Actin Polymerization in Apicomplexan: A Structural, Functional and Evolutionary Analysis [1st ed. 2019] 978-981-13-7449-4, 978-981-13-7450-0

Table of contents :
Front Matter ....Pages i-xi
Actin Polymerization: A Cellular Perspective for Motility (Samridhi Pathak, Sarita Tripathi, Ricka Gauba, Sarath Chandra Dantu, Avinash Kale)....Pages 1-14
Actin: The Central Ubiquitous Player in the Phenomenon (Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Avinash Kale)....Pages 15-28
Formin: The Multidomain Elongator of Actin Polymer (Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Dhriti Sheth, Avinash Kale)....Pages 29-38
Profilin: The Associates of Formin (Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Avinash Kale)....Pages 39-50
ADF (Actin Depolymerizing Factor): The Breaker of the Polymer in Homeostasis (Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Avinash Kale)....Pages 51-62
CAP (Cyclase-Associated Protein): The Silent Worker (Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Avinash Kale)....Pages 63-68
Capping Protein (CP): The Formin Competitor (Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Avinash Kale)....Pages 69-75
Coronin: An Overview (Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Avinash Kale)....Pages 77-83
Evolution: The Hallmarks of Gliding Motility in Apicomplexan (Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Avinash Kale)....Pages 85-91
Actin Polymerization in Apicomplexans: A Novel Perspective (Samridhi Pathak, Ricka Gauba, Avinash Kale)....Pages 93-97
Back Matter ....Pages 99-101

Citation preview

Avinash  Kale Editor

Actin Polymerization in Apicomplexan A Structural, Functional and Evolutionary Analysis

Actin Polymerization in Apicomplexan

Avinash Kale Editor

Actin Polymerization in Apicomplexan A Structural, Functional and Evolutionary Analysis

Editor Avinash Kale School of Chemical Sciences UM-DAE Centre for Excellence in Basic Sciences (CEBS) Mumbai, Maharashtra, India

ISBN 978-981-13-7449-4 ISBN 978-981-13-7450-0 https://doi.org/10.1007/978-981-13-7450-0

(eBook)

# Springer Nature Singapore Pte Ltd. 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Apicomplexa is the largest phylum of parasitic protozoans. Some of the apicomplexan parasites cause life-threatening diseases such as malaria, cyclosporiasis, isosporiasis, and toxoplasmosis in humans, babesiosis in cattle, and other animals. The global mortality due to malaria in 2015 was 4.38 billion, while 3.2 billion people are at risk of malaria (http://www.who.int). Other apicomplexans such as Toxoplasma gondii and Cryptosporidium parvum are relatively benign in individuals with robust immune system. However, these organisms can be a serious threat and potentially life threatening in immunocompromised patients suffering from AIDS, organ transplant recipients, and immunosuppressed cancer patients. Other members of apicomplexans, such as Babesia, Eimeria tenella, and Theileria annulata, which infect cattle and poultry, are also serious nuisance-causing organisms in the livestock industry and also are opportunistic parasites on humans. Apicomplexans invade the host cells by employing a unique mechanism known as “Gliding Motility.” Following attachment to the host surface, the organism “crawls” over the surface of the host cell to find a specific receptor and subsequently punctures into it. Gliding motility is executed by actin-myosin cytoskeletal elements. The significance of myosin motor in promoting motility is well-studied and documented. However, actin monomers are the building blocks of gliding motility. Yet, paradoxically, little is known about the detailed mechanism of actin polymerization, which essentially sets the gliding motility in motion. Actin, being a ubiquitous protein and moreover a cytoskeletal housekeeping protein, is definitely not a good choice for therapeutic applications. Developing efficient drug strategies using actin and its regulators requires understanding the mechanism of actin polymerization and de-polymerization and the differences between apicomplexan and their host actin regulation mechanisms. Among the various organisms of the apicomplexan phylum, despite the sequential and structural differences in actin, in all of them, actin is regulated by a minimal set of seven regulators, namely, formin, profilin, actin depolymerization factor (ADF), capping proteins (CPα and CPβ), cyclase-associated protein (CAP), and coronin. In higher eukaryotes, actin is regulated by a multitude of 150–200 regulators. Some of the actin regulators from apicomplexans show structural differences in comparison to their counterparts in their respective hosts, suggesting that such regulators can be pursued for drug development. v

