision. In general, the vast interconnected biochemical pathways that make up the metabolism of a living cell can appear fuzzy because they are complex and hard to predict, and we have incomplete and not yet accurate knowledge.
    [Show full text]
  • Structures, Functions, and Mechanisms of Filament Forming Enzymes: a Renaissance of Enzyme Filamentation
    Structures, Functions, and Mechanisms of Filament Forming Enzymes: A Renaissance of Enzyme Filamentation A Review By Chad K. Park & Nancy C. Horton Department of Molecular and Cellular Biology University of Arizona Tucson, AZ 85721 N. C. Horton ([email protected], ORCID: 0000-0003-2710-8284) C. K. Park ([email protected], ORCID: 0000-0003-1089-9091) Keywords: Enzyme, Regulation, DNA binding, Nuclease, Run-On Oligomerization, self-association 1 Abstract Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI system are also highlighted. 2 Contents INTRODUCTION…………………………………………………………..4 STRUCTURALLY CHARACTERIZED ENZYME FILAMENTS…….5 Acetyl CoA Carboxylase (ACC)……………………………………………………………………5 Phosphofructokinase (PFK)……………………………………………………………………….6
    [Show full text]
  • First Structure of Full-Length Mammalian Phenylalanine Hydroxylase Reveals the Architecture of an Autoinhibited Tetramer
    First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer Emilia C. Arturoa,b, Kushol Guptac, Annie Hérouxd, Linda Stitha, Penelope J. Crosse,f,g, Emily J. Parkere,f,g, Patrick J. Lollb, and Eileen K. Jaffea,1 aMolecular Therapeutics, Fox Chase Cancer Center, Temple University Health Systems, Philadelphia, PA 19111; bBiochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102; cBiochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; dEnergy Sciences Directorate/Photon Science Division, Brookhaven National Laboratory, Upton, NY 11973; eBiomolecular Interaction Centre, University of Canterbury, Christchurch 8041, New Zealand; fDepartment of Chemistry, University of Canterbury, Christchurch 8041, New Zealand; and gMaurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1142, New Zealand Edited by Judith P. Klinman, University of California, Berkeley, CA, and approved January 21, 2016 (received for review August 27, 2015) Improved understanding of the relationship among structure, dy- There are >500 disease-associated missense variants of human namics, and function for the enzyme phenylalanine hydroxylase PAH; the amino acid substitutions are distributed throughout (PAH) can lead to needed new therapies for phenylketonuria, the the 452-residue protein and among all its domains (Fig. 1A) most common inborn error of amino acid metabolism. PAH is a (7–9). Of those disease-associated variants that have been stud- multidomain homo-multimeric protein whose conformation and ied in vitro (e.g., ref. 10), some confound the allosteric response, multimerization properties respond to allosteric activation by the and some are interpreted as structurally unstable. We also sug- substrate phenylalanine (Phe); the allosteric regulation is neces- gest that the activities of some disease-associated variants may be sary to maintain Phe below neurotoxic levels.
    [Show full text]
  • Allosteric Modulation of Protein Oligomerization: an Emerging Approach to Drug Design
    REVIEW ARTICLE published: 24 March 2014 doi: 10.3389/fchem.2014.00009 Allosteric modulation of protein oligomerization: an emerging approach to drug design Ronen Gabizon † and Assaf Friedler* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel Edited by: Many disease-related proteins are in equilibrium between different oligomeric forms. The Youla S. Tsantrizos, McGill regulation of this equilibrium plays a central role in maintaining the activity of these University, Canada proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins Reviewed by: by molecules that bind preferentially to a specific oligomeric state is emerging as a Lee Fader, Boehringer Ingelheim Pharmaceuticals, Inc., USA potential therapeutic strategy that can be applied to many biological systems such Wolfgang Jahnke, Novartis as cancer and viral infections. The target proteins for such compounds are diverse Institutes for Biomedical Research, in structure and sequence, and may require different approaches for shifting their Switzerland oligomerization equilibrium. The discovery of such oligomerization-modulating compounds *Correspondence: is thus achieved based on existing structural knowledge about the specific target proteins, Assaf Friedler, Institute of Chemistry, The Hebrew University as well as on their interactions with partner proteins or with ligands. In silico design and of Jerusalem, Safra Campus, Givat combinatorial tools such as peptide arrays and phage display are also used for discovering Ram, Jerusalem 91904, Israel compounds that modulate protein oligomerization. The current review highlights some e-mail: [email protected] of the recent developments in the design of compounds aimed at modulating the †Present address: oligomerization equilibrium of proteins, including the “shiftides” approach developed in Ronen Gabizon,California Institute our lab.
