Professional Documents
Culture Documents
1
CONTENTS
List of abbreviation
List of figures
List of tables
1. Introduction 9
2. Review of Literature 15
5. Conclusion 51
6. References 52
2
ABBREVIATIONS
3D 3-dimensional
DC Dendritic Cell
GN Gram-negative
GPI Glycosylphosphatidyllinositol
IFN Interferon
Ig Immunoglobulin
IKK I B kinase
IL Interleukin
LP Lipoprotein
LPS Lipopolysaccharide
3
NK Natural killer
PG peptidoglycan
TH T-helper
4
LIST OF FIGURES
5
Figure 4.9 Ramachandran Plot of TIR domain of TLR6. 31
Figure 4.12 Amino acid residues involved in interactions between TLR2 and 37
TLR6.
Figure 4.13 Interaction between TIR domains of TLR2 and TLR6 after 39
Protein-Protein Docking.
Figure 4.14 Region of interaction between TIR domains of TLR2 and TLR6. 40
6
LIST OF TABLES
Table 4.3 Amino Acid residues involved in interaction between TLR2 and 38
TLR6.
7
ABSTRACT
Toll-like Receptors (TLRs) not only recognize pathogens but also, upon ligand binding, initiate
a cascade of cellular signaling that direct the subsequent immune responses. TLR6 has been
shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived
from Mycoplasma and to activate the NF- B signaling cascade in conjunction with TLR2. In
this study, we have produced a heterodimer of TLR2/TLR6 and a TIR-TIR platform formed by
heterodimerization of TLR2/TLR6 by using computer assisted homology modelling and
protein-protein docking. We have done a detailed analysis of selected TLR2 and TLR6 docked
complex and TIR-TIR docked complex to find out the interacting amino acid residues between
both the molecules. We have found that heterodimerization of TLR2 with TLR6 is an
evolutionary process which enhance the ligand recognition capacity to enable the innate
immune system. TLR2 and TLR6 are interacting with each other at three points and TIR
domains of both TLR2 and TLR6 are interacting at one point. These interactions are very
crucial for activation of pro- and anti-inflammatory cytokine cascade and T cell proliferation to
stimulate immune responses.
8
Chapter 1
INTRODUCTION
Vertebrate immunity can be broadly categorized into adaptive and innate immunity (Janeway
et al., 2002). Adaptive immune responses are mediated by clonally distributed B and T
lymphocytes and are characterized by specificity and memory. Recognition relies on the
generation of a random and highly diverse repertoire of antigen receptors, the T- and B-cell
receptors, followed by clonal selection and expansion of receptors with relevant specificities
(Janssens and Beyaert, 2003). This mechanism accounts for the generation of immunological
memory, an important advantage, but has the main limitation that specific clones need to
expand and differentiate into effector cells before they can participate in host defense.
Therefore, adaptive immune responses are typically delayed for 4 to 7 days (Janeway et al.,
2002).
To control the infection during the first days, our body relies on the evolutionarily ancient and
more universal innate immune system. Its main functions include opsonization, activation of
complement and coagulation cascades, phagocytosis, activation of proinflammatory signaling
cascades, and apoptosis (Medzhitov, 2001). The innate immune system also has an important
function in activation and shaping of the adaptive immune response through the induction of
co-stimulatory molecules and cytokines (Medzhitov and Janeway, 1997). In contrast to the
9
clonotypic receptors, expressed by B and T lymphocytes, the innate immune system uses
nonclonal sets of recognition molecules, called Pattern Recognition Receptors (PRRs). Pattern
recognition receptors bind conserved molecular structures found in large groups of pathogens,
termed Pathogen-Associated Molecular Patterns (PAMP) (Medzhitov and Janeway, 1997).
There are various groups of pattern recognition receptors, which can be secreted, expressed on
the cell surface, or resident in intracellular compartments (Medzhitov, 2001). The Toll-like
receptors (TLRs) are one of the most important pattern recognition receptor families.
Toll-like receptors are a recently discovered family of cell surface receptors that have received
considerable attention because they are helping to unravel the details of the immune response to
infection. They are key components of the innate immune response, the arm of the immune
system that provides a rapid frontline attack against organisms to contain infection while the
adaptive arm generates an antigen-specific response. It is now appreciated that through Toll-
like receptors, the innate immune system demonstrates substantial specificity and controls
activation of the adaptive immune system with precision (Kang et al., 2006).
The TIR-TIR platform formed by the dimerization of TLR2 and TLR6 promotes homotypic
protein-protein interactions with additional cytoplasmic adapter molecules to form an active
signaling complex resulting in the expression of pro- and anti-inflammatory cytokine genes.
Historical clinical observations suggested that cellular immunity is central outcome of deep
fungal infections, and experimental observations later proved this. Adaptive immunity is
influenced by the generation of TH (helper T cell) cell and continuous production of
proinflamatory cytokine Naїve TH cell when stimulated with antigen captured by antigen
presenting cell (APC) differentiated into two T cell subset i.e. TH1, TH2. TH1 cell secrete IFN-γ
10
and mainly promote cellular immunity whereas TH2 cell produce mainly IL-4, IL-5, IL-10 and
also IL-12 drives TH1 differentiation and IL-4 drives TH2 differentiation (Barton et al., 2002;
Trinchieri, 2003, Iwasaki and Medzhitov, 2004). The balance between TH1 TH2 determines
the onset and outcome of allergic as well as autoimmune disease. A tilt towards type1 T-helper
cell pathway seems essential in antifungal host defenses.
It has been reported that TLR-2/6 recognizes some components of Zymosan of yeast which
result in production of cytokines and chemokines. Recent studies have demonstrated a crucial
involvement of TLRs in the recognition of fungal pathogens such as Candida albicans,
Aspergillus fumigatus, and Cryptococcus neoformans (Netea et al., 2004). Through the study
of fungal infection in knock-out mice deficient in either TLRs or TLR-associated adaptor
molecules, it became apparent that specific TLRs such as TLR2 and TLR6 play differential
roles in the activation of the various arms of the innate immune response. Recent data also
suggest that TLRs offer escape mechanisms to certain pathogenic microorganisms, especially
through TLR2-driven induction of anti-inflammatory cytokines. These new data have
substantially increased our knowledge of the recognition of fungal pathogens, and the study of
TLRs remains one of the most active areas of research in the field of fungal infections.