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Preface

This book helps us to better our understanding about the regulation of actin polymerization by plausibly the minimal machinery of regulatory proteins by discussing the structural, functional, and evolution perspective in apicomplexan. This updated knowledge might lead us to better comprehend other cellular process driven by actin polymerization. Chapter 1 discusses, in general, the role of central protein, actin, in various cellular motility processes. Also, we had briefed the problems that arise due to the misregulation of actin polymerization phenomenon. We further introduce the actin-mediated gliding motility in apicomplexans by summarizing the known model and mechanism. Chapter 2 details the role of actin as a central player in polymerization cascade, following the general introduction about the protein. The role of multi-domain, megadalton formin in the process is discussed in detail in Chap. 3. Importance of profilin, as detailed in Chap. 4, reveals its probable multifunctional role that has not been known earlier. Actin depolymerization factors (ADFs) are described in Chap. 5. Known facts about the cyclaseassociated protein (CAP), as depicted in Chap. 6, reveal interesting implications about this partner being a special player in the acting polymerization cascade. Chapter 7 talks about the potential competitor role of capping protein (CP), whereas Chap. 8 provides an overview of coronin and its role in actin polymerization. Chapter 9 deals with the evolutionary perspective of actin and the regulators involved in gliding motility. The evolutionary analysis was carried out to discern whether these proteins have evolved individually or as a matter of the entire phenomenon from the previously existing prokaryotes or archaea. We have deduced the plausible evolutionary pathway for the actin and its regulators. Finally, in Chap. 10 we provide a brief about the significant finding/facts of the individual chapters in the summary section. It also presents the “holistic mechanism” of actinmediated gliding motility by reporting the cross-talks between actin and its regulators in Plasmodium. Mumbai, Maharashtra, India

Avinash Kale

Contents

1

Actin Polymerization: A Cellular Perspective for Motility . . . . . . . . . Samridhi Pathak, Sarita Tripathi, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

1

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Actin: The Central Ubiquitous Player in the Phenomenon . . . . . . . . 15 Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

3

Formin: The Multidomain Elongator of Actin Polymer . . . . . . . . . . . 29 Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, Dhriti Sheth, and Avinash Kale

4

Profilin: The Associates of Formin . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

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ADF (Actin Depolymerizing Factor): The Breaker of the Polymer in Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

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CAP (Cyclase-Associated Protein): The Silent Worker . . . . . . . . . . . 63 Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

7

Capping Protein (CP): The Formin Competitor . . . . . . . . . . . . . . . . 69 Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

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Coronin: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

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Contents

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Evolution: The Hallmarks of Gliding Motility in Apicomplexan . . . . 85 Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

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Actin Polymerization in Apicomplexans: A Novel Perspective . . . . . . 93 Samridhi Pathak, Ricka Gauba, and Avinash Kale

Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

About the Editor

Dr. Avinash Kale is a Reader at the School of Chemical Sciences, UM-DAE Centre for Excellence in Basic Sciences, Mumbai, India. His research interests are in the area of integrative structure biology and proteomics (ISBAP). He has more than 16 years of teaching experience in computational biology, biotechnology, molecular modeling and drug design, group theory, spectroscopy, and bioinformatics. He has served as a reviewer for a number of international journals, including the Journal of Biomolecular Structure and Dynamics (USA) and Nature Scientific Reports (USA).

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Abbreviations

PCOS EMT ABP AD TgFRM2 TgFRM3 FH1 domain FH2 domain DAD PAMP ADF ADF-H domain PfADF1 PfADF2 HsCof TgADF CAP C-CAP HFD WH2 domain CP GBD VASP N-WASP WAVE

Polycystic ovarian syndrome Epithelial mesenchymal transition Actin binding proteins Alzheimer’s disease Toxoplasma gondii formin 2 Toxoplasma gondii formin 3 Formin homology-1 domain Formin homology-2 domain Diaphanous autoregulatory domain Pathogen-associated molecular pattern Actin depolymerization factor ADH-homology domain Plasmodium falciparum ADF-1 Plasmodium falciparum ADF-2 Human Cofiin Toxoplasma gondii ADF Cyclase-associated protein C-terminal domain containing CAP Helical folded domain Wiskott-Aldrich syndrome protein homology 2 domain Capping protein GTPase-binding domain Vasodilator-stimulated phosphoprotein N-terminal Wiskott-Aldrich syndrome protein WASP family proteins

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Actin Polymerization: A Cellular Perspective for Motility Samridhi Pathak, Sarita Tripathi, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

Abstract

The first chapter introduces the importance of actin and its polymerization dynamics in general. It briefs about the major cellular processes that are controlled by the virtue of controlled regulation of actin polymerization process. It also summarizes different pathophysiological conditions arising due to impaired regulation of actin. Further, we also discuss the importance of acting polymerization in motility in different prokaryotic and eukaryotic cellular organisms. Finally, we conclude by introducing the phenomenon of gliding motility in apicomplexans, where actin which is the central player is tightly controlled by a minimal set of regulatory proteins. This is quite unlike higher eukaryotes where the number of similar actin-binding accessory proteins is fairly high.

1.1

Introduction

Actin is a ubiquitous cytoskeleton protein constitutively expressed in nearly all cells. The spontaneous polymerization of monomeric actin into its filamentous state forms a dense network providing mechanical support and characteristic shape to the cells. The controlled regulation of actin assembly/disassembly is fundamental to many cellular processes like embryo morphogenesis, mitosis, cytokinesis, vesicular transport and immunoregulation [1–5]. Any perturbation in actin remodelling has been S. Pathak · S. Tripathi · R. Gauba · A. Kale (*) School of Chemical Sciences, UM-DAE Centre for Excellence in Basic Sciences (CEBS), Mumbai, Maharashtra, India e-mail: [email protected]; [email protected]; [email protected] S. C. Dantu Department of Computer Science, Synthetic Biology Theme, Brunel University London, Uxbridge, UK e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 A. Kale (ed.), Actin Polymerization in Apicomplexan, https://doi.org/10.1007/978-981-13-7450-0_1

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Fig. 1.1 Major processes/phenomenon affected by controlled and impaired regulation of actin polymerization dynamics in general

linked to several diseases like cancer metastasis and angiogenesis, neurodegenerative diseases, impaired learning and memory, pathogenic infections, immunodeficiencies, etc. [5–8], as depicted in Fig. 1.1. Some of the major actinrelated disorders are described below.

