How Structural Modifications Affect the Regulation and Activation Mechanisms of CD44 and JAK2

Cell surface receptor proteins are important gatekeepers. They enable cells to react to their surroundings by receiving and propagating chemical signals through the cell membrane. Countless such reactions occur in the cells of our bodies every moment to keep us alive. Hence, understanding how these molecules operate helps us to improve our health and the quality of our life. In this Thesis, we discuss the activation of two cell signaling-related proteins, CD44 and JAK2. CD44 is a cell surface receptor for a carbohydrate called hyaluronan. Through hyaluronan binding, CD44 is involved in various signaling cascades that regulate, e.g., cell–cell interactions as well as cell differentiation, proliferation, and survival. JAK2 is a non-receptor tyrosine kinase — an intracellular signaling protein that binds to cytokine receptors, forming a receptor–JAK signaling complex. Through this interaction, JAK2 mediates central physiological functions, including hematopoiesis and immune response. Despite their physiological significance, the understanding of these proteins is incomplete because their atomic-level operating principles have not yet been fully elucidated. Therefore, we use biomolecular simulations to shed light into the function and regulation of these proteins and their cognate molecules. In the case of CD44, we expand the current knowledge on the details of its hyaluronan binding. We also show how N -glycosylation of the receptor can modulate the ligand binding by altering its binding site preference. In the case of JAK2, we find how the activation of the signaling complex is controlled by intracellular dimerization and proper orientation through specific membrane binding. Our work provides novel atomic-level insight into the functions and interactions of the studied proteins. The results can be useful in drug development — especially in the search for new drug binding sites, for example, at glycosylation, dimerization, or membrane binding interfaces of proteins. Finally, this work highlights the added value gained by bridging computer simulations with experimental techniques.Solupinnan reseptoriproteiinit ovat tärkeitä portinvartijoita. Ne vastaanottavat solunulkoisia signaaleja ja välittävät niitä eteenpäin käynnistämällä solujen sisäisiä kemiallisia kaskadeja. Lukemattomia tällaisia reaktioita tapahtuu kehomme soluissa joka hetki, jotta pysyisimme hengissä. Siksi näiden molekyylien ja niiden toiminnan ymmärtäminen on keskeistä esimerkiksi sairauksien hoidossa. Tässä väitökirjassa käsittelemme kahden solun signalointiin liittyvän proteiinin, CD44:n ja JAK2:n, aktivaatiota. CD44 on luonnollisen hiilihydraattipolymeerin, hyaluronihapon, solupintareseptori. Hyaluronihapon sitoutumisen kautta CD44 osallistuu erilaisiin signalointikaskadeihin, jotka säätelevät esimerkiksi solujen välistä vuorovaikutusta sekä solujen kasvua, lisääntymistä ja eloonjäämistä. JAK2 puolestaan on tyrosiinikinaasi, joka ei kuitenkaan itse toimi reseptorina. Se on signalointiproteiini, joka sitoutuu sytokiinireseptoreihin muodostaen signalointikompleksin, johon kuuluu sekä JAK2 että reseptori. Tämän vuorovaikutuksen kautta JAK2 säätelee monia kehomme prosesseja, kuten hematopoieesia ja immuunivastetta. Käsitys näiden proteiinien toimintaperiaatteista on edelleen puutteellista, sillä niiden molekyylitason toimintaa ei vielä täysin ymmärretä. Tämän vuoksi käytämme tietokonesimulaatioita valaisemaan kyseisten proteiinien toimintaa ja säätelyä nanomittakaavassa. CD44:n tapauksessa laajennamme nykyistä käsitystä sen ja hyaluronihapon vuorovaikutuksesta. Näytämme myös, kuinka CD44:n glykosylaatio voi moduloida hyaluronihapon sitoutumista muuttamalla sen sitoutumispaikkaa. JAK2:n tapauksessa osoitamme, kuinka sekä dimerisaatio että sitoutuminen solukalvoon ohjaavat signalointikompleksin aktivaatiota. Työmme avulla saavutettiin uutta tietoa tutkittujen proteiinien toiminnasta ja vuorovaikutuksista. Tuloksista voi olla hyötyä lääkekehityksessä — erityisesti etsittäessä uusia lääkeaineiden sitoutumiskohtia esimerkiksi proteiinien glykosylaatio-, dimerisaatio- tai kalvoonsitoutumisrajapinnoista. Työ korostaa myös tietokonesimulaatioiden ja kokeellisten tekniikoiden yhdistämisen avulla saavutettua lisäarvoa tieteellisessä tutkimuksessa

