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5.5: Complementary Interactions between Proteins and Ligands - The Immune System and Immunoglobulins

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    14942
  • Introduction to the Immune System

     

    Now let's consider the daunting task faced by the immune system - to recognize all possible "foreign" molecules and react to them, either by targeting them for elimination, or, paradoxically, to recognize them but not react to them (a process called tolerance). The same can be said of "self-molecules". The immune system must recognize them but not respond to them, otherwise autoimmune disease might arise in which the body's powerful immune system targets self. 

    It is virtually impossible to give an in-depth description of the immune system in a short section. Our goal is simply to illustrate how the immune system recognizes such a myriad of molecules. First we will briefly cover the innate and adaptive immune system and their differences, and how some cells (macrophages in particular) in the innate immune system and cells (B and T cells) in the adaptive immune response recognize and response to target molecules and cells. Finally we'll discuss how the immune system can respond to a myriad of different molecules through recognition of common molecular patterns. Emphasis will be given to recognition. Ways to simplifying the complexities of the immune system have been presented in a fantastic book written by Lauren Sompayrac, How the Immune System Works. (2003, Blackwell Publishing. ISBN: 0-632-04702-X) and adopted here 

    Before we start, think of the variety of chemical species that the immune system should recognize as foreign:

    • a bacterial glycan or glycolipid on the outside of the cell
    • a viral surface protein, such as the spike protein of the SARS Coronavirus 2
    • bacterial dsDNA (and not host dsDNA)
    • viral dsRNA (which is not common in host systems
    • a self protein that has been modified in a tumor cell
    • a crystal of urea
    • extracellular ATP (a place where it is not usually found)
    • a silica particle found in particles like asbestos.

    How would you design an immune system to bind each of the "enemy" targets above?  That is what we will explore in this sections - the binding interactions.  What happens after the binding is beyond the scope of this section and fall generally in the field of signal transduction - how binding events at the self surface are transfer into intracellular responses.

    Three lines of defense protect us from the "enemies", foreign substances (bacteria, viruses and their associated proteins, carbohydrates, and lipids) collectively called antigens.

    • physical barriers of cells that line our outside surface and our respiratory, GI tract, and reproductive system
    • the innate immune system (IS) that all animals have. Composed of scavenger cells like macrophages (MΦ), neutrophils, dendritic cells, and natural killer cells (NK) that can move around the body through the blood and lymph systems and burrow into tissue to meet the enemy where they can engulf and destroy bacteria and "cellular debris". Macrophages start off as immature circulating monocytes which enter tissues by slipping through blood vessel walls, and in the process they differentiate into macrophages. There they lie in wait ready for the enemy.
    • the adaptive immune system, which, as its name implies, can change and adapt to new molecular threats. This branch is better at dealing with viruses which do their damage inside of host cells.. The adaptive IS is comprised of B cells that make and secret protein antibodies that recognize specific foreign molecules, and T cells.

    In a world experiencing the most deadly pandemic of the last 100 years, and with more to come, it's important that immune recognition be an important part of any biochemistry text. The chapter section should be a whole chapter, but we'll leave it as a very large section.  Let's start with the adaptive immune system, which we can coopt to make vaccines to the major threats we face.

    B Cells and Antibodies

    B cells and other differentiated forms of them such as B memory and plasma cells, make antibodies. Antibodies just bind to foreign molecules (proteins, glycans, lipids, etc), which might neutralize their effects, such as binding to the hemagglutinin molecule of the influenza virus and preventing its entry into the cell. We all are now familiar with the utility of vaccines that create antibodies to recognize the spike protein of the acute respiratory syndrome coronavirus 2 (SARS-CoV-2). They also bind to foreign cells like bacteria which signals other host immune proteins and cells to come in for the kill. Antibodies are secreted by B cells, which also have a membrane bound form of the antibody on their surface. This antibody acts as a receptor which recognizes antigen and through a signal transduction process, helps to activate the B cell. Mature B cells (those that have seen the antigen before) can churn out lots of antibodies quickly. Different surface antibodies, as well as secreted antibodies, can recognize and bind to almost any kind of molecules. 

    There are many forms for antibody, also called immunoglobins (Ig).  These include IgA, IgG, IgM and IgD. We will concentrate on the structure of IgG.  It consists of 4 chains (a tetramer) of two light chains and two heavy chains.  The light chains form disulfide links with the heavy chains and disulfides also link the heavy chains.  Effectively, it's one big protein molecule (about 160 K).  Figure \(\PageIndex{1}\) below shows a spacefill, secondary structure, and geometric cartoon rendering of a mouse IgG protein (pdb ID 1IGT).

    AntibodyStructureIgG_renderings.svg

    Figure \(\PageIndex{1}\): Renderings of an IgG antibody

    The antibody is shaped like a Y.   Foreign molecules (antigens) bind at the end of the top tips of the Y with both chains contributing to antigen binding.  The structures of both are dominated by antiparallel beta sheets. 

    Each chain consists of a single N-terminal variable domain (VL or VH) which participate in antigen recognition.  The light chains have an additional constant domain (CL) while the heavy chains have 3 constant domains (CH1-CH3).  The constant domains are not involved in antigen recognition but there are involved in effector functions (such as the binding of other immune molecules like complement proteins) to the antigen-bound antibody heavy chain constant regions.  Each of the domains is about 100 amino acids.  A cartoon structure with  the domain structures is shown in Figure \(\PageIndex{2}\) below. 

    AntibodyStructureIgG _fragments2.svg

    Figure \(\PageIndex{2}\): IgG antibody and fragments

    Two other features are depicted in the above figure.  In each variable region of both light and heavy changes, there are hypervariable regions, which contribute to the unique binding features of the a given antibody.  The regions are also called complementarity determining regions (CDRs).  Membrane bound forms of antibodies that serve as "receptor" proteins have additions domains now shown in the figure above.  The binding site on the antigen recognized by the antibody is called the epitope.  The corresponding binding site on the Y shaped ends of the antibody that recognized the antigen is called the paratope.

    In Figure \(\PageIndex{2}\) above, you can see that the intact full IgG molecule has in total 12 variable V and constant C domains called the immunoglobulin domain.Each has about 110 amino acid in length, two layers of β-sheets each with 3-5 antiparallel β-strands with a disulfide bond connecting the two layers.  