1.1.1

Cancer and Polycystic Ovarian Syndrome (PCOS)

Aberrant regulation of cellular motility leads to tumour cell invasion and metastasis making cancer a dreadful disease [9–11]. The transformed cells employ a process called epithelial-mesenchymal transition (EMT) wherein GTPase family proteins (Rho, Rac and Cdc42) transmit extracellular signals in response to several chemoattractants to bring about extensive reorganization of actin cytoskeleton [12–17]. Such proteins that induce these signalling pathways are highly expressed in cancerous cells. The de novo polymerization of F-actin at the leading edge of the cell leads to the formation of membranous protrusions to obtain migratory and invasive properties. These include lamellipodia to generate force for cell migration

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and invadopodia/podosomes to promote migration by degrading ECM (extracellular matrix) [18]. Studies have identified several pharmacological inhibitors that target actin polymerization and function, as a potent drug for cancer therapy [19–22]. Hippo signalling pathway is a tumour suppressor pathway responsible for restricting the size of organ. This pathway is highly conserved from Drosophila to humans [23–25]. Any alteration in this signalling cascade might lead to development of many pathological states including cancer and polycystic ovarian syndrome (PCOS) [26–28]. Apart from several factors such as cell-to-cell contact, cell polarity and architecture and various physical and hormonal stimuli cues, it has been found that actin cytoskeleton also regulates the Hippo signalling [29–31]. Imbalances in F-actin organization, especially capping proteins, disrupt the Hippo signalling by activation and nuclear localization of YAP, key effector molecule of the cascade, thereby increasing the expression of growth factors [32]. Polycystic ovarian syndrome (PCOS) showing follicle arrest can be targeted by actin polymerizationpromoting drugs which in turn would promote growth of follicles and development of mature oocytes by disrupting the Hippo signalling rather than currently used disruptive measures like ovarian wedge resection or laser drilling [27, 33].

1.1.2

Age-Related Neurodegenerative Disorders

The structural and functional integrity of dendritic spines is largely dependent on actin cytoskeleton [34]. The dynamic equilibrium between G-actin/F-actin brings about activity-dependent structural enlargement/retraction at pre- and postsynaptic ends of dendritic spines known as synaptic plasticity. The strength of the synapse is critical for immediate processing and transmission of signals in neuronal circuits [35–37]. Any alteration in these dynamics is associated with the synaptic degeneration and ultimate loss, typical in Alzheimer’s disease (AD) [38]. A recent study by Kommaddi et al. (2018) showed that the accumulation of β-amyloid peptides mediates the disruption of outwardly polymerizing F-actin nano-architecture leading to synaptic shrinkage and loss in early AD mice [39, 40]. In neurodegenerative diseases, the cytoskeleton is abnormally assembled, and impairment of neurotransmission occurs. The progression of neurodegenerative diseases is associated with the formation of intra-neuronal Hirano bodies and ADF/cofilin rods [41]. Hirano bodies are paracrystalline inclusions formed due to clustering and aggregation of actin filaments and actin-binding proteins [42–44]. Yun Dong et al. showed that de novo actin polymerization is essential for the formation of Hirano bodies in Dictyostelium [45]. The sequestration of proteins that sever actin filaments, actindepolymerizing factor (ADF) and cofilin, in Hirano bodies clearly indicates the critical role of these proteins in maintaining actin homeostasis and preventing accumulation of such cross-linked structures [46].

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Actin: A Vital Player in Cell Motility

Actin is also a key player in cellular motility in wide variety of organisms. Different organisms adopt different modes of cellular motility to survive. These include either the use of discrete locomotory organelles or continuous reorganization of cytoskeletal and motor proteins bridging the cell membrane and substrate. The locomotory function of any cell is fundamental for growth and development, transport of food and oxygen, migration to an optimal surrounding and wound repair. Despite of vast diversity of life forms, there are relatively fewer forms of motility seen.

1.2.1

Swimming: A Cellular Motility Phenomenon

Swimming forms one of the modes of cellular locomotion in primitive eukaryotes, ciliates, bacteria, green alga Chlamydomonas, Tetrahymena, etc. These organisms make use of their appendages, namely, cilia and flagella, specialized in locomotion [47–50]. These organelles rely on dynein motor proteins residing with the microtubules meshwork to help the organisms propel through the aqueous environment [51, 52]. The bacterial flagellum, most studied of all the prokaryotic motility structures, aids in whole cell locomotion as well as helps the pathogenic bacteria to anchor onto and colonize the host cells, thereby transmitting the diseases [53, 54]. Archaeal flagella differ slightly from their bacterial counterparts [55]. The motility apparatus of sperm flagella comprises of highly organized microtubule-based structures called axonemes. This helps the sperm to swim through the fluid to fertilize the egg [56–58]. Other forms of motility also swarming, twitching/gliding and sliding or spreading are also observed in different prokaryotic organisms [59].