1.3 Bioinformatics
11
1.4 Homology Modelling
1.5 Docking
Molecular docking is a study of how two or more molecular structures, for example drug and
enzyme or receptor of protein, fit or interact together in 3D space. If the three-dimensional
structure of the target protein is known, docking algorithm can be applied to virtually search for
potential leads. The two main questions arising are what the complex between a protein and a
potential leads looks like and how strong the binding affinity of the lead is with respect to other
candidates (Rarey, 2002). Protein-Protein Docking is also known as the molecular docking
technique. Protein receptor-ligand docking is used to check the structure, position and
orientation of a protein when it interacts with small molecules like ligands.
1.6 Objective
1. To predict the 3-D structures of protein of TLR2, TLR6 and TIR domains of both TLR2
and TLR6 by Homology modelling.
2. To find out the amino acid residues involved in Heterodimerisation of TLR2/TLR6 and in
the TIR-TIR platform formed by Heterodimerisation of TLR2/TLR6 by protein-protein
docking.
12
Chapter 2
REVIEW OF LITERATURE
The first member of the TLR family identified was a Drosophila protein implicated in
dorsoventral patterning during embryonal development (Hashimoto et al., 1988). Gay and
Keith were the first to realize that the intracellular domain of Drosophila Toll showed striking
similarities to the intracellular domain of the mammalian interleukin-1 (IL-1) receptor, and
Lemaitre et al. demonstrated that Drosophila Toll was also involved in the immune response
of the adult fly. Different human homologues of Drosophila Toll were identified and shown to
induce activation of the transcription factor nuclear factor- B (NF- B) upon overexpression,
revealing that TLRs and IL-1 receptors trigger similar signal transduction cascades (Medzhitov
et al., 1997; Rock et al., 1998).
In 1997, Janeway et al. discovered the first human homologue of the drosophila Toll receptor,
now known as Toll-like receptor 4. It contained the Toll-like receptor/IL-1 receptor
intracytoplastic domain (Slack et al., 2000), but instead of an immunoglobulin (Ig)
extracellular domain like the IL-1 receptor, it showed a structure similar to that of the fly
receptor, composed of leucine-rich repeats. This similarity represented the ancient strategy of
pattern recognition receptors conserved throughout evolution and utilized by both human
beings and insects. In 1998, Poltorak et al. discovered by positional cloning that the lps gene in
the lipopolysaccharide (LPS)-nonresponsive mouse strain CH3/HeJ encoded a murine member
of the TLR family, providing the first clue of a function as pattern recognition receptors for
13
mammalian TLRs. Once Toll-like receptors were discovered to recognize pathogen-associated
molecular patterns, they became the most important group of pattern recognition receptors in
the innate immune system.
The structure of the Toll-like receptor has now been well characterized and has provided useful
information about the downstream cellular signaling that occurs after ligand binding (Kang et
al., 1996) (Figure 2.1). Toll-like receptors are transmembrane proteins with a series of leucine-
rich repeats in the N-terminal extracellular domain and a cytoplasmic portion greatly similar in
structure to that of IL-1 receptor (Bowie and O’Neill, 2000). This intracellular region is hence
referred to as the Toll-IL-1 receptor homology domain (Slack et al., 2000). The Toll-IL-1
receptor motif is also found in a number of important adaptor proteins that recruit downstream
kinases and transcription factors, such as myeloid differentiation factor 88, Toll-IL-1 receptor
domain containing adaptor protein, and Toll-IL-1 receptor domain containing adaptor inducing
interferon-β (IFN-β) (Kang et al., 1996).
Fungi
Extracellular
Leucine
rich TLR2 TLR6
repeats
Transmembrane Domain
TIR domain
(Highly conserved
cytoplasmic domain)
Intracellular
SIGNALING PATHWAY
14
It is the physical interaction between the Toll-IL-1 receptor domains of the Toll-like receptor
and the adaptor proteins that form a structural platform on which other downstream signaling
molecules dock to initiate the signaling cascade. This area is so crucial that even single point
mutations in the Toll-IL-1 receptor domain have been shown to abolish the host immune
response to pathogen-associated molecular pattern stimulation of some Toll-like receptors
(Kang et al., 1996).
Although the exact nature of the physical interaction between the Toll-like receptor Toll-IL-1
receptor domain and adaptor proteins has not been fully clarified, structure and function studies
have provided important information regarding the molecular basis of Toll-IL-1 receptor
signaling. A large, conserved area containing a special configuration of amino acids called the
„„BB loop‟‟ was shown to protrude away from the rest of the Toll-IL-1 receptor domain that
may mediate interactions with downstream adaptor molecules (Xu et al., 2000).
Also, evidence suggests that multiple signaling pathways are dependent on common Toll-IL-1
receptor residues, and the differential outcomes of Toll-like receptor activation probably reflect
diverging signaling pathways downstream of the Toll-IL-1 receptor domain (Ronni et al.,
2003). It is uncertain whether these structurally critical areas participate in the oligomerization
of the Toll-like receptors, in the interactions with adaptor proteins, or with the subsequently
recruited kinases. Attempts to isolate the critical amino acid residues within the Toll-IL-1
receptor domain involved in Toll-like receptor signaling continue.
The toll-like receptor (TLR) signaling pathway is the front-line subsystem against invasive
microorganisms for both innate and adaptive immunity (Iwasaki and Medzhitov, 2004). To
sense innumerable and various pathogenic threats, TLRs have evolved to recognize pathogen-
associated molecular patterns (PAMPs), which represent molecular features on the surface of
pathogens. The TLR gene family and their pathways have been evolutionarily well conserved
in both invertebrates and vertebrates (Hoffmann and Reichhart, 2002; Roach et al., 2005).