    When we discussed domain structure, we indicated that proteins with multiple binding domains can often be selectively cleaved with protease, with cleaved fragments often retaining binding and other functions properties.  The same as true with antibodies.  Cleavage with either the proteases pepsin or papain forms fragments with binding activities as illustrated in the figure above.  Selected protease digestion was used to clarify structure/function relationships in antibody recognition.

    When antibodies targeting different antigens were sequenced, it was clear that the much variability was found among antibodies in the variable domains of both the light and heavy chains. In those domains there were also hypervariable regions. The origin of the variability and hypervariability arise mostly from an extremely large number of gene segments (also exons) in the gene encoding the variable domains.  The exons can be spliced together at both the DNA and RNA levels to produce an incredible variety of different DNA/RNA sequences that are decoded into the variable and hypervariable regions of the light and heavy changes.  Somatic mutations are also enhanced in this region.  

    In-depth: Generation of Antibody Diversity

    We mentioned above that both DNA and RNA splicing occurs as B cells mature to become antibody secreting cells (plasma cells).  For those who have already had course that cover the Central Dogma of Biology, splicing for primary RNA transscripts should come as no surprise.  What's is surprising is that the DNA genome of B cells changes on their maturation due to splicing of a multiple of exons within the variable chain genes to produce unique coding sequences for each clone of a given B cell.  There are sets of exons (V, D and J) or segments within the genes for the variable chain.  As the immune cells terminally differentiates, a unique combination of a VDJ segment forms in the DNA genome, os each terminally differentiated B cell is different. When needed (i.e.  when their unique antigen binds to membrane forms of the antibody), the cell secretes a monoclonal antibody.

    Figure \(\PageIndex{3}\) below shows how the different segments become linked in the DNA and how they can be uniquely spiced in the RNA to produce a unique, monoclonal antibody. 

    generationAbDiversityFig1-01.svg

    Figure \(\PageIndex{3}\) Generation of Antibody Diversity.  Oliver Backhaus DOI: 10.5772/intechopen.72818.  Creative Commons Attribution 3.0 License,

    The first antibodies produced by the immune system are often of low affinity. Over time, high affinity (low KD) antibodies are produced.  What differentiates high and low affinity binding at the molecular level? Do high affinity interactions have lots of intramolecular H-bonds, salt bridges, or are hydrophobic interactions most important? Crystal structures of a variety of antibody-protein complexes were determined in order to study the basis of affinity maturation of antibody molecules. Clones of antibody-producing cells with higher affinity are selected through binding and clonal expansion of these cells. Investigators studied the crystal structure of four different antibodies which bound to the same site or epitope on the protein antigen lysozyme. Increased affinity was correlated with increased buried apolar surface area and not with increased numbers of H bonds or salt bridges as described in  Table\(\PageIndex{1}\) below.

    Antibody H26-HEL H63-HEL H10-HEL H8-HEL
    Kd (nM) 7.14 3.60 0.313 0.200
    Intermolecular Interactions
    H bonds 24 25 20 23
    VDW contacts 159 144 134 153
    salt bridges 1 1 1 1

    Buried Surface Area

    ΔASURF (A2) 1,812 1,825 1,824 1,872
    ΔASURF-polar (A2) 1,149 1,101 1,075 1,052
    ΔASURF-apolar (A2) 663 724 749 820

    Table \(\PageIndex{1}\): Characteristics of Antibody:Hen Egg Lysozyme Complexes (HEL).  Data from Y. et al. Nature: Structural Biology. 6, pg 484 (2003)

    Many crystal structures of antibody-antigen complex have been determine. Especially interesting are those in which the antigen is a protein.  Understanding antibody:protein antigen interacts is important in the development of vaccines against key epitopes in protein such as the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes Coronavirus disease 2019 (COVID-19).    Let's look in more detail at the antibody against the hen egg white lysozome (HEWL).   The crystal structure of many different IgG antibodies against HEWL have been solved.  One in particular recognizes a discontinous epitope on lysosome consisting of the following amino acids:  H15, G16, Y20, R21, T89, N93, K96, K97, I98, S100, D101, G102, W63, R73 AND L75.  Most of these amino acids are polar and five are charged.  

    Figure \(\PageIndex{4}\) below shows the interaction of part of the Fab fragment of an antibody that binds to the HEWL epitope just mentioned (3hfm). The light chain is shown in magenta, the heavy chain in dark blue, and the antigen lysozyme in gray.  The side chains of the amino acids in the epitope of HEWL are shown in sticks.  Note the complete complementary of HEWL and Fab surfaces.  Water is excluded form the interface.

     

    3hfm epitopeBindSiteSurface.svg

    Figure \(\PageIndex{4}\):  Surface interactions between a hen egg while lysozome epitope and an IgG Fab antibody fragment

     

    Figure \(\PageIndex{5}\) below shows an interactive iCn3D model of the same HEWL:Fab complex (3hfm).  Lysozyme is shown in black. 

    HenEggWhiteLysozome_FabComplex(3HFM).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{5}\): Hen egg white lysozme:Fab complex. (3hfm) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...agQkuAKhgfs177

    Here is an external link to an interactive iCn3D model showing a detailed view of the multiple interactions (salt bridges, hydrogen bonds, pi-cation)

    T Cells

    What happens if a virus makes it into a cell? Antibodies can not bind to them anymore to prevent their entry. Something must be able to recognize a virally-infected cells and eliminate it. What about a cancer cell. Wouldn't it be nice if something could recognize a tumor cell as foreign and eliminate it before it divides too much and metastasizes? Those "something" are T cells. There are many T cells in a person, and many different kinds, including T helper cells (Th), cytotoxic lymphocytes (CTL) and even suppressor T cell. The express different subsets of proteins that differentiate them and their functions.

    T cells also recognize antigens but unlike B cells, these antigens can only be proteins fragments. The membrane proteins that recognize proteins fragments are called T cell receptors. In addition, they don't recognize protein antigens in isolation. They must be bound to a protein on the surface of an "antigen" presenting cell (such as a macrophage or dendritic cell). The T cell receptor recognizes and binds simultaneously to the foreign protein fragment and to the self "antigen presenting" protein on the surface of the antigen-presenting cells. The self protein which binds and presents the foreign protein fragments (peptides) is called a Major Histocompatability Complex (MHC) protein.  