1.2.2

Actin-Driven Cellular Motility

Another kind of cell locomotion observed is of amoeboid type driven by reorganization of cytoskeletal protein called actin. Actin is a building block protein, and its association with actin-binding proteins generates diverse structure each serving a unique function. The polymerization of monomeric globular actin (F-actin) into filamentous form (F-actin) drives the crawling locomotion in amoeboid and eukaryotic cells. Constant remodelling of actin cytoskeleton has been implicated in several cellular events, including cell motility, cytokinesis and intracellular transport [60]. This actin cycling is tightly controlled by a large number of actin-binding proteins (ABPs) which include actin-nucleation machinery (Arp2/3 complex and formins), actin disassembly machinery (ADF/Cofilin) and actin contractile machinery (myosins) [61–65]. The detailed insights on how actin helps in different cellular functions are given below.

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1.2.2.1 Directional Motility On establishment of polarity in response to different signals, a cell rearranges its cytoskeleton components accordingly. Directional motility is then achieved by forming of protrusions at the leading edge of the cell, adhering of cell membrane to the substrate, translocation via actin-myosin contractile forces followed by retraction of the trailing edge [66]. The formation of protrusions is mediated by polymerization of actin filaments by addition of monomeric subunits to their barbed (fastgrowing, plus) ends. Moreover, studies have shown that these filaments orient their barbed ends preferentially in the outward directions [60, 67, 68]. The polymerization of actin filaments and their association with actin regulatory proteins produce a variety of architectures like branched or cross-linked meshwork in the lamellipodia, parallel bundles in filopodia and antiparallel structures in contractile fibres, as a function of time and space [69]. Depending upon their cellular environment and type, cells organize different protrusions to fulfil their roles, for example, lamellipodium in epithelial keratocytes for gliding towards the wounded area for its healing, pseudopods of neutrophils for locating and destroying pathogens or cancer cells and filopodia of growth cones of neurons for wiring of neuronal circuits [70–72]. 1.2.2.2 Cytokinesis Cytokinesis and wound closure in many eukaryotes require the formation of contractile rings in order to separate the dividing cells into two [73, 74]. These rings are composed of actin filaments cross-linked myosin II motor proteins and many actinbinding proteins [75, 76]. The Rho family of GTPases are associated with the regulation of initiation, formation and restriction of the actin ring. The actomyosin ring drives the constriction of the ring forming the cleavage furrow, segregating the cell into two halves [77, 78]. 1.2.2.3 Organelle Transport and Protein Trafficking Actin cytoskeleton along with molecular motor proteins are essential for transport of various organelles – Golgi apparatus, endoplasmic reticulum (ER), mitochondria, endosomes and lysosomes – as well as their retention at specific locations in cell, directed transport of proteins within subcellular organelles and release of exocytic vesicles in secretory cells [79–81]. The orientation of plus and minus ends of actin and microtubules determines the direction of transport. Establishment of polarity activates formin which associates with each of the fast-growing ends (plus end) of actin and promotes nucleation and elongation. The rapid polymerization of actin forms tracks on which myosin motors walk along to transport organelles and protein vesicles. Several studies have also shown that actin filaments are also required for proper organization of Golgi stacks [82]. The actin-binding proteins and other cellular factors mediate interaction with motor proteins to organize vesicles and cargo transport. There are several examples of different myosin proteins implicated in the transportation of transport vesicles on actin meshwork. These include myosins I and VI in the transport of Golgi-derived vesicles to plasma membrane, myosin II in

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transport of Golgi stacks and Golgi-derived vesicles and myosin V for transport of ER vesicles in nerve cells [83]. Apart from eukaryotic cells, actin cytoskeleton is also taken over by numerous pathogens like Listeria monocytogenes, Shigella flexneri and vaccinia virus to infect eukaryotic cells [84–86]. These bacteria induce massive rearrangements of actin cytoskeleton to facilitate their own movement within the host cells. Bacterial nucleation-promoting surface proteins (ActA in L. monocytogenes and IcsA of S. flexneri) recruit Arp2/3 complex to initiate polymerization of actin filaments forming a dense network of actin bundles called comet-like tail. The contraction of these actin bundling propels the bacteria in forward direction [87–90]. The intercellular spread and subsequent infection is mediated by forming membrane protrusions containing bacterial cells at the tip actin tails. Even actin-depolymerizing protein ADF/cofilin, severing protein profilin and α-actinin have shown to participate in efficient bacterial movement by maintaining the association and dissociation of actin monomers at plus and minus ends [91–96].