Each TLR binds to a variety of PAMPs that work as molecular markers of potential pathogens
that the host shall be defended against. For example, TLR4 was found to be a receptor for
15
lipolysaccharide (LPS) and essential to generate responses to Gram-negative bacteria in which
LPS is a part of the outer membrane (Poltorak et al., 1998), TLR9 responds to DNA-
containing unmethylated CpG motifs (Hemmi et al., 2000), TLR3 is activated by double-
stranded RNA (Alexopoulou et al., 2001), and bacteria flagellin activates TLR5 (Hayashi et
al., 2001).
TLRs and interleukin 1 receptors (IL-1Rs) have a conserved region of amino acids, which is
known as the toll/IL-1R (TIR) domain (Slack et al., 2000). Signaling of the TLR/IL-1R
superfamily is mediated through myeloid differentiation primary response gene 88 (MyD88),
IL-1R-associated kinases (IRAKs), transforming growth factor beta-activated kinase 1 (TAK1),
TAK1-binding protein 1 (TAB1), TAB2, tumor necrosis factor (TNF) receptor-associated
factor 6 (TRAF6), etc. (Akira and Takeda, 2004).
It should be mentioned that TLR1, TLR2, TLR6, TLR4, and TLR5 are located on the plasma
membrane, whereas TLR3, TLR7, and TLR9 are not located on the cell surface (Akira and
Takeda, 2004). While ligands for each TLR and interactions downstream of receptors are now
being identified at a dramatic pace, doubt is now being cast on the global logic behind all TLR
pathways. It was argued that the TLR pathway forms an hourglass structure (Beutler, 2004),
but the precise shape of the global TLR signaling network and its functional implications has
not been elucidated. Since TLRs activate innate immunity and influence the nature of adaptive
immunity (Hoebe et al., 2004), understanding the logic behind TLR signaling is the most
important topic in immunology.
Lipoprotein
Imidazoquinolines
Triacylated Zymosan Double stranded
ss RNA
Lipoprotein GPI Anchors RNA LPS Flagellin CpG DNA
MD2
CD 14
16
Table 2.1 Toll-like receptors and their characteristics
(Kang et al., 2006)
17
2.4 Receptors of the Adaptive versus those of the Innate Immune System
To appreciate the significance of Toll-like receptors and their pivotal role in immunology, it is
helpful to review the differences between adaptive and innate immunity (Kang et al., 1996).
The innate immune system is charged with curbing the proliferation of an invading pathogen
during the initial stages of an infection, before the lymphocyte expansion that characterizes the
adaptive immune response. The hallmark of the innate immune system is the rapidity with
which it responds to microbes and orchestrates the appropriate cellular response to defend the
host. For this response to occur, a foreign organism must be quickly recognized and identified
as a threat. At the heart of innate immunity is professional antigen presenting cells (APCs),
such as macrophages and dendritic cells (DCs) that provide continual surveillance of the
environment and are prepared to rapidly alert other vital components of the immune system to
perilous substances.
Instead of differentiating the countless potential microorganisms, APCs recognize patterns that
are common and indispensable among pathogen classes, termed „„pathogen-associated
molecular patterns.‟‟ A classic example of a PAMP is the lipopolysaccharide (LPS) of gram-
negative (GN) bacteria, which serves as a vital structural component of the cell wall and is
found across all GN bacterial species. The receptors that recognize PAMPs are known as
pattern-recognition receptors and are found both on cellular membranes and as circulating
plasma proteins. These receptors are germ-line encoded, that is, they rely on inherited genetic
material to provide the different receptor specificities. It is estimated that the receptors of the
innate immune system number only in the hundreds and each type of receptor is identical for
each individual (Medzhitov and Janeway, 2000). Thus, the recognition of molecular patterns
that are vital and common to large groups of pathogenic organisms confers an evolutionary
advantage to offspring and is an efficient use of a finite genome.
18
must reinvent their own defense (Kang et al., 1996). The innate immune system has a limited
variety of highly evolved receptors that have been retained through generations. Also
contributing to the complementary functions are the differences in onset of action. In the
adaptive immune response, time is required for the selected cell to clonally proliferate and
mature into a fully functional effector cell, while cells of the innate immune system are able
immediately to mount an effective immune response. Toll-like receptors represent a class of
membrane bound pattern recognition receptors that not only recognize common pathogens but
also, upon ligand binding, initiate a cascade of cellular signaling that directs the subsequent
immune responses.
The TLR signaling through different intracellular molecules, such as MAP kinases and IκB
kinases which are conserved signaling elements for many receptors, leads to a distinct set of
proinflammatory gene expressions (Jayalakshmi et al., 2007).
The signaling pathways activated by TLRs are broadly classified into MyD88-dependent and
independent pathways (Takeda and Akira, 2005) as MyD88 is the universal adapter protein
recruited by all TLRs except TLR3. The major pathways activated by TLR engagement are
19
passed through I B kinase (IKK), MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt
pathways. These pathways regulate the balance between cell viability and inflammation. The
signaling pathways activated by a specific TLR are largely dictated by the adapter proteins
recruited to the intracellular domain of the TLR upon ligand binding (Akira and Takeda,
2004). There are currently four cytosolic adaptor proteins that are thought to play a crucial role
in specificity of individual TLR-mediated signaling pathways. Amongst them, TLR4 signaling
involves all four adapter proteins, MyD88 (myeloid differentiation primary response gene 88),
MyD88 adapter like [MAL; also known as TIRAP (TIR domain-containing adapter protein)],
TIR domain-containing adapter protein inducing IFN- β
domain-containing adapter molecule 1)], and TRIF-related adapter molecule [TRAM; also
known as TICAM2 (TIR domain-containing adapter molecule 2)] (McGettrick and O'Neill,
2004). The differential recruitment of these adapter proteins by different TLRs form the basis
for the specificity in the signaling process activated by them.