    Antigen presenting cells like macrophages and dendritic cells  have MHC Class II molecules on their surface. These bind protein fragments from engulfed bacteria, for example, and present them on the surface. T cell receptors bind to the peptide:MHC II complex. All cells in the body have MHC Class I proteins on their surface. If a cell is infected with a virus, protein fragments from the virus end up bound to the MHC Class I protein on the surface. Now a T cell can bind through its T cell receptor to the peptide:MHC Class I complex. By displaying a viral protein fragment on the surface, the immune cell can recognize a virally-infected cell without getting inside of the cell where the virus is. Sompayrac describes MHC molecules as looking like a hot dog bun. In the grove of the bun lies the peptide fragment - like the hot dog. The T cell receptor recognizes both the bun and the hot dog!

    Figure \(\PageIndex{6}\) below shows an interactive iCn3D model of a MHC Class Class I  heavy chain complexed with beta-2-microglobulin with a peptide fragment of the vesicular stomatitis virus nucleoprotein (2VAA). 

     

    MHCClass I_HC_Bmc_peptide_ VS viru nucleoprotein (2VAA).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{6}\): MHC Class I heavy chain - beta-2-microglobulin - vesicular stomatitis virus nucleoprotein peptide complex (2VAA). (Copyright; author via source). Click the image for a popup or use this external link  https://structure.ncbi.nlm.nih.gov/i...NPS9QfLeghCmo8

     

    The T-cell receptor consists of two transmembrane protein chains, alpha and beta, each containing a single variable and constant domain, followed by a transmembrane domain.  Hence they are less complicated than an antibody chain.  They bind through their extracellular variable domains a peptide fragment bound to a MHC Class I or Class II membrane protein in the target cell. 

    In-depth:  Generation of T-Cell Receptor Diversity

    We described above how an undifferentiated B cell has the potential to produce an incredibly diversity of different antibodies from a starting genetic sequence.  This occurs through both DNA and RNA splicing.  The same process occurs with both the alpha and beta chains of T-cell receptors.  This is illustrated in the Figure \(\PageIndex{7}\) below.  Note that the alpha chains has no D (diversity) coding sequences.

     

    Molecular_T-Cell_Repertoire_Analysis_as_Source_of_Fig2_Diversity-01.svg

    Figure \(\PageIndex{7}\): The diversity of T-cell receptor (TCR)αβ is a result of genetic recombination and diversification mechanisms occurring at the α and β TCR chain loci. Diversity is first created in the germline via recombination of variable V, diversity D (for β chain), and joining J segments. Further diversification occurs through imprecise junctions of these gene segments (addition of P- and N-nucleotides adjacent to the D segment), and the combination of α and β chains

    Molecular T-Cell Repertoire Analysis as Source of Prognostic and Predictive Biomarkers for Checkpoint Blockade Immunotherapy.  International Journal of Molecular Sciences 21(7):2378 (2020).  DOI: 10.3390/ijms21072378.  License CC BY

    Figure \(\PageIndex{8}\) below shows an interactive iCn3D model of the T-cell receptor alpha and beta chains binding to MHC Class 1 protein with a bound peptide (6rp9).  The MHC protein complex consists of the histocompatibility antigen, A-2 alpha chain and β-2-microglobulin, an 11K subunit of MHC Class I proteins but not Class II MHC proteins .  Bound to it is the 9 amino acid cancer/testis antigen 1 (shown in spacefill).  The peptide is sandwiched between the MHC protein complex and the T-cell receptor α and β chains. 

    T-cell_alpha_ beta_MHC Class 1- bound peptide (6rp9).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{8}\): T-cell receptor alpha and beta chains binding to MHC Class 1 protein with a bound peptide (6rp9) (Copyright; author via source). Click the image for a popup or use this external link:   https://structure.ncbi.nlm.nih.gov/i...txUWWfbNhNXwQ6

    The actual functional structure in vivo is actually more complicated. The T cell receptor is found within the much larger T Cell receptor complex (TRC), which contains two copies of the CD3 complex, which itself consists of γ, δ, ε and ζ chains, as shown in Figure \(\PageIndex{9}\) below (A).   Part A shows the variable and C domains of the α and β chains of the T-cell receptor in green and dark.  The rest of the T-cell receptor complex include two copies each of the CD3 complex, which consist of one copy of εδζ chains and one copy of εγζ chains. 

    TCellReceptorFig2_Crop-01.svg

    Figure \(\PageIndex{9}\):  T cell receptor structor.  Kumaresan Pappanaicken R., da Silva Thiago Aparecido, Kontoyiannis Dimitrios P.  Methods of Controlling Invasive Fungal Infections Using CD8+ T Cells.  
    Frontiers in Immunology, 8, 1939 (2018). https://www.frontiersin.org/article/...mmu.2017.01939.    DOI=10.3389/fimmu.2017.01939.  Creative Commons Attribution License (CC BY).
     

    As mentioned earlier, one of the functions of the MHC Class I molecules is to present peptides derived from tumor antigens to T-cells, which leads to activation of other immune cells and hopefully destruction of the tumor cells displaying the tumor antigen.  Much work has gone into the study of immune surveillance and ultimate destruction of tumor cells with the hopes of improving on our own immune response to cancer cells. In early work, T-cells that had infiltrated tumors were isolated from a patient, amplified in the lab by adding a cytokine (ex interleukin 2 - IL2), a protein growth factor released by activated immune cells, and then reinfusing the tumor-specific T cells along with IL2 back into the patients.  This adoptive cell transfer (ACT) therapy led to remissions in some patients but the therapy also could be lethal. 

    One promising type of immune therapy is chimeric antigen receptor (CAR) T cell therapy or (CAR T) in which patients are treated with modified versions of their own T-cells. T cells are removed from a cancer patient's own blood. A gene is constructed to mimic the V and C domains of the alpha and beta chain of the T-cell receptor and inserted into the patient's T-cell using a viral vector.  The gene construct contains as their tumor antigen binding motif the V and C domain of an antibody gene made to recognize the tumor antigen.  The receptor is hence a chimeric (formed from parts of different proteins) antigen receptor (CAR) with antibody and T cell parts.  The genetically modifed cells are amplified and reinfused back into the patient.   Once the collected T cells have been engineered to express the antigen-specific CAR, they are "expanded" in the laboratory into the hundreds of millions.  