Binding of Hyaluronic Acid to Its CD44 Receptor

CD44 is a transmembrane receptor protein binding its carbohydrate ligand, hyaluronic acid (HA), in a reversible fashion. In addition to enabling normal cell migration, such as the rolling of white blood cells, these carbohydrate-protein interactions are exploited by malignant cancer cells metastasizing through the blood stream. A normal cell therefore requires effective regulatory mechanisms for controlling the binding affinity between CD44 and HA. Earlier studies addressing this topic have, however, been unable to identify these regulatory mechanisms. More precisely, traditional wet-lab experiments, limited by their spatial and temporal resolution, have been inadequate in describing transient nanoscale phenomena, such as the ligand binding. Previous computer simulations have, on the other hand, been limited by both veracity of simulation models and availability of computational resources. In this Thesis, we use all-atom explicit-solvent molecular dynamics (MD) simulations to study the adsorption of HA oligomer to a human wild-type CD44 HA binding domain (HABD). In practice, we explore the role of three potential regulation mechanisms: size of the ligand, glycosylation of the protein, and conformation of the protein. First, free energy profiles for the adsorption of HA octamer, revealing the strength of an individual CD44-HA interaction to be over 25 kJ/mol, suggest ligand binding to be irreversible. Second, by glycosylating the HABD at residues Asn25, Asn100, and Asn110, we show the first of these residues to block most of the native binding interactions. As a result, the strength of the adsorption is, at least when using charge-neutral core pentasaccharides as the attached carbohydrates, decreased by 40 %. More surprisingly, the simulation data reveal a conformation transition previously correlated with high-affinity binding to, in fact, act as a molecular mechanism repelling the bound HA oligomer, and thereby dynamically regulating the biological activity of the CD44. The findings of this study uncover how the binding of HA to its CD44 receptor is regulated. This information may facilitate the design and targeting of novel drugs and therapies against conditions, such as cancer. Lastly, the insight from this study is of potential value when considering the carbohydrate-protein interactions of other cell surface receptors

Atomistic fingerprint of hyaluronan-CD44 binding

Hyaluronan is a polyanionic, megadalton-scale polysaccharide, which initiates cell signaling by interacting with several receptor proteins including CD44 involved in cell-cell interactions and cell adhesion. Previous studies of the CD44 hyaluronan binding domain have identified multiple widespread residues to be responsible for its recognition capacity. In contrast, the X-ray structural characterization of CD44 has revealed a single binding mode associated with interactions that involve just a fraction of these residues. In this study, we show through atomistic molecular dynamics simulations that hyaluronan can bind CD44 with three topographically different binding modes that in unison define an interaction fingerprint, thus providing a plausible explanation for the disagreement between the earlier studies. Our results confirm that the known crystallographic mode is the strongest of the three binding modes. The other two modes represent metastable configurations that are readily available in the initial stages of the binding, and they are also the most frequently observed modes in our unbiased simulations. We further discuss how CD44, fostered by the weaker binding modes, diffuses along HA when attached. This 1D diffusion combined with the constrained relative orientation of the diffusing proteins is likely to influence the aggregation kinetics of CD44. Importantly, CD44 aggregation has been suggested to be a possible mechanism in CD44-mediated signaling.Peer reviewe

Maturation of the SARS-CoV-2 virus is regulated by dimerization of its main protease

SARS-CoV-2 main protease (Mpro) involved in COVID-19 is required for maturation of the virus and infection of host cells. The key question is how to block the activity of Mpro. By combining atomistic simulations with machine learning, we found that the enzyme regulates its own activity by a collective allosteric mechanism that involves dimerization and binding of a single substrate. At the core of the collective mechanism is the coupling between the catalytic site residues, H41 and C145, which direct the activity of Mpro dimer, and two salt bridges formed between R4 and E290 at the dimer interface. If these salt bridges are mutated, the activity of Mpro is blocked. The results suggest that dimerization of main proteases is a general mechanism to foster coronavirus proliferation, and propose a robust drug-based strategy that does not depend on the frequently mutating spike proteins at the viral envelope used to develop vaccines. (c) 2022 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Peer reviewe

Dynamics and energetics of the mammalian phosphatidylinositol transfer protein phospholipid exchange cycle

Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITP alpha, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITP alpha lipid exchange cycle: (i) interaction of PITP alpha with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITP alpha to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITP alpha; (iv) the trajectory of PtdIns or PtdCho into and through the lipidbinding pocket is chaperoned by sets of PITP alpha residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITP alpha PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family.Peer reviewe

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin -binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.Peer reviewe

Molecular basis of JAK2 activation in erythropoietin receptor and pathogenic JAK2 signaling

Janus kinase 2 (JAK2) mediates type I/II cytokine receptor signaling, but JAK2 is also activated by somatic mutations that cause hematological malignancies by mechanisms that are still incompletely understood. Quantitative superresolution microscopy (qSMLM) showed that erythropoietin receptor (EpoR) exists as monomers and dimer-izes upon Epo stimulation or through the predominant JAK2 pseudokinase domain mutations (V617F, K539L, and R683S). Crystallographic analysis complemented by kinase activity analysis and atomic-level simulations revealed distinct pseudokinase dimer interfaces and activation mechanisms for the mutants: JAK V617F activity is driven by dimerization, K539L involves both increased receptor dimerization and kinase activity, and R683S prevents autoinhibition and increases catalytic activity and drives JAK2 equilibrium toward activation state through a wild-type dimer interface. Artificial intelligence–guided modeling and simulations revealed that the pseudokinase mutations cause differences in the pathogenic full-length JAK2 dimers, particularly in the FERM-SH2 domains. A detailed molecular understanding of mutation-driven JAK2 hyperactivation may enable novel therapeutic approaches to selectively target pathogenic JAK2 signaling.Peer reviewe

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.</p

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo

Atomistic Fingerprint of Hyaluronan-CD44 Binding: Weak E-field Simulations, Upright Mode

Simulation files (Gromacs 4.6.7 format) for the "E-field weak, upright mode" simulations in Ref. [1]. There are 20 replicas marked with "_1" , "_2", etc. Files include: -trajectories (.xtc) that are saved every 100ps -initial structures (.gro), -run input files (.tpr) -checkpoint files (.cpt) -simulation parameter files (.mdp) -system topology file (.top) -topology files included in the system topology file (.itp) [1] Vuorio J. et al., Atomistic Fingerprint of Hyaluronan-CD44 Binding, PLOS Comp. Biol., 2017. (Submitted