Every TLR member differentially utilizes adapters, but MyD88 (296 amino acid protein) seems
to be the widely used adapter molecule. MyD88 harbors a TIR domain as well as a death
domain. The carboxy terminal of TIR domain interacts with the cognate domains in the
cytoplasmic tails of the TLRs, and the amino terminal death domain mediates the interaction
with the corresponding domain of interleukin 1 receptor-associated kinase 4 (IRAK4) (Wesche
et al., 1997; Li et al., 2002). MyD88 was originally isolated as a myeloid differentiation
primary response gene that is rapidly induced upon IL-6 stimulated differentiation of M1
myloleukemic cells into macrophages (Lord et al., 1990).
Most of the TLRs seem to be absolutely dependent on the expression of MyD88 for all of their
functions. MyD88-independent signaling events are controlled by TRIF/TRAM (for TLR4 and
TLR 2,6) and induce IRF3-dependent type I interferon production (Fitzgerald et al., 2003;
Hoebe et al., 2003; Oshiumi et al., 2003; Yamamoto et al., 2003).
20
2.6.2 Kinases involved in signaling from adapters to transcription factors
The next component of downstream TLR signaling is the IRAK family members. IRAKs are
important mediators in the signal transduction of the TLR family as they may act to potentiate
the downstream signaling. So far, four IRAKs have been identified, such as IRAK1, IRAK2,
IRAK4 and IRAKM. IRAK1 and IRAK4 possess intrinsic serine/threonine protein kinase
activities, whereas IRAK2 and IRAKM lack this activity, that may negatively regulate TLR
mediated signaling. IRAK1 has three TRAF6 (tumor necrosis factor receptor associated factor
6) binding motifs to mediate the interaction with TRAF6 (Ye et al., 2002) and undergoes
autophosphorylation. IRAK4 and IRAK1 are sequentially phosphorylated and dissociated from
MyD88, which results in activation of TRAF6.
TRAF6 belongs to an E3 ubiquitin ligase family, which facilitates the synthesis of lysine 63
linked polyubiquitin chains (Chen, 2005). TRAF6 is the activator of canonical NF- B
pathway (Hayden and Ghosh, 2004). TRAF6 is ubiquitinated at K63 chains and this K63
polyubiquitinated TRAF6 mediates activation of the next component in the pathway, which is
most likely to be TGF-β activated kinase-1 (TAK1) (Sun et al., 2004). In fact, the TAK1
associated proteins, TAB2 and TAB3, contain a domain that interacts specifically with K63-
ubiquitin chains. This model for TLR signaling predicts that the TAK1-TAB complex
associates with K63-ubiquitinated TRAF6 to activate TAK1 kinase, which then activates the
IKK complex as well as the JNK kinases. Sato et al., 2003 reported that TRAF6 is involved in
TRIF mediated IRF3 activation and NF- B activation during TLR signaling. However, a recent
paper delineated the involvement of TRAF6 in TLR signaling, where TRAF6 is involved in
MyD88 mediated NF- B activation but not TRIF mediated NF- B activation (Gohda et al.,
2004).
21
in resting cells. The multi-transcription factor binding sites in the promoter of a given gene lead
to this highly specific activation (Jayalakshmi et al., 2007). The multistage gene regulation by
this interaction and the specific transcription factors activated is discussed below.
The continued research on TLRs has led to the delineation of specificity in the regulation and
interaction of transcription factors upon stimulation leading to a highly specific gene
expression. NF- B is the major transcription factor, which functions on TLR signaling to
control/elicit inflammation. NF- B was first described as a B cell specific transcription factor
that binds the B site in the Ig light chain enhancer (Sen and Baltimore, 1986). Viral
promoters contain NF- B binding sites making it advantageous for its replication. So it is not
exaggerating to say that cells which have NF- B as a sword against the viral infection turn
back against to them. NF- B has often been called a „central mediator of the immune
response‟. MAL-MyD88 and TRAM-TRIF pathways stimulate NF- B activation albeit with
different kinetics (Selvarajoo, 2006). NF- B activity was found to be inducible in all cell types
and it is now known that members of the NF- B/Rel family regulate many genes involved in
immune and inflammatory responses (Pahl, 1999; Hayden and Ghosh, 2004).
The JNK and p38 cascades are activated first and foremost in response to inflammatory
cytokines, bacterial products, and various stress factors. Activation of TAK1 during TLR
signaling results in the activation of MAPKs, including JNK/p38, leading to the activation of
AP-1 (Ninomiya et al., 1999; Akira and Takeda, 2004; Sato et al., 2005), which together
with NF- B governs the production of inflammatory cytokines and chemokines (Kawai and
Akira, 2006). Activation of these JNK/p38 cascades is associated with selective activation of
different AP-1 subunits and transcription factors interacting with AP-1 (Johnson and Lapadat,
2002). This activation via p38 is necessary for the full induction of TNF-α -12 as
inhibition of p38 abrogates this biological response. All these studies together indicate that it is
the differential activation and binding of AP-1 subunits, which contribute to the inflammation.
22
LIPOPROTEIN,
LIPOPOLYSACCHARIDES ZYMOSAN
LPS
TLR4 TLR2
MD2 CD14 PLASMA MEMBRANE
TLR6
TIRAP
TIR
TOLLIP MyD88
T IRAK4
TRAM
R
IRAK1
A
F
TRIF 6 IRF5
P
Myd88 Independent MKK4 MKK7
Pathway
IKKγ P P
MKK3 MKK6
IKKα IKKβ
JNK
P38
P105 P
TPL2
P P
IкB MEK1 MEK2
NF-кB
DEGRADATION
(Ubiquitin Mediated MAPK AP1
Proteolysis) Signaling
ERK
Pathway
P
IкB
NUCLEUS
AP1
NF-кB
IRF5 IRF5
CELL
MEDIATED INFLAMMATORY CYTOKINES
IMMUNITY (IL-12, IL-1, IL-6, TNF-α)
BACTERIAL
T CELL DEATH
23
2.7 TLRs bridge the gap between innate and adaptive immunity
Identification of the different ligands of the Toll like receptors has allowed the study of the
cellular signaling that occurs after ligand engagement. The downstream signaling has revealed
the previously unrecognized role of the innate immune system as a regulator of the adaptive
immune response at several steps along the path from Toll-like receptor engagement to the
resultant inflammatory response. The first example of the impact of Toll-like receptors in
controlling the adaptive immune system is illustrated by the events that take place during the
physical interaction between APCs and T cells in the lymphoid organs (Kang et al., 1996).