1.3

Gliding Motility

This is a substrate-dependent cell locomotion empowered by either actin or microtubule, depending on the cell type. This kind of motility is observed in raphid diatoms [97], flagellated green alga Chlamydomonas [98], net slime mould Labyrinthula [99] and even some bacteria [100, 101]. In addition to these pathogens, a large group of parasites, belonging to phylum Apicomplexa, also harness actin cytoskeleton of the host cells to establish a successful infection [102, 103] as explained in the subsequent section.

1.3.1

Gliding Motility in Apicomplexan

Apicomplexan belong to a large group of obligate intracellular parasites and are responsible for some of the life-threatening diseases in humans (malaria, cyclosporiasis, isosporiasis, toxoplasmosis, etc.) and in cattle and poultry animals (babesiosis, theileriases, coccidiosis, etc.). These protozoans are known to have complex life cycles with sexual and asexual stages [104–109]. Depending upon the differences in their life cycle, apicomplexans are classified into three different classes: Hematozoa (Plasmodium and Toxoplasma), Coccidia (Eimeria tenella and Theileria annulata) and Gregarinia (Gregarina niphandrodes) [110– 114]. According to the WHO statistics for the year 2015, the global mortality due to malaria was 4.38 million, while 3.2 billion people are at risk of malaria (http:// www.who.int). Other apicomplexans such as Toxoplasma gondii and Cryptosporidium parvum are relatively benign in individuals with robust immune system. However, these organisms can be a serious threat and potentially life-threatening in immunocompromised patients suffering from AIDS, organ transplant recipients and immunosuppressed cancer patients [115].

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Other members of apicomplexans such as Babesia, Eimeria tenella and Theileria annulata, which infect cattle and poultry, are also serious nuisance-causing organisms in the livestock industry and are opportunistic parasites on humans [115]. Some apicomplexans such as Cryptosporidium adopt a single host system (monoxenous) [116], while others such as Plasmodium possess a two-host system (heteroxenous), where the intermediate host, also known as the vector, serves as a carrier vehicle for transfecting the definitive host with the parasite [117]. Despite the difference in the host systems of the apicomplexan parasites, the molecular aspect of invasion remains highly conserved throughout the different classes of the phylum [102, 104, 112, 113, 118]. Apicomplexans invade the host cells by employing a unique mechanism known as ‘gliding motility’. Following attachment to the host surface, the organism ‘crawls’ over the surface of the host cell to find a specific receptor and subsequently punctures into it [2–5]. Gliding motility is executed by actin-myosin cytoskeletal elements [1–6]. The significance of myosin motor in promoting motility is well-studied and documented [6, 7]. This myosin motor is in turn governed by the treadmilling phenomenon exhibited by actin monomers. However, the polymerization regulation of actin responsible for aforementioned phenomenon is least understood. The entire machinery responsible for the gliding motility is located in a space of approximately 20 nm between plasma membrane and the inner membrane complex (IMC) of the parasite [104, 119]. Owing to the serious threats from apicomplexan parasites to humans, animals and livestock, drug development against these parasites is of medical and economical importance. Developing drugs against these organisms poses a challenge, as these parasites share many of the metabolic pathways with their hosts [120]. Reasonable success was achieved in regulating malarial outspread using insecticides such as pyrethroids [121]. Currently, the antiparasitic drug targets the plastid structure found commonly in all apicomplexans [106]. Most of the Plasmodium parasites have developed high resistance against the most commonly available antimalarial drugs such as quinine, chloroquine and sulfadoxine-pyrimethamine [122]. Recent studies have also showed emerging of drug-resistant variants of T. gondii and Babesia [123]. Actin is a ubiquitous cytoskeletal housekeeping protein [124] and therefore is not the ideal choice for therapeutic applications. Developing efficient drug strategies, using actin and its regulators, requires understanding the mechanism of actin polymerization and depolymerization and the differences between apicomplexan and their host actin regulation mechanisms. Among the various organisms of the apicomplexan phylum, despite the sequential and structural differences in actin, in all of them, actin is regulated by a minimal set of seven regulators, viz. formin, profilin, actin depolymerisation factor (ADF), capping proteins (CPα and CPβ), cyclase-associated protein (CAP) and coronin [118, 125, 126]. In higher eukaryotes, actin is regulated by a multitude of 150–200 regulators [126]. Some of the actin regulators from apicomplexans show structural and sequential differences in comparison to their counterparts in their respective hosts, suggesting that such regulators can be pursued for drug development [126].