    [Show full text]
  • PKM2, a Central Point of Regulation in Cancer Metabolism
    Hindawi Publishing Corporation International Journal of Cell Biology Volume 2013, Article ID 242513, 11 pages http://dx.doi.org/10.1155/2013/242513 Review Article PKM2, a Central Point of Regulation in Cancer Metabolism Nicholas Wong,1,2,3,4 Jason De Melo,1,2,3,4 and Damu Tang1,2,3,4 1 Division of Nephrology, Department of Medicine, McMaster University, Hamilton, ON, Canada L8S 4L8 2 Division of Urology, Department of Surgery, McMaster University, Hamilton, ON, Canada L8S 4L8 3 Father Sean O’Sullivan Research Centre, St. Joseph’s Healthcare Hamilton, Hamilton, ON, Canada L8N 4A6 4 The Hamilton Center for Kidney Research, St. Joseph’s Healthcare Hamilton, Hamilton, ON, Canada L8N 4A6 Correspondence should be addressed to Damu Tang; [email protected] Received 2 December 2012; Revised 11 January 2013; Accepted 13 January 2013 Academic Editor: Claudia Cerella Copyright © 2013 Nicholas Wong et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aerobic glycolysis is the dominant metabolic pathway utilized by cancer cells, owing to its ability to divert glucose metabolites from ATP production towards the synthesis of cellular building blocks (nucleotides, amino acids, and lipids) to meet the demands of proliferation. The M2 isoform of pyruvate kinase (PKM2) catalyzes the final and also a rate-limiting reaction in the glycolytic pathway. In the PK family, PKM2 is subjected to a complex regulation by both oncogenes and tumour suppressors, which allows for a fine-tone regulation of PKM2 activity.
    [Show full text]
  • A Renaissance of Enzyme Filamentation
    Biophysical Reviews (2019) 11:927–994 https://doi.org/10.1007/s12551-019-00602-6 REVIEW Structures, functions, and mechanisms of filament forming enzymes: a renaissance of enzyme filamentation Chad K. Park1 & Nancy C. Horton1 Received: 23 August 2019 /Accepted: 24 October 2019 /Published online: 16 November 2019 # The Author(s) 2019 Abstract Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI endonuclease system are also highlighted. Keywords Enzyme . Regulation . DNA binding . Nuclease . Run-on oligomerization . Self-association Introduction phenomenon and its role in regulation of their particular en- zyme systems (Kessler et al. 1992; Cutler and Somerville For over 50 years, it has been known that many enzymes form 2005; Korennykh et al. 2009; Ingerson-Mahar et al. 2010; filamentous structures in vitro as assessed by various biophys- Kim et al. 2010; Park et al.
    [Show full text]
  • Regulation of Cellular Glucose Metabolism by Hiv-1 Infection
    REGULATION OF CELLULAR GLUCOSE METABOLISM BY HIV-1 INFECTION A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY By SATARUPA SEN MAY 2014 Examining Committee Members: SHOHREH AMINI, Advisory Chair, BIOLOGY PRASUN K. DATTA, NEUROSCIENCE KAMEL KHALILI, NEUROSCIENCE ANTONIO GIORDANO, BIOLOGY MICHAEL NONNEMACHER, External Member, DREXEL UNIVERSITY ABSTRACT REGULATION OF CELLULAR GLUCOSE METABOLISM BY HIV-1 INFECTION SATARUPA SEN DOCTOR OF PHILOSOPHY TEMPLE UNIVERSITY 2014 DOCTORAL ADVISORAL COMMITTEE CHAIR: DR. SHOHREH AMINI, PH.D. Regulation of Glucose metabolism is known to play an important role in pathogenesis of many diseases. Primarily because deregulation of this metabolic pathway can lead to either apoptosis or extended life span of the cells involved. Viruses are parasitic in nature, they utilize the host cellular pathways to support their own progeny; hence it is expected that viruses would regulate the central glucose metabolism of infected host cells. Human immunodeficiency virus type 1 (HIV-1) causes acquired immune deficiency syndrome, and it uniquely infects both activated CD4+ T cells and terminally differentiated macrophages during the course of HIV-1 pathogenesis. While HIV-1 infection of CD4+ T cells induces G2 arrest and cell death within 2–3 days, HIV-1 infection of macrophages results in longer survival of infected cells and low constitutive viral production, generating viral reservoirs. Our studies show that HIV-1 infection lead to significant changes in the glycolytic pathway of infected cells by altering the enzymatic activity and protein expression of various glycolytic components. The data suggests that the two HIV- 1 target cell types exhibit very different metabolic outcomes.