The „„2-signal hypothesis‟‟ states that when a circulating T cell encounters a captured antigen
on the surface of an APC in the context of a major histocompatibility complex, a second co-
stimulatory signal must be seen by the T cell for it to become activated and to clonally expand.
These essential co-stimulatory molecules include B7-1 (CD80) and B7-2 (CD86) on the
surfaces of APCs that engage their cognate receptors on T cells (CD28 and CD152) at the time
of antigen presentation. Engagement of Toll-like receptors by microbial products initiates the
expression of these second signals. If this critical communication between the T cell and the
APC does not occur, the T cell will invariably meet a fate of apoptosis or permanent anergy to
the antigen stimulus. This phenomenon constitutes a valuable safety mechanism to prevent an
inadvertent expansion of a T-cell clone; it requires that a pathogen must be recognized by the
Toll-like receptors of the innate immune system before a fully developed adaptive
immunologic reaction can occur (Kang et al., 1996).
Secondly, the innate immune system directs the type of adaptive immune response that is
waged against a stimulus. Naїve T cells have the potential to differentiate toward one of the
two mutually antagonistic poles of helper T (TH) cell types termed TH1 or TH2 subsets. The
principal function of the TH1 subset is to stimulate phagocyte-mediated defense against
intracellular organisms, whereas the TH2 cells promote IgE, eosinophil, and mast-cell-mediated
immune responses against extracellular pathogens (Barton et al., 2002; Trinchieri, 2003,
Iwasaki and Medzhitov, 2004). The APCs produce cytokines that instruct the expanding clone
of T cells to differentiate toward either a TH1 or a TH2 profile. It is becoming increasingly clear
that the nature of the antigen and the Toll-like receptor to which it binds can determine the
24
specific cytokine milieu that the APC will produce to influence the polarity of the TH response
(Kang et al., 1996).
Lastly, APCs regulate a specialized subset of T cells known as regulatory T cells. Ordinarily
the peripheral effector T cells remain in a quiescent state owing to their suppression by
regulatory T cells. This mechanism of peripheral tolerance is important in protecting the host
against the development of potentially auto reactive cells, but the presence of these regulatory
cells also means that the concomitant, bystander suppression of the T cell bearing a useful
receptor specific for the pathogen-associated molecular patterns of an invading organism could
result in detrimental consequences to the host in the setting of an infection. There is now
evidence that IL- 6 secreted from Toll-like receptor activated DCs can relieve this suppression,
allowing the activation of the antigen-specific T cell during antigen presentation (Pasare and
Medzhitov, 2003). Therefore, the Toll-like receptor expressing APCs not only provide the
necessary co-stimulatory signals, while presenting antigens to the naïve T cells and cytokines
that instruct TH1 or TH2 differentiation but also suppress the inhibitory regulators of the T cells
in the appropriate setting, thereby permitting the mounting of an effective adaptive immune
response.
25
During infection, CD4+ TH cell responses polarize to become primarily TH1 or TH2. TH1 cells,
which make IFN- , are crucial for immunity to many bacterial and protozoal infections,
whereas TH2 cells, which make IL-4, IL-5, and IL-13, are important for resistance to Helminth
infections. Polarized TH1 responses are induced by dendritic cells (DCs), which respond to
pathogen-derived TLR ligands to produce IL-12 and related cytokines that are instrumental in
TH1 cell outgrowth and coordinately process and present Ag in the context of MHC class II to
activate naїve TH cells. It has become clear recently that TLR-activated DCs generally favor
the development of TH1 responses due in large part to the fact that TLR ligation usually induces
the production of IL-12, a cytokine that plays a pivotal role in TH1 cell differentiation (Barton
et al., 2002; Trinchieri, 2003, Iwasaki and Medzhitov, 2004). TLR ligands can activate
dendritic cells to provide a MyD88-dependent negative signal for TH2 cell development (Jie, et
al., 2005).
Toll-like receptor-1 (TLR1) and TLR6 are receptors of the TLR family that form heterodimers
with TLR2. Netea et al., 2008 investigated the role of TLR1 and TLR6 for the recognition of
the fungal pathogen Candida albicans. TLR1 is not involved in the recognition of C. albicans,
and TLR1 knock-out (TLR1−/−) mice showed a normal susceptibility to disseminated
candidiasis. In contrast, recognition of C. albicans by TLR6 modulated the balance between
TH1 and TH2 cytokines, and TLR6 knock-out mice displayed a defective production of IL-10
and an increased IFN-γ release. Production of the monocyte-derived cytokines tumor necrosis
factor, IL-1, and IL-6 was normal in TLR6−/− mice, and this was accompanied by a normal
susceptibility to disseminated candidiasis. In conclusion, TLR6 is involved in the recognition of
C. albicans and modulates the TH1/TH2 cytokine balance, but this results in a mild phenotype
with a normal susceptibility of TLR6−/− mice to Candida infection (Netea et al., 2008).
26
Chapter 3
MATERIALS AND METHODS
3.1 MATERIALS
3.1.1 Amino Acid Sequence of Toll-Like Receptor 2 in FASTA Format
27
3.2 TOOLS USED
3.2.1 FASTA
It was the first widely used program designed for database similarity searching (Lipman and
Pearson, 1988; Pearson, 1990). FASTA enables the user to compare a query sequence against
large databases, and various versions of the program are available. FASTA determines all
overlapping words of a certain length in both the query sequence and in each of the sequences
in the target database, creating two lists in the process.
3.2.2 BLAST
In bioinformatics, Basic Local Alignment Search Tool, or BLAST, (Altschul et al., 1900) is
an algorithm for comparing biological sequences, such as the amino acid sequences of different
proteins or the DNA sequences. A BLAST search enables a researcher to compare a query
sequence with a library or database of sequences, and identify library sequences that resemble
the query sequence above a certain threshold. BLAST searches for high scoring sequence
alignments between the query sequence and sequences in the database, using a heuristic
approach that approximates the Smith-Waterman algorithm. Protein-protein BLAST (blastp)
program, given a protein query, returns the most similar protein sequences from the protein
database that the user specifies.