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In the following chapters, we have individually discussed about actin and the role of each of its regulators in apicomplexan along with their structural and sequential analysis. From the available pool of data, we have compared and contrasted the structure and molecular functions of actin and its regulators with their respective counterparts across the wide spectrum of eukaryotic organisms. In our study, we also report on the evolution of the actin protein sequence back from prokaryotes. Further, using actin as an evolutionary marker, we tried to trace the evolution of its regulators involved in the gliding motility in apicomplexans. Our bioinformatics analysis also offers novel insights into the functional aspects of regulators mediating actin polymerization. Based on the available kinetic data on these actin regulators across various eukaryotes, we further propose a mathematical model to explain the actin treadmilling mechanism for Plasmodium parasites. Apart from polymerization and depolymerization, a number of regulators (profilin, FH (formin), ADF, CAP, capping protein (α and β), coronin) play an important role to control actin treadmill. However, the mechanistic detail of the control and cross-talk among regulators is not well understood. Theoretical methods were used to study filament size at steady state, nucleation time and elongation time in order to understand the control of treadmill in detail. From the current analysis, we propose that there might be a strong synergistic cross-talk between these actin regulators and this plays a vital role in the entire mechanistic process. Additionally, we also observe that with the help of multitasking capability and strong cross-talk among regulators, actin treadmill emerges as a robust mechanism to estimate a broad range of environments thus, providing an accurate motility to Plasmodium.

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Actin: The Central Ubiquitous Player in the Phenomenon Samridhi Pathak, Ricka Gauba, Sarath Chandra Dantu, and Avinash Kale

Abstract

Actin is a ubiquitous cytoskeletal protein and is a central player in the actinmediated gliding motility phenomenon. In this chapter, we have explored the structural aspect of actin in general and various aspects of actin polymerization. We briefly discuss the different forms of actin found in apicomplexans. Further we present the various structural details known about the molecule and have summarized the functional role of every section of the protein and its metal ions. We also have discussed about the actin polymerization process in general by providing the brief of conformational changes of actin molecule (G-actin). We also provide an overview of the actin polymerization process followed by kinetics and thermodynamics of actin polymers (F-actin). Finally, we conclude by summarizing the interactions of actin regulatory drugs and by discussing their plausible modes of action on actin.

Electronic Supplementary Material The online version of this chapter (https://doi.org/10.1007/ 978-981-13-7450-0_2) contains supplementary material, which is available to authorized users. S. Pathak · R. Gauba · A. Kale (*) School of Chemical Sciences, UM-DAE Centre for Excellence in Basic Sciences (CEBS), Mumbai, Maharashtra, India e-mail: [email protected]; [email protected] S. C. Dantu Department of Computer Science, Synthetic Biology Theme, Brunel University London, Uxbridge, UK e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 A. Kale (ed.), Actin Polymerization in Apicomplexan, https://doi.org/10.1007/978-981-13-7450-0_2

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Introduction

Actin (molecular weight ¼ 43,000) [1] is a housekeeping protein, involved in cellular processes such as cytokinesis, cell locomotion, transport of organelles and morphogenesis [2–5]. It belongs to ATPase superfamily. Actin is the most abundant intracellular protein, ubiquitously present in all eukaryotes. In higher eukaryotes, it exists in two types: muscle actin and non-muscle actin. In both types, monomeric, globular actin (G-actin) polymerizes to form filamentous actin (F-actin). In this chapter, we are referring to non-muscle actin from higher eukaryotes [6]. A fundamental difference between the actin from higher eukaryotes and that in apicomplexans is that the latter cannot form long filamentous fibres as the former [7]. Actin in apicomplexans forms short, wobbly filaments as observed in Plasmodium [8–10] and T. gondii [11]. Theileria annulata is an exceptional apicomplexan, which does form long filaments [10, 12]. Moreover, only 2% of actin is predicted to polymerize into filaments in apicomplexans, while the rest of the actin pool exists in monomeric form [7, 13].

2.2

Sequence Analysis of the G-Actin

Actins are highly conserved across the eukaryotic species, except T. annulata, Aedes aegypti, Necator americanus, Amphimedon queenslandica and Microcystis aeruginosa (Supplementary table ST_ACT-1) [10]. Vahokoshi et al. [10] proposed that all Plasmodium species have two actin isoforms: actin1 (PfACT1) and actin2 (PfACT2). PfACT1 is expressed throughout the life cycle [14], and PfACT2 is expressed during the sexual stage (both male gamete and zygote) of the organism [10]. PfACT1 and PfACT2 show 78% similarity, yet are functionally different. Replacement of PfACT1 for PfACT2 does not compensate for the function of the latter during gametogenesis [10]. Non-Plasmodium apicomplexans, on the other hand, consist of only one actin, which is similar to PfACT1. It is argued that since PfACT1 shows about 80% similarity with actin from higher eukaryotes, it may differ functionally from the latter [7, 10]. However, we point in opposition since 80% similarity provides high enough confidence in the homology of PfACT1 with actins from higher eukaryotes (Supplementary table ST_ACT-1).

2.3

Structure of G-Actin

Actin monomer has five central β-sheets and three α-helixes connected via X loops, and many of these loops have functional roles. Atomic resolution structures of PfACT2 and PbACT1 are not known. Therefore, we will consider PfACT1 and PbACT2 for our further structural analysis. The secondary structure of PfACT1 contains 57% α-helix and 22% β-sheet, whereas Plasmodium berghei actin2 (PbACT2) contains 47% α-helix and 27% β-sheet, which is quite similar to those of actin crystal structure elucidated from other eukaryotic species [15].