    Compare the structure of the chimeric antigen receptor (CAR) in Figure XX-C with normal T-cell receptors shown in A.  The CAR contains two, single-chain variable fragment (scFv) derived from combining the variable domains of the light (VL) and heavy (VH) chains of an antibody recognizing the tumor cell.   These domains are connected by a linker peptide (10-25 amino acids) enriched in glycine for flexibility and serines/threonines for hydrogen bonding interactions. This can be then attached to a FC fragment and other intracellular effector domains to create the receptor.  Figure \(\PageIndex{10}\) belows shows the scFv structure.  We'll discuss the addition cytoplasmic CD28 domain in a bit.

    SC-FV_CAR receptor.svg

    Figure \(\PageIndex{10}\): Single-chain variable fragment structure used in CART

    You can image this whole T-cell receptor complex involved in the binding a tumor peptide antigen presented by a MHC I transmembrane protein on a tumor cell, as illustrated in Figure \(\PageIndex{11}\) below (in different colors).  Such an interaction is also called the T cell immunological synapse.

    TcellReceptorWithPepMHCFig5.png

    Figure \(\PageIndex{11}\): T cell immunological synapse of T cell with a cancer cell. Zhao Lijun, Cao Yu J. Engineered T Cell Therapy for Cancer in the Clinic. Frontiers in Immunology, 10, 2250 (2019) https://www.frontiersin.org/article/...mmu.2019.02250.  Creative Commons Attribution License (CC BY).
          
    Protection Against Autoimmune Recognition - Coreceptors

    How can the immune system recognize and bind to any foreign molecule but not self?  The subject of immune tolerance is perhaps too specialized to include here. But there are a few features of immune protection against self that we will discuss.

    tMHC Class I proteins do present "self" peptides in their binding pocket, since self proteins also are degraded in the cell by proteasomes, the site for peptide creation. However, the T cell receptor does not recognize and bind to the self-peptide fragment bound to the MHC Class 1 protein. Hence T cells do not recognize self and turn against the bodies own cells. Once and a while they do, however, and autoimmune disease like MS, rheumatoid arthritis, and lupus result.

    B cells and T cells must be "turned on" before the can carry out their function. It is important to regulate the on switch. If the cells were to be become active without need, the cells might turn against self, which would be a big problem. It turns out that it is not enough that the T cell receptor complex binds to foreign peptide:MHC complexes for immune cell activation. They must bind yet another protein on the antigen-presenting cell.

    In the case of T helper cells. a protein on the T cell, CD28, must also bind a protein, B7, on an antigen-presenting cell like a macrophage expressing an MHC II protein:foreign peptide complex. Hence there is one specific signal (the peptide:MHC complex binding to the T cell receptor complex) and a nonspecific signal (B7 binding CD28). Why are two signals needed for activation? Again Sompayrac has a great analogy - that of a safety deposit box (if you have one) at a bank. It takes two keys (a specific key which you own and a nonspecific key (which the bank opens and which opens all the boxes) to open the box. Think of it as double security. You don't want to activate immune cells for killing unless you really need to do so.

    Yet other proteins are involved to ensure correct T-cell activations.  We'll consider T-cells expressing either the proteins CD4 or CD8.  T-cells expressing these expand after antigen stimulation (infection or immunization).  It depends on the subtype of T-cell.  Let's consider two here:

    T cells expressing the protein CD4:    After initial simulation, the differentiate and proliferate in T helper cells (TH1 if they produce the cytokine interferon (IFN)-γ) and T helper type 2 (TH2) cells (if they produce the cytokine IL4). CD4 is an integral membrane protein and acts as a co-receptor for MHC Class II:peptide complex found on cells like macrophages. .

    T-cells expressing CD8:  These cells produce cytokines (IFN-γ and tumor necrosis factor (TNF)-α) or secrete protein which form pore-forming complex on foreign cells, leading to their lysis of cells such as pathogens or tumor cells. The CD8 protein has an alpha and beta subunit.  The serve as co-receptors for MHC Class I:peptide complex found on tumor cells for example. Cytotoxic T-cells (a type of T-cell) express CD8. 

    Figure \(\PageIndex{12}\) below shows the multiple co-signals that are required to activate the CD4 T-cell (blue sphere), which has the T-cell receptor complex, the co-receptor CD4 and the CD28 protein.  It also displays a cytokine receptor which binds cytokines released by the antigen presenting cells (macrophage shown in pink), which leads to proliferation and differentiation of the activate T-cell.

    CD4Tcells_3SignalsRequired_Fig1 copy-01.svg

    Figure \(\PageIndex{12}\): Co-signals that are required to activate the CD4 T-cell (blue sphere) Salmonella as a Model for Non-Cognate Th1 Cell Stimulation.  Frontiers in Immunology 5(621):621 (2014) DOI: 10.3389/fimmu.2014.00621.  CC BY 4.0

    Sompayrac asks another interesting question. Why is antigen presentation by MHC proteins necessary at all? B cells don't really need presentation since they can bind antigen with membrane antibody molecules. Why do T cells need it. He gives different reasons for Class I and Class II presentations:

    Class I MHC (found on most body cells): T cells need to be able to "see" what is going on inside the cell. When virally-infected cells bind foreign peptide fragments and present them on the surface, they can be "seen" by the appropriate T cell. It's a way to get a part of the virus, for example, to the surface. They can't hide out in the cell. T cells don't need to recognize extracellular threat since antibodies from B cells can do that. Presentation is also important since viral protein fragments that might be found outside of the cell might bind to the outer surface of a noninfected cell, that would then be targeted for killing by the immune system. That wouldn't be good. It also helps that peptide fragments are presented on the surface. This allows parts of the protein that are buried and not exposed on the surface, which would be hidden from interaction with outside antibodies, to be used in signaling infection of the cell by a virus.