28
SIB Swiss Institute of Bioinformatics by Manuel Peitsch, Nicolas Guex and Torsten
Schwede. Since 2001, SWISS-MODEL is being developed by Torsten Schwede’s Structural
Bioinformatics Group at the SIB & Biozentrum (University of Basel). Computational resources
for the SWISS-MODEL server are provided in collaboration by the Biozentrum (University
Basel), the Swiss Institute of Bioinformatics and the Advanced Biomedical Computing Center
(NCI Frederick, USA) (Guex and Peitsch, 1997; Schwede et al., 2003; Arnold et al., 2006).
29
VERIFY_3D- Determines the compatibility of an atomic model (3D) with its own amino
acid sequence (1D) by assigned a structural class based on its location and environment and
comparing the results to good structures (Bowie et al., 1991; Luethy et al., 1992).
PROVE- Calculates the volumes of atoms in macromolecules using an algorithm which
treats the atoms like hard spheres and calculates a statistical Z-score deviation for the model
from highly resolved (2.0 Å or better) and refined (R-factor of 0.2 or better) PDB-deposited
structures.
In Hex's docking calculations, each molecule is modeled using 3D parametric functions which
are used to encode both surface shape and electrostatic charge and potential distributions. By
writing an expression for the overlap of pairs of parametric functions, one can derive an
expression for a docking score as a function of the six degrees of freedom in a rigid body
docking search (Ritchie and Kemp, 2000).
30
3.3 METHODOLOGY
As the object was to find the region of interaction between TLR2/TLR6 and TIR domains of
TLR2 and TLR6, these were the target proteins.
The first and foremost step was searching for the protein through NCBI that provide us with the
basic properties and the ID for the protein along with the FASTA format. The primary
sequences was retrieved from NCBI using the following link: http://ncbi.nlm.nih.gov/
The FASTA format of the protein was noted and was given as input for NCBI protein- protein
BLAST (blastp). The results showed a number of homologous proteins, which were similar in
their protein sequence with the target protein. The extent of similarity was judged on the basis
of certain parameters like E-value, Identities and Positives.
Homologous sequences of TLR2 were retrieved from Protein Data Bank. PDB files (PDB ID-
2Z7X and 1FYW) were homologous to TLR2 protein sequences.
The 3 D structure of TLR 6 and TIR domain of TLR6 were predicted by using the SWISS
MODEL server. We had performed homology modelling for TLR2 also by using the SWISS
MODEL server. The steps performed for homology modelling are as follows:
i. Identify homologous proteins and determine the extent of their sequence similarity with
one another and the unknown protein sequence.
ii. Align the sequences.
iii. Identify structurally conserved and structurally variable regions.
iv. Generate coordinates for core (structurally conserved) residues of the unknown
structure from those of the known structure(s).
v. Generate conformations for the loops (structurally variable) in the unknown structure.
31
vi. Build the side-chain conformations.
vii. Refine and evaluate the unknown structure.
All models were then uploaded to SAVS Server to analyze predicted protein structures for
validity and assessing. All models were submitted to PROCHECK to check the stereo-chemical
quality of a protein structure by analyzing residue-by-residue geometry and overall structure
geometry. PROCHECK summary of all predicted models show the disallowed regions
according to the RAMACHANDRAN Plot and the bad contacts of the model.
We had submitted all models to Verify-3D to determine the compatibility of an atomic model
(3D) with its own amino acid sequence (1D) by assigned a structural class based on its location
and environment (alpha, beta, loop, polar, nonpolar etc) and comparing the results to good
structures.
Deep View (Swiss PDB viewer) was then used to visualize and to rectify the bad contacts by
Energy Minimization after removing the disallowed regions.
The program HEX was used for rigid body protein-protein docking between TLR2 and TLR6.
The docking was also carried out between TIR domains of both TLR2 and TLR6. For the
docking of TLR2/TLR6 and TIR domains of TLR2 and TLR6, we employed grid dimension of
0.6, twist range of 15, distance range of 15, scan step of 1, sub steps 2, steric scan 16, and final
search 25 and 500 solutions. The correlation type used was the „shape and electrostatic‟.
The program generated 500 lowest energy matches. Post processing involved bumps removal
and NEWTON like energy minimization. Automated selection of docked structure was based
upon threshold of RMS within 2Å. Selection from subsequent output was done on the basis that
only those docked solutions, which had their TIR domains in the same plane and their
respective N-terminals facing the cell membrane. Out of these, the docked complex having the
least energy was selected.
32
3.3.8 Analysis of region of interaction
Selected docked complex of TLR2 and TLR6 and TIR domains of both TLR2 and TLR6 were
analyzed to find out the regions of interaction among them. Swiss PDB viewer was used to find
out the interacting amino acid residues between two molecules.
33
Chapter 4
4.1.1 TLR2
The following 3-D structure of TLR2 was obtained by using SWISS MODEL server. The
generated model was based on template PDB ID 2Z7X (chain A) and had residue range from
27 to 553 and sequence identity of 92.15%.
34
Figure 4.1 Structure of TLR2 predicted by using SWISS MODEL server
35
4.1.2 TIR-TLR2
The model of TIR domain of TLR2 generated by submitting its FASTA format to SWISS
MODEL server. The generated model of TIR domain of TLR2 was based on template PDB ID
1O77 (chain A) and had residue range from 639 to 784 and sequence identity of 95.89%.
36
37
Figure 4.2 Structure of TIR domain of TLR2 predicted by using SWISS MODEL server
38
4.1.3 TLR6
The following 3-D structure of TLR6 was obtained by using SWISS MODEL server. The
generated model was based on template PDB ID 2Z7X (chain B) and had residue range from
33 to 556 and sequence identity of 49.62%.