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We used the sequence of actin to identify the existence of function-related conserved motifs using ScanProsite [16] (Supplementary table ST_ACT-1). However, it did not reveal any motif, due to the overall sequential resemblance of actin across the eukaryotic species.

2.3.1

3D Structure of G-Actin

2.3.1.1 Architecture of G-Actin We further analysed the available tertiary structures (Supplementary table ST_ACT-3) of actin to understand the organization of the protein and how it supports the functional mechanism of actin. We examined the domain organization and structure-function relationship of actin. Actin consists of two major domains, viz. a small domain and a large domain, separated by a hinge region. The small domain consists of subdomain 2 (SD2) and subdomain 4 (SD4), whereas the subdomain 1 (SD1) and subdomain 3 (SD3) constitute the large domain [17]. The domain architecture of actin is conserved across the whole eukaryotic species. SD2 and SD4 form the pointed end, and SD1 and SD4 form the barbed end. Monomeric actin, i.e. G-actin, can bind to both ATP and ADP mediated by Mg2+/ 2+ Ca , which is essential to continue actin polymerization at a steady state [18, 19]. ATP or ADP bind to the nucleotide-binding cleft, located at the interface between the domains (Fig. 2.1). Loss of nucleotide causes an irreversible denaturation of actin [20]. Polymerization of actin has two processes, nucleotide exchange of ADP to ATP in G-actin and ATP hydrolysis, while binding to various regulators. Different structural elements of actin have specific roles to carry out the abovelisted functions. S-loop, G-loop and H-loop are involved in ATP hydrolysis; W-loop is a regulator binding loop; and H-plug, loop 60–69 and D-loop are involved in lateral and longitudinal interactions. These loops are discussed in detail below. 2.3.1.2 Loops 2.3.1.2.1 S-Loop (Residues 11–16) and G-Loop (Residues 154–161) These loops are present in the vicinity of the nucleotide-binding cleft and are involved in direct ATP/ADP binding. The incoming nucleotide, bound to the MG2+/Ca2+, is clamped between S-loop and G-loop, which are also known as P1 and P2 loops, respectively, as it binds to the α-phosphate and β-phosphate (Kudryashov and Reisler, 2012). S14 in the S-loop is considered as a sensor for ATP hydrolysis. This residue binds to the β-phosphate and drifts away from γ-phosphate to avoid steric hindrance [21, 22]. 2.3.1.2.2 H-Loop (Residues70–78) The shift in the G- and S-loops is propagated in the H-loop, which contains an important residue H73 [23]. Methylation of H73 is considered an important activity, favouring delayed Pi release from the actin subunits [22, 24], ascertained by the fact

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Fig. 2.1 Actin monomer is depicted as cartoon. Subdomains 1, 2, 3 and 4 are shown as transparent surface and are demarked with different colours of blue-white, green, pink and wheat, respectively. Nucleotide (ADP) bound to actin is shown as sticks. All the prominent loops are highlighted with distinct colours. The figure is made from the structure adopted from Protein Data Bank with Pdb id: 1J6Z

that yeast actin, lacking methylated H73, experiences undulated Pi release during actin polymerization [22, 25, 26]. PfACT1 is also reported to be unmethylated at H73 [8, 11]. The S-loop, G-loop and H-loop work in a synchronized manner to release the γ-phosphate through a back-door exit. The S-loop, G-loop and prolinerich region (consist of P109 and P112) constitute the back-door exit region for γ-phosphate. Methylated H73 is known to aid in the exit [24].

2.3.1.3 Dnase-Binding Loop (D-Loop, Residues 38–52) This loop is located in SD2 and is a disordered loop in G-actin and an ordered helix in F-actin [27]. It is called the Dnase-binding loop because of its interaction with DNase-I. Transition of G-actin to F-actin involves small domain movements, except SD2 (containing D-loop), which is twisted by 150 in the F-actin [28]. Structural data

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suggest that SD2 is drifted away from SD4, which allows a larger area for nucleotide exchange [29]. In F-actin, it is also known to participate in longitudinal interactions [30], by intercalating in the hydrophobic groove between SD1 and SD3 of the adjacent protomer [29]. This interaction is stabilized by V43 and M44 of the D-loop and L346 and F375 of the adjacent protomer [31]. Such interaction is absent in G-actin, which may account for its disordered structure. No direct participation of the D-loop is observed in ATP hydrolysis. However, while examining the structural data of G-actin, we observed that, in the open state of actin, there is a significant shift in the position of D-loop due to shifting in the loop preceding it. Also, studies with TMR-labelled G-actin demonstrate that in ADP-bound G-actin, D-loop adopts a helical conformation, while ATP-bound G-actin is in the coil state [19, 32]. But Rould et al. in 2006 reported that the D-loop is in the coiled state in ADP- and ATP-bound state [21]. A P38A mutation in the D-loop led to a decrease in the rate of actin translocation in vitro assay [30].