    MHC Class II (found on antigen-presenting cells like macrophages): In this way two different cells (the presenting cell and the T helper cell) must interact for a signal for immune system activation to be delivered to the body. Again it is a safety mechanism to prevent nonspecific activation of immune cells. Also, as in the case above, since fragments are presented, more of the foreign "protein" can contribute to the signal to activate the immune system.

     

    Recognition and Response in the Innate Immune System

    The B and T cell part of the immune system represents the more sophisticated branch of the immune system call the adaptive immune system.  It can be trained to recognize any foreign chemical/cellular species.  The other branch of the immune system is the innate immune system. The system recognizes common molecular structures found all many different organisms, so in this branch there is no need to adapt to each foreign species individually. The adaptive immune response also must be activated by cells of the innate immune system.

    The innate immune system recognizes common structural features in  viruses and living cells like bacteria, fungi, and protozoans like amoebas. The cells of the innate system (dendritic cells, macrophages, eosinophils, etc, which we talked about as antigen presenting calls above) have receptors called Toll-like Receptors 1-10 (TLRs) that recognize the common pathogen associated molecular patterns (PAMPs) , which leads to binding, engulfment, signal transduction, maturation (differentiation), antigen presentation, and cytokine/chemokine release from these cells. Take for example dendritic cells, which reside in the peripheral tissues and act as sentinels. They can bind PAMPs which include:

    • CHO/Lipids on bacteria surface (LPS)
    • mannose (CHO found in abundance on bacteria,
    • yeast dsRNA (from viruses)
    • nonmethylated CpG motiffs in bacterial DNA

    Bacterial and viral nucleic acids are recognized by intracellular TLRs in the cell after the they been taken up into the cells by endocytosis. Dendritic cells phagocytize microbial and host cells killed through programmed cell death (apoptosis). In the process of maturation, surface protein expression is altered, allowing the cells to leave the peripheral tissue and migrate to the lymph nodes where they activate T cells through the antigen presentation methods described above. They also control lymphocyte movement through release of chemokines. Figure \(\PageIndex{13}\) below shows the TLR family, their binding signals, and intracellular adapter proteins used to transmit signals into the cell.

     

    TLRsFig1-01.svg

    Figure \(\PageIndex{13}\): TLR family, their binding signals, and intracellular adapter proteins Ji-Yoon Noh, Suk Ran Yoon, Tae-Don Kim, Inpyo Choi, Haiyoung Jung, "Toll-Like Receptors in Natural Killer Cells and Their Application for Immunotherapy", Journal of Immunology Research, vol. 2020, Article ID 2045860, 9 pages, 2020. https://doi.org/10.1155/2020/2045860.  This is an open access article distributed under the Creative Commons Attribution License

     

    Figure \(\PageIndex{14}\) below shows an interactive iCn3D model of the mouse Toll-like receptor 3 ectodomain (sticks out into the cytoplasmic space from an internal organelle) complexed with double-stranded RNA (3CIY).

     Mouse TLR3 ectodomain  double-stranded RNA_complex (3CIY).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{14}\): Mouse Toll-like receptor 3 ectodomain complexed with double-stranded RNA (3CIY) (Copyright; author via source). Click the image for a popup or use this external link:  https://structure.ncbi.nlm.nih.gov/icn3d/share.html?otC8JXNQe45xBFx47

    Double-stranded RNA is found in the life cycle of many viruses so it makes great sense for evolution to create a binding protein to recognize this common structure (PAMP).  The TLR3 ectodomains (ECDs) forms dimers when the dsRNA is at least 40-50 nucleotides long.  The dsRNA is shown in spacefill (cyan and magenta).  Note the extensive glycosylation (colored cubes) in the structure.  The protein looks like a "horseshoe-shaped solenoid " with lots of beta structure.  It has 23 leucine-rich repeats (LRRs) with some conserved asparagines allowing for extensive hydrogen-bonding.  One face appears to be free of carbohydrate and may be important in dimerization and function. 

     

    TLRS and mRNA vaccines

    Messenger RNA vaccines against the Sars-Cov2 spike protein have probably saved millions of lifes durin the COVID-19 pandemic. The development of mRNA vacccines will go down as one of the greatest scientific achievement in the service of human kind.  The road to vaccine took decades, large number of scientists and some creative thinking.

    Vaccine usually are composed of target proteins from a virus, for example.  Instead of delivering an actual protein, whose actualdevelopment and mass production takes years, why not use mRNA that encodes a viral protein or fragments of it?  The idea has been around for a long time.  The problem is that RNAs, with their 2'-OH on the ribose ring, are very labile and degrade easily.  In addition, injecting RNA into a patient causes a significant immune response to the RNA.  We mount immune responses to foreign viral RNA through our TLR receptors (TL3, 7 and 8) but what is needed is an immune response to the protein made from the inject mRNA, not to the RNA.  Yet we don't make an immune response against our own RNA. What the difference?

    There are two problems that needed to be solved (an a host of others as well) to make mRNA vaccines, the problem of stability and our immune response against them.   A hint comes from the observation that TLRs recognize non- or undermetylated DNA found in acteria.  Methylated CpG motifs in DNA to do not stimulate an immune response.  Katalin Karikó,Michael Buckstein,  Houping Ni and Drew Weissman reported in 2005 that incorporation of methylated (m) and modified nucleosides m5C, m6A, m5U, s2U, and pseudouridine ablated the immune response to the RNA.  This opened the door to mRNA vaccines. The paper was rejected by Nature and Science but published in Immunity (Vol. 23, 165–175, August, 2005.  DOI 10.1016/j.immuni.2005.06.008).  The last line from the paper was truely prophetic:  "Insights gained from this study could advance our understanding of autoimmune diseases where nucleic acids play a prominent role in the pathogenesis, determine a role for nucleoside modifications in viral RNA, and give future directions into the design of therapeutic RNAs".  

    Katalin Karikó and Drew Weissman where awarded the 2021 Lasker–DeBakey Clinical Medical Research Award (often a prelude to the Nobel Prize) for their fundamental research that has saved so many of us.  Many thought they would also receive the Nobel Prize in 2021 but they were overlooked.  Surely they will win in 2022.