39
Figure 4.3 Structure of TLR6 predicted by using SWISS MODEL server
40
4.1.4 TIR-TLR6
The model of TIR domain of TLR6 generated by submitting its FASTA format to SWISS
MODEL server. The model generated of TIR domain of TLR6 was based on template PDB ID
1FYV (chain A) and had residue range from 630 to 786 and sequence identity of 87.90%.
Figure 4.5 Structure of TIR domain of TLR6 predicted by using SWISS MODEL server
41
4.2 Ramachandran Plot
4.2.1 TLR2
Ramachandran plot of TLR2 shows 68.18% amino acid residues in the core region, 31.5% in
the allowed region and 0.4% in the generously allowed region.
4.2.2 TIR-TLR2
Ramachandran plot of TIR domain of TLR2 shows 83.1% amino acid residues in the core
region, 14.7% in the allowed region and 2.2% in the generously allowed region.
42
4.2.3 TLR6
Ramachandran plot of TLR6 shows 78.8% amino acid residues in the core region, 19.8% in the
allowed region and 1.4% in the generously allowed region. Comparatively more residues form
β–pleated sheet.
4.2.4 TIR-TLR6
Ramachandran plot of TIR domain of TLR6 shows 81.9% amino acid residues in the core
region, 17.4% in the allowed region and 0.7% in the generously allowed region.
43
4.3 PROCHECK Summary
All predicted models were submitted to PROCHECK to check the stereochemical quality of
modelled protein structures by analyzing residue-by-residue geometry and overall structure
geometry. The PROCHECK results of all models are as follows:
4.3.1 TLR2
+----------<<< P R O C H E C K S U M M A R Y >>>----------+
| |
| /var/www/html/Services/SAVES_3/jobs/1287804/TLR2.pdb 2.0 527 residues |
| |
*| Ramachandran plot: 68.1% core 31.5% allow 0.4% gener 0.0% disall |
| |
*| All Ramachandrans: 44 labelled residues (out of 525) |
+| Chi1-chi2 plots: 9 labelled residues (out of 354) |
| |
+| Main-chain params: 5 better 0 inside 1 worse |
| Side-chain params: 5 better 0 inside 0 worse |
| |
*| Residue properties: Max.deviation: 4.1 Bad contacts: 0 |
*| Bond len/angle: 5.5 Morris et al class: 2 2 2 |
+| 1 cis-peptides |
| G-factors Dihedrals: -0.36 Covalent: 0.26 Overall: -0.12 |
| |
| M/c bond lengths:100.0% within limits 0.0% highlighted |
| M/c bond angles: 97.6% within limits 2.4% highlighted |
*| Planar groups: 80.6% within limits 19.4% highlighted 7 off graph |
| |
+----------------------------------------------------------------------------+
+ May be worth investigating further. * Worth investigating further.
4.3.2 TIR-TLR2
+----------<<< P R O C H E C K S U M M A R Y >>>----------+
| |
| /var/www/html/Services/SAVES_3/jobs/1731225/TLR2_TIR.p 2.0 146 residues |
| |
+| Ramachandran plot: 83.1% core 14.7% allow 2.2% gener 0.0% disall |
| |
*| All Ramachandrans: 10 labelled residues (out of 144) |
+| Chi1-chi2 plots: 2 labelled residues (out of 108) |
| |
| Main-chain params: 6 better 0 inside 0 worse |
| Side-chain params: 5 better 0 inside 0 worse |
| |
*| Residue properties: Max.deviation: 5.6 Bad contacts: 0 |
*| Bond len/angle: 6.7 Morris et al class: 1 1 2 |
+| 2 cis-peptides |
| G-factors Dihedrals: -0.20 Covalent: 0.27 Overall: -0.01 |
| |
| M/c bond lengths:100.0% within limits 0.0% highlighted |
| M/c bond angles: 97.2% within limits 2.8% highlighted |
*| Planar groups: 69.7% within limits 30.3% highlighted 6 off graph |
| |
+----------------------------------------------------------------------------+
+ May be worth investigating further. * Worth investigating further.
44
4.3.3 TLR6
+----------<<< P R O C H E C K S U M M A R Y >>>----------+
| |
| /var/www/html/Services/SAVES_3/jobs/3875898/TLR6.pdb 2.0 472 residues |
| |
*| Ramachandran plot: 78.8% core 19.8% allow 1.4% gener 0.0% disall |
| |
*| All Ramachandrans: 22 labelled residues (out of 465) |
+| Chi1-chi2 plots: 3 labelled residues (out of 324) |
| |
| Main-chain params: 6 better 0 inside 0 worse |
| Side-chain params: 5 better 0 inside 0 worse |
| |
+| Residue properties: Max.deviation: 11.1 Bad contacts: 0 |
+| Bond len/angle: 4.8 Morris et al class: 1 1 2 |
| |
| G-factors Dihedrals: -0.23 Covalent: 0.21 Overall: -0.05 |
| |
| M/c bond lengths: 100.0% within limits 0.0% highlighted |
| M/c bond angles: 96.7% within limits 3.3% highlighted |
*| Planar groups: 77.1% within limits 22.9% highlighted 10 off graph |
| |
+----------------------------------------------------------------------------+
+ May be worth investigating further. * Worth investigating further.
4.3.4 TIR-TLR6
+----------<<< P R O C H E C K S U M M A R Y >>>----------+
| |
| /var/www/html/Services/SAVES_3/jobs/2535652/TLR6_2.pdb 2.0 157 residues |
| |
+| Ramachandran plot: 81.9% core 17.4% allow 0.7% gener 0.0% disall |
| |
*| All Ramachandrans: 7 labelled residues (out of 155) |
+| Chi1-chi2 plots: 2 labelled residues (out of 117) |
| |
| Main-chain params: 6 better 0 inside 0 worse |
| Side-chain params: 5 better 0 inside 0 worse |
| |
+| Residue properties: Max.deviation: 4.9 Bad contacts: 0 |
+| Bond len/angle: 3.4 Morris et al class: 1 2 2 |
| |
| G-factors Dihedrals: -0.22 Covalent: 0.33 Overall: 0.00 |
| |
| M/c bond lengths:100.0% within limits 0.0% highlighted |
| M/c bond angles: 98.1% within limits 1.9% highlighted |
*| Planar groups: 75.4% within limits 24.6% highlighted 2 off graph |
| |
+----------------------------------------------------------------------------+
+ May be worth investigating further. * Worth investigating further.