2.3.1.4 Loop 60–69 This loop is located at the top of the nucleotide-binding cleft, and its sensitivity to trypsinization is dependent on the nucleotide and the divalent cation bound to actin such that it is accessible for proteolyses in Ca+2-ATP-G-actin and Mg+2-ADP-Gactin state, less accessible in Mg+2-ATP-G-actin and completely inaccessible in filamentous actin. It is suggested that ADF/cofilin performs the function of depolymerization of actin by targeting the trypsinization of this loop [33]. 2.3.1.5 W-Loop (Residues 165–172) The W-loop is the binding site for profilin, cofilin and twinfilin [22, 34]. Moreover, it also plays a role in longitudinal and lateral actin interaction [22, 35, 36]. This loop is the interaction site for ATP/ADP. MD simulations suggest that W-loop of actin is in a β-sheet conformation when bound to ADP or ADP-Pi, due to hydrogen bonding between the backbones of Y116 and Y169. However, it adopts a loop conformation in ATP state [37, 38]. Studies by other investigators suggest that ATP- and ADP-Pibound actins are closer in structure and more stable that ADP-actin. Kudryashov et al., 2010, also suggest that functional properties of W-loop are similar in ATP- and ADP-Pi-actin and differ from ADP-actin. The loop-to-sheet conformation apparently originates from the structural changes in the H-loop which further causes structural rearrangements at the beginning (G158 and V159) and end (R177 and E179) of the W-loop [39, 40]. 2.3.1.6 Hydrophobic Plug (H-Plug) (Residues 264–273) This loop generates a finger-like projection, which docks the hydrophobic plug of F-I-G-M from the loop in the adjacent subunit (Y166, A169, L171, H173, C285, I289, G63, I64 and residues 40–45) [28]. Without this interaction, the stability of the actin filament is challenged. Electron microscopy and MD studies suggest that, at the pointed end, the terminal subunit is tilted towards the penultimate subunit by 120 . This requires the

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interaction of the H-plug of the terminal subunit with the D-loop of the penultimate subunit. In order for an actin monomer to polymerize at the pointed end, it must break this interaction [41]. Moreover, the surface of D-loop which does not interact with the H-plug forms a tight intermolecular loop-to-loop interaction with the terminal subunit. Such interactions are not observed at the barbed end of the filament as the loops are buried in the structure. These tight interactions are also suggested to be responsible for the slow association and dissociation of subunits at the pointed end of the filament [41].

2.3.1.7 C-Terminal (Residues 349–375) and N-Terminal (Residues 1–10) Loops These loops are considered as regions of highest structural flexibility. MD simulation studies suggest a 5 Å shift in the position of the C-term loop from ADP to ATP state. C-term loop is also structurally connected to the nucleotide-binding cleft which facilitates an allosteric relationship between the two loops, such that in a polymorphic F-actin, these two loops form alternative contacts between SD1 and SD2 of the longitudinal protomers. The N-tem loop is also highly unstable and the least conserved part of the molecule [40].

2.4

Role of Metal Ions

Metal ions play a very significant role in the actin polymerization dynamics. Actin binds to Mg2+ and Ca2+. These divalent cations tightly bind to the nucleotide in the nucleotide-binding cleft [1]. Absence of the divalent cation leads to denaturation of the actin monomer [42], and actin polymerization comes to a standstill [1]. Actin is known to possess six sites of interaction with Mg2+. Actin nucleation occurs faster in the presence of MgCl2 than CaCl2 [43, 44]. Thus, polymerization is better favoured by Mg2+. It is also observed experimentally that Ca2+ promotes monomer state, while Mg2+ promotes filamentous state of actin [1, 42]. Actin binds with higher affinity to Mg2+ (10 nM) than Ca2+ (2 nM) at neutral pH [29, 45]. ATP binds Mg-Gactin with a higher affinity (1.2 nM) than Ca-G-actin (0.12 nM) [29]. An in vitro experiment, reported by Carlier et. al, suggests that replacement of Ca2+ with Mg2+ activates polymerization. This is favoured because of the smaller size of Mg2+, which eliminates one water molecule from the coordination sphere [46], that allows considerable flexibility to the protein. Its interactions with all the four subdomains of actin aid in its longitudinal and lateral interactions [24]. Plasmodium G-actin binds divalent cations, mainly Ca2+, whose removal reduces the polymerization rates of actin and, however, does not lead to irreversible denaturation [46, 47].

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Conformational States of Actin

From the structures solved by various X-ray studies, structures of actin can be classified into three states based on the relative rotation of the small and the large domain, i.e. wide open, partially open and closed [17, 48–52]. Transition from one conformational state to another requires the participation of various loops of the actin molecule as extensively (Fig. 2.1) [8, 11, 21, 23–26, 30, 31, 33–36, 38, 41, 51, 53– 57]. In the closed conformation, the nucleotide is state of bovine actin, the two major domains are rotated by 9 in comparison to open state actin, while a rotation of 4.7 is observed between the major domains in the closed state, in comparison to the partially open state [30]. In the closed conformation, the nucleotide is buried in the cleft, stabilized by 10–12 hydrogen bonds, while in the open state, it is almost outside the nucleotide pocket, and the stabilizing bonds are disrupted, leaving