    Inflammasome

    Think of the things that you would want your immune system to protect you from. Of course, there are the pathogens like viruses, bacteria, and fungi. And of course, you want to be protected from yourself in that you don't want to activate your immune system with self-antigens. But what about "non-biological" molecules like silica or asbestos whose presence might be deleterious? What about normal biomolecules (proteins, nucleic acids) that suddenly find themselves in the wrong cellular location due to cell death by necrosis or physical injury?

    In the previous section, we discussed how innate system immune cells (dendritic cells, macrophages, eosinophils, etc) have receptors that recognize common pathogen associated molecular patterns (PAMPs) such as lipopolysaccahrides (LPS) on the surface of bacteria, mannose on bacteria and yeast, flagellin from bacterial flagella, dsRNA (from viruses) and nonmethylated CpG motiffs in bacterial DNA. These antigens are recognized by pattern recognition receptors (PRRs) - specifically the Toll-like Receptors (TLRs) 1-10. These include plasma membrane TLRs (TL4 for LPS, TL5 for flagellin, TLR 1, 2 and 6 for membrane and wall components of fungi and bacteria) and intracellular endosomal TLRs (TLR3 for dsRNA, TLR 7 and 8 for ssRNA and TLR9 for dsDNA)

    Damage associated molecular patterns (DAMPs) are typically found on molecules released from the cell or intracellular compartments on cellular damage (hence the name DAMP). Many are nuclear or cytoplasmic proteins released from the cells. These would now find themselves in a more oxidizing environment which would further change their properties. Common DAMP proteins include heat shock proteins, histones and high mobility group proteins (both nuclear), and cytoskeletal proteins. Think what non-protein molecules might be released from damaged cells that might pose problems? Here are some other common non-protein DAMPS: ATP, uric acid , heparin sulfate , DNA and cholesterol crystals. In the wrong location, these can be considered danger signals.

    If TLRs recognize PAMPs, what recognizes DAMPs? They are recognized by another type of intracellular pattern recognition receptor (PRR) called NOD (Nucleotide binding Oligomerization Domain (NOD)- Like Receptors or NLRs. NLRs also recognize PAMPs. The proteins also are named as the Nucleotide-binding domain (NBD) and Leucine-Rich repeat (LRR)–containing proteins (NLR)s. This family of proteins participates in the formation of a large protein structure called the inflammasome. (Sorry about the multiple abbreviations and naming systems!)

    As both PAMPs and DAMPs pose dangers, it would make sense that once they recognize their cognate PRRs (TLRs and NLRs, respectively), that pathways leading from the occupied receptors might converge in a common effector system for release of inflammatory cytokines from immune cells. Given that uncontrolled immune effector release from cells in an inflammatory response might be dangerous, it would be sometimes helpful to require two signals to trigger cytokine release from the cell. We've seen this two-signal requirement for the activation of T cells.

    Two such inflammatory cytokine are Interleukin 1-beta (IL 1-b) and IL-18. Activation of TLRs by a PAMP leads to activation of a potent immune cell transcription factor, NF-kbeta, which leads to transcription of the gene for the precursor of the cytokine, pro-interleukin 1-beta. Without a specific proteolytic cleavage, the active cytokine will not be released from the cell. 

    The protease required for this cleavage is activated by a signal arising when a DAMP activates a NLR, which then through a sequence of interactions leads to the proteolytic activation of another inactive protease, procaspase 1, on a large a multi-protein complex called the inflammasome. (In later chapters we will see other such protein complexes with targeted activities - including the spliceosome, which splices RNA to produce mRNA and the proteasome which conducts controlled intracellular proteolysis). The activated inflammasome activates procaspase to produce the active protein caspase (a cysteine-aspartic protease). 

    The convergence of the signals from the PAMP activation of a TLR and DAMP activation of a NLD at the inflammasome is shown in Figure \(\PageIndex{15}\) below. 

    TLR_NLR_Inflam_IL1B

    Figure \(\PageIndex{15}\): Signals from PAMP activation of a TLR and DAMP activation of a NLD at the inflammasome

    The active cytokine interleukin 1-beta helps recruit innate immune cells to the site of infection. It also affects the activity of immune cells in the adaptive immune response (T and B cels). Active IL-18 leads to the increase of another cytokine, interferon gamma and it also increases the activity of T cells that kill other cells. 

    The focus of this chapter is on binding interaction and their biological consequences. From that perspective, this section will address the structure and activity of caspases which activate the pro-cytokine prointerleukin 1 beta, the structure and ligands for the NLRs, the structure and properties of the inflammasome, and finally how "danger" molecules such as ATP and crystals (cholesterol, silica) activate the inflammasome. Unfortunately, there are a myriad of proteins involved with crazy acronyms for names. These proteins have multiple domains and many of the proteins often have multiple names. Sorry in advance!

    A. Caspases

    Caspases (Cys-asp-proteases), not to be confused with Cas9 (CRISPR associated protein 9, an RNA-guided DNA endonuclease) is a protease which when active can lead to cell death, or in a less austere fashion initiate the inflammatory response (sometimes good, often bad or even fatal). They have an active site nucleophilic Cys and cleave peptide bonds after an Asp in target proteins. All caspases (13 in humans) have an N-terminal pro-domain followed by large and small protease catalytic domain subunits. As with other proteases, it is found as an inactive zymogen. Why is this important?

    To become activated they are recruited to a scaffolding protein where they are activated by removal of the N-terminal pro domain of the zymogen and then a second cut between the large and small catalytic subunits. The enzyme that does this is caspase itself in an autocatalytic step. . There are 3 kinds of caspases, two of which are involved in programmed cell death. We'll discuss the inflammatory cytokine processing Caspase-1. Once activated, the initiators activate other effector (executioner) caspases in the cell) . Caspase 1 is activated by the inflammasome.