    [Show full text]
  • Wrangling Shape-Shifting Morpheeins to Tackle Disease and Approach Drug Discovery
    fmolb-07-582966 November 23, 2020 Time: 16:31 # 1 REVIEW published: 27 November 2020 doi: 10.3389/fmolb.2020.582966 Wrangling Shape-Shifting Morpheeins to Tackle Disease and Approach Drug Discovery Eileen K. Jaffe* Fox Chase Cancer Center, Philadelphia, PA, United States Homo-multimeric proteins that can come apart, change shape, and reassemble differently with functional consequences have been called morpheeins and/or transformers; these provide a largely unexplored context for understanding disease and developing allosteric therapeutics. This article describes such proteins within the context of protein structure dynamics, provides one detailed example related to an inborn error of metabolism and potential herbicide development, and describes the context for applying these ideas for understanding disease and designing bioactive molecules, such as therapeutics. Keywords: protein structure dynamics, allostery, morpheein, fifth level of protein structure, drug discovery INTRODUCTION Edited by: Guang Hu, A great number of medically relevant proteins are homo-multimers, some of which exist as Soochow University, China an equilibrium of alternate assemblies that are both non-additive and functionally distinct. The Reviewed by: phenomenon wherein protein homo-multimers can come apart, change shape while dissociated, Jian Zhang, and reassemble into an architecturally and functionally different assembly has been called the Shanghai Jiao Tong University, China morpheein model of protein allostery (Jaffe, 2005; see Morpheein in Wikipedia1). A key to this Claire Lesieur, UMR 5005 Laboratoire Ampère protein structure dynamic is that the required conformational change is spatially forbidden within (Ampère), France the context of either assembly. Proteins with this capacity can be called morpheeins and the Athi N.
    [Show full text]
  • ARTICLE ALAD Porphyria Is a Conformational Disease
    ARTICLE ALAD Porphyria Is a Conformational Disease Eileen K. Jaffe and Linda Stith ALAD porphyria is a rare porphyric disorder, with five documented compound heterozygous patients, and it is caused by a profound lack of porphobilinogen synthase (PBGS) activity. PBGS, also called “d-aminolevulinate dehydratase,” is encoded by the ALAD gene and catalyzes the second step in the biosynthesis of heme. ALAD porphyria is a recessive disorder; there are two common variant ALAD alleles, which encode K59 and N59, and eight known porphyria-associated ALAD mutations, which encode F12L, E89K, C132R, G133R, V153M, R240W, A274T, and V275M. Human PBGS exists as an equilibrium of functionally distinct quaternary structure assemblies, known as “morpheeins,” in which one func- tional homo-oligomer can dissociate, change conformation, and reassociate into a different oligomer. In the case of human PBGS, the two assemblies are a high-activity octamer and a low-activity hexamer. The current study quantifies the morpheein forms of human PBGS for the common and porphyria-associated variants. Heterologous expression in Escherichia coli, followed by separation of the octameric and hexameric assemblies on an ion-exchange column, showed that the percentage of hexamer for F12L (100%), R240W (80%), G133R (48%), C132R (36%), E89K (31%), and A274T (14%) was appreciably larger than for the wild-type proteins K59 and N59 (0% and 3%, respectively). All eight porphyria- associated variants, including V153M and V275M, showed an increased propensity to form the hexamer, according to a kinetic analysis. Thus, all porphyria-associated human PBGS variants are found to shift the morpheein equilibrium for PBGS toward the less active hexamer.
    [Show full text]
  • Exploration of the Interplay Between Allosteric
    EXPLORATION OF THE INTERPLAY BETWEEN ALLOSTERIC INTERACTIONS OF PHOSPHOFRUCTOKINASE FROM RAT LIVER A Dissertation by DAVID ALAN HOLLAND III Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Gregory Reinhart Committee Members, Ry Young Hays Rye Coran Watanabe Head of Department, Gregory Reinhart May 2018 Major Subject: Biochemistry Copyright 2018 David Alan Holland III ABSTRACT The key metabolic enzyme phosphofructokinase (PFK) catalyzes the phosphorylation of fructose-6-phosphate (Fru-6-P) by MgATP in the first committed step of glycolysis. PFK’s phosphorylation activity is tightly regulated by allosteric effectors who act by either increasing or decreasing its affinity to substrate Fru-6-P. Liver PFK from rats (RLPFK) is allosterically regulated by several metabolic byproducts including MgATP, citrate and AMP. Despite the importance of precise regulation of this enzyme, the interplay between the different allosteric effectors has not been thoroughly characterized. Presented here are the effects of MgATP on the ability of allosteric activator (AMP) or inhibitor (citrate) to modulate the activity of RLPFK. We see that MgATP dramatically decreases the ability of both AMP and citrate to allosterically regulate RLPFK. RLPFK has additionally been implemented to be subjected to activation through a novel self-association mechanism. This is difficult to prove unequivocally as the kinetic assays used to measure enzyme activity are performed (by necessity) at concentrations thought to be too low for self-association to occur. It has been demonstrated that at a physiological enzyme concentration Fru-6-P promotes self- association.
    [Show full text]
  • Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity
    Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity Irene N. Kiburu, Nicole LaRonde-LeBlanc* Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, and the University of Maryland Marlene and Stewart Greenebaum Cancer Center, University of Maryland, College Park, Maryland, United States of America Abstract Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 A˚ and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.
    [Show full text]