    Two major domains are found in Caspase 1, the caspase recruitment domain (CARD) which mediate self-interaction with scaffold and adaptor proteins in the inflammasome for activation, and a proteolytic catalytic domain, as shown in Figure \(\PageIndex{16}\) below. All domain structures in the section were obtained using Conserved Domains from the NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) or the Simple Modular Architecture Research Tool (SMART) at the EMBL ( http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1 ). Uniprot was used for protein (FASTA) sequences (http://www.uniprot.org/uniprot/). We will see the CARD domain often.

    caspase 1 domain structure

    caspase1domainb

    Figure \(\PageIndex{16}\):  Domains structure of Caspase 1

     

    B:  NOD like receptor proteins (NLRPs)

    The NOD like receptor protein (NLRPs) are a family of proteins with similar domain structure. The structures and abbreviations used for the molecular players in inflammasome activation are very complicated and confusing.  Different programs show different domains, which adds to the complexity. We will attempt to reduce the confusion by just showing domain structure diagrams, even if some show different domains for the same protein.  Remember domains are calculated from structure so different algorithms using different data bases might return different domain structures.  Table \(\PageIndex{2}\) belows shows the domain structure for NLRPs.

    NLRP1,2,3 NLRP domain structure
    NLRP1 (NALP1) NLRP1_DOMAINB
    NLRP3 (NALP3) NLRP3-DOMAIN-B
    NLRP4 NLRP4-DOMAINB
    NAIP1

    NAIP1_domain

    NAIP1_DOMAINB

    NAIP2

    NAIP2DOMAIN

    NAIP2DOMAINB

    Table  \(\PageIndex{2}\): Domain structures of the NLRPs

    Another protein in the NLR family is NAIP (neuronal apoptosis inhibitor protein). The domain structure for NAIP1 is shown in Table \(\PageIndex{2}\) above. In contrast to the other NLR for which specific ligands have not yet been found, several NAIPs have been shown to bind specific PAMPs. NAIP1 binds the needle protein CprI from C.violaceum which starts to drive the assembly of the NLRC4 inflammasome. NAIP2 binds the inner rod protein of the bacterial type III secretion system (which for Salmonella typhimurium is the protein PrgJ). NAIP5 and NAIP6 bind bacterial flagellin (which for Salmonella typhimurium is the protein FliC).  AAA is the second domain representation stands for ATP associated activities in the cell (otherwise denoted as the NACHT domain in the top representation).

    NAIP2 interacts with another adapter NLR family protein, NLRC4 (NLR family CARD domain-containing protein), to form the inflammasome. The domain structure of NAIP2 is shown in Figure \(\PageIndex{x}\) below:

    Note that many of these proteins share common domains:

    • Pyrin-NALP - Pyrin domains on different proteins self-associate through inter-protein Pyrin:Pryin interactions
    • NACHT - This domain contains about 300-400 amino acids and can bind ATP and may cleave it (i.e. act as an ATPase)
    • LRR - for Leucine Rich Repeat. These 20-30 amino acid repeats may occur up to 45 times in a given protein. They fold into an arc shape and seem to facilitate protein:protein interactions. On the concave side of the arc they have a parallel beta sheet while on the convex side they have an alpha helix. They also appear to be involved in the binding of PAMPS and DAMPs;
    • CARD - for caspase activation and recruitment. CARD domains on different proteins self-associate through inter-protein CARD:CARD interactions;
    • BIR - Baculoviral inhibition of apoptosis protein repeat;
    • ASC - Apoptosis-associated speck-like protein containing a CARD Adapter domain, allowing it to interact with other proteins with a CARD domain.

    C. ASC Adaptor Protein

    Small adapter proteins like ASC with a CARD domain mediate binding of caspases in the apoptosome (involved in apoptosis or programmed cell death) and in the inflammasome. This smaller protein has two domains, a pyrin domain and a CARD domain as shown in Figure \(\PageIndex{17}\) below. It is required for recruitment of caspase-1 to some inflammasomes (for examples, ones that contain NLRP2 and NLRP3

    ASC domain structure

    Figure \(\PageIndex{17}\): Domain structure of the small adapter proteins ASC

    The Inflammasome

    The active inflammasome, in general, consists of three different kinds of proteins, some present in multiple copies: NLRPs, adapter proteins like ASC, and procaspases. They may also contain additional recruitment and ligand sensor proteins. We'll discuss two types using different NLRPs, the NLRP4 and NLRP3 inflammasome.

    a. NLRP4 Inflammasome

    At present, the best structural information (obtained by cryomicroscopy) is for the NAIP2:NLRP4 inflammasome.  Figure \(\PageIndex{18}\) below show part of the complex consisting of 11 NLRP4 subunits arranged in a large ring. The actual biological complex has 1 NAIP2 subunit and 10 NLRP4s.

    11_NLRP4_inflammasometop

    11-NLRP4-INFLAMMASOME-SIDE

    Figure \(\PageIndex{18}\): Part of the complex of the NAIP2:NLRP4 inflammasome showing 11 NLRP4 subunits arranged in a large ring

    Here is a link to a iCn3D model of the NAIP2-NLRC4 inflammasome since it loads very slowly (3jbl) 

    How does this structure arise? Presumably it would not exist in the absence of a PAMP or DAMP so as to minimize immune-mediated inflammatory damage. Data suggests the the bacterial protein PrgJ (denoted PrgX in the figure below) binds to its receptor, NAIP2, altering its conformation as shown in Figure \(\PageIndex{19}\) below. This binary complex presents an asymmetric electrostatic surface which allows a lose association with NLRP4, which leads, after a conformational change, to a tighter binding interaction. Nine more NLRP4 bind in a similar fashion to form the 11-subunit ring structure. 

    11ringNAIP2_NLRP4

    Figure \(\PageIndex{19}\): Formation of the  NAIP2:NLRP4 ring

    The complementary electrostatic interactions between two of the many NLRC4 subunit monomers in the NLRP4 inflammasome are depicted in Figure \(\PageIndex{20}\) below.

    NLRC4 chain electrostatic interactions

    Figure \(\PageIndex{20}\): Complementary electrostatic interactions between two of the many NLRC4 subunit monomers in the NLRP4 inflammasome

    The top right panel shows two of the NLRC 4monomers (of the 12 in the Jsmol model) bound to each other, with the concave inner face of the A subunit interacting with the convex outer face of the B subunit. For simplicity, only the interactions at the top of the dimers are highlighted. The other panels show the electrostatic potential surface (red indicating negative and blue positive) for each monomer on a sliding scale of -5 to +5 (images created using the PDB2PQR Server and Pymol). 

    The depicted negative (red) electrostatic potential outer surface on one NLRC4 monomer that is complementary to the positive (inner) surface on the other NLRC4 subunit are outlined in each panel in red or blue ellipses, respectively. The curved tertiary structure of the proteins and the opposing electrostatic surface potentials of opposite faces commit the subunits to form a large ringed 12-mer core of the nucleosome.

    Note that this assembled ring brings together the CARD (caspase recruitment domain, yellow circles/spheres) which can interact with the CARD domain of the procaspase protein through CARD:CARD inter-protein interactions (think of a stack of playing cards all stuck together in a deck of cards) as shown in Figure \(\PageIndex{21}\) below.  

    fullinflammasome

    Figure \(\PageIndex{21}\):  Interactions of procaspase with the NAIP2:NLRP4 ring

    Once assembled, proximal procaspases autocatalytically convert procaspase 1 into active caspase 1, which can activate, by proteolysis, the cytokines interleukin 1 beta and interleukin 18 to form active cytokines which are released from the cell. Remember, the procytokines are present only if their genes from been transcribed by activation of the transcription factor NF-kappa beta through PAMP binding to a TLR.

    B. NLRP3 Inflammasomes

    In contrast to NLRP4 inflammasomes which require a defined PAMP/DAMP for activation, NLRP3 inflammasomes seem to be activated by cellular distress as wells as cell exposure to pathogens. It is one of the main responders to a variety of microbial infections. Given the large number of microbes that lead to NLRP3 inflammasome activation, it has been suggest that the actual signal that triggers NLRP3 is indirect. One such indirect signal is K+ ion levels in cells.

     In normal cells, K+ ions are higher in the cytoplasm than in the outside of the cell (see Chapter 9B: Neural Signaling). Potassium ion decreases in cells caused by efflux can activate NLRP3 inflammasomes. Other conditions include the rupture of lysosomes (perhaps associated with the cellular uptake of particles like silica, uric acid, cholesterol crystals and other "nanoparticles"), altered mitochondrial metabolism (which can lead to reactive oxygen species within the cell), etc. Obviously, all of these danger triggers don't bind to NLPR3 but somehow lead to down stream activation of it. NLRP3 hence probably works by being a general sensor for cell stress.

    Inappropriate and chronic activation of inflammation has been associated with many disease such as cancer, cardiovascular disease, diabetes and autoimmune diseases. Given the multiple types of signals that can activate the NLRP3 inflammasome, this complex is the focus for active drug development to finds inhibitors that would stop undesired inflammation. These inflammasomes are found in granulocytes, monocytes (macrophages), megakaryocytes, and dendritic cells.

    Activated NLRP3 recruits the ASC Adaptor Protein, which then leads to recruitment and activation of procaspase 1. NLRP3 has a pyrin, NACHT, and LRR domain. ASC has a pyrin and CARD domain. Active LRP3 can then recruit ASC through pyrin:pyrin inter-protein domain interactions. This then allows the CARD domain of bound ASC to recruit procaspase through CARD:CARD interactions (remember that procaspase has a CARD domain as well), forming the active NLRP3 inflammasome. An added feature of NLRP3 inflammasome activation occurs when the transcription factor NFkb, which is activated by PAMPs (signal 1), leads to the transcription of both the procytokines (IL-1 beta and IL 18) and of NLRP3 itself.

    Hence two signals are again needed:

    Signal 1

    The first signals are the bacterial and viral (influenza virus, poliovirus, enterovirus, rhinovirus, human respiratory syncytial virus etc) PAMPs which bind to TLRs and lead to the activation of the NFkb transcription factor. This activates not only the transcription of pro-interleukin 1-beat and interleukin 18, but also to the transcription of the NLRP3 sensor itself.

    Signal 2

    Signal 2 is delivered through PAMPs and DAMPs indirectly to the sensor NLRP3 which leads to the assembly of the inflammasome. These DAMPs appear to prime the activation of NLRP3 protein and subsequent formation of the active NLRP3 inflammasome. But what activates NLR3P3? After many studies, it became clear that the the typical bacterial ligands that would activate TLRs and perhaps NLRs only prime NLRP3 for activation. They don't bind to it directly. 

     Extracellular ATP is a major activator of NLRP3. Nanoparticles are known to release ATP as well. Most studies shown the K+ efflux from the cell is an early signal and that the protein NEK7, a protein that phosphorylates other proteins, binds to NLRP3 after potassium ion efflux and activates it. Removing NEK7 stopped NLRP3 but not NLRP4 inflammasome activation. Although NLRP3 bound to NEK7 through the NEK7 catalytic domain, the activity of the catalytic domain of NEK7 was not needed. 

    What leads to K+ efflux? Let's backup to find upstream events that could lead to efflux and try to find a link to ATP. The background for some of this material will be explored in future chaperters. The following steps occur as shown in the figure and information below:

    - solids such as silica, cholesterol crystals, uric acid crystals, and even aggregated proteins such as prions can be engulfed by monocytes/macrophages (much as they engulf bacteria as part of their immune function) in a process call phagocytosis. The particles are enveloped in plasma bilayer-derived membrane which buds off into the cell. This vesicle merges with a lysosome which gets damaged in the process. They then release ATP into the cytoplasm;

    - cytoplasmic ATP can then move outside of the cell through the glycoprotein membrane channel called pannexin 1;

    - extracellular ATP can bind to another membrane protein called the P2X7 purinoceptor. This protein now becomes a cation channel which allows K+ efflux since the ion has a higher concentration inside the cell than outside, as illustrated in Figure \(\PageIndex{22}\) below. The extracellular ATP "gates" open the P2X7 cation channel. The pore-forming toxin nigericin from Streptomyces hygroscopicus also leads to potassium ion efflux. Likewise, pore-forming proteins from S. aureus (hemolysins) lead to potassium ion efflux and activation of the NLRP3 inflammasome.  We will disuss membrane protein in great detail in a latter chapter.

     

    Pannexin1_P2X7_Kionefflux

    Figure \(\PageIndex{22}\):Extracellular ATP triggers K+ efflux from cell

    Other signals also activate the NLRP3 inflammasome. The include mitochondrial damage and release of reactive oxygen species.