45
4.4 Verify- 3D Results
Table 4.1 Verify-3D results
46
4.5 Docking Results
4.5.1 Docking Result of TLR2 and TLR6
TLR2 and TLR6 were submitted for docking to Hex that generated 500 models. The following
model was selected on the basis of certain criteria and had least energy.
TLR2
TLR6
Figure 4.10 Interaction between TLR2 and TLR6 after Protein-Protein Docking
47
Table 4.2 Docking correlation summary of TLR2/TLR6
Three regions of interaction were identified in docked complex of TLR2 and TLR6.
TLR2 TLR6
1
48
i. First region of interaction between TLR2 and TLR6
TLR2 TLR6
TLR2 TLR6
ASN248 ASP233
ASP233 ASN248
TLR2
TLR6
TLR2 TLR6
LEU250
LEU250
ASN231
ASN231
LYS339 LYS338
Figure 4.12 Amino acid residues involved in interactions between TLR2 and TLR6
49
Table 4.3 Amino Acid residues involved in interaction between TLR2 and TLR6
Abbreviations: ASN- Asparagine, ASP- Aspartic Acid, B- Beta, C- Carbon, D- Delta, E- Epsilon, G-
Gamma, LEU- Leucine, LYS- Lysine, N- Nitrogen, O- Oxygen, Z- Zeta.
50
4.5.3 Docking Result of TIR Domains of TLR2 and TLR6
The following docked complex of TIR domains of both TLR2 and TLR6 was selected as both
protein were in the same plane and had their N-terminal towards the cell membrane.
Region of interaction
Figure 4.13 Interaction between TIR domains of TLR2 and TLR6 after Protein-Protein
Docking
51
Table 4.4 Docking correlation summary of TIR domains of TLR2 and TLR6
PRO680
GLN747 PRO680 GLN747
Figure 4.14 Region of interaction between TIR domains of TLR2 & TLR6
Table 4.5 Amino Acid residues involved in interaction between TIR domains of TLR2 &
TLR6
Abbreviations: A- Alpha, C- Carbon, D- Delta, GLN- Glutamine, ILE- Isoleucine, PHE- Phenylalanine,
PRO- Proline.
52
4.6 DISCUSSION
As of now, it is understood that TLR2 signaling complex functions via forming a heterodimer
either with TLR1 to recognize Tri-acylated LP, or with TLR6 to recognize Di-acylated LP,
Zymosan of yeast and GPI anchors of T. cruzi. Previous in vitro studies as well as
computerized docking studies suggest that MyD88 and TIR domain of TLR2, which is
activated by formation of heterodimer with either TLR1 or TLR6 (Gautam et al., 2006). For
the fungal infection to occur it is essential that a heterodimer forms between TLR2 and TLR6
and TIR domains of both TLR2 and TLR6 to activate the signaling. In the consequence of an
ability to form such a heterodimer, a decreased activity is observed in innate immune response.
Separate molecular modeling studies were carried out for the interaction of TLR2 and TLR6
molecules and TIR domains of both TLR2 and TLR6. Previously resolved crystal structures of
TLR2, TLR1 and their TIR domains exist, but none of TLR6. Structures of TLR6 and TIR
domain of TLR6 were modeled by using SWISS MODEL.
In our project work studies were corroborated using computer assisted docking methodology of
the two TIR domains interaction of TLR2 and TLR6. We used remodeled structure of TLR2,
its TIR domain, modeled structure of TLR6, and its TIR domain as initiating molecules. As
described in methodology all protein molecules were individually structurally refined and
energy minimized. Hex program was run for rigid protein- protein docking between TLR2 and
TLR6 and between TIR domains of both TLR2 and TLR6. The correlation type „shape and
electrostatic‟ was used. We employed grid dimension of 0.6Å, twist range of 15Å, distance
range of 15Å, scan step of 1, sub steps 2, steric scan 16, and final search 25 and 500 solutions.
The program had generated 500 lowest energy matches. The Hex program selected the docked
structures, which had threshold RMS within 2Å. From these combinations only those docked
structure were selected which had their N-terminal facing towards cell membrane and were in
same plane. This complex represented the functionally meaningful orientations between TLR2
and TLR6 (Figure 4.10) and TIR domains of both TLR2 and TLR6 (Figure 4.13). The docked
complex, which had least energy score was selected for further analysis. From the table 4.2 and
table 4.4 we can see the energy scores and correlation summary of docked complex of
TLR2/TLR6 and TIR domains of both TLR2 and TLR6, respectively.
53
We have done detail analysis of selected TLR2 and TLR6 docked complex to find out the
interacting amino acid residues between both molecules. From the figure 4.11 we can see that
TLR2 and TLR6 are interacting with each other at three points. The figure 4.12 shows that in
first region amino acid residues ASN248 of TLR2 interacts with ASP233 of TLR6. In second
region LEU250 of TLR2 interacts with ASN231 of TLR6 and in third region LYS338 of TLR2
interacts with LEU318 of TLR6 (Table 4.3).
Study of selected docked complex (Figure 4.13) of TIR domains of TLR2 and TLR6 was also
carried out. From the figure 4.14 we can see that amino acid residues ILE745 and GLN747 of
TIR domain of TLR2 are interacting with PRO680 and PHE678 of TIR domain of TLR6. The
three bonds have been emerged as the prominent factor for the stabilization of this region in the
docked complex of TIR domains of TLR2 and TLR6. These bonds are formed between the
residues ILE745(CA)---PRO680(C), GLN747(CA)---PRO680(CD), and GLN747(CD)---
PHE678(C) which have distances of 2.37Å, 2.55Å and 2.84Å, respectively (Table 4.5). These
interactions are crucial for TLR2/TLR6 mediated responses.
This Heterodimerization of TLR2 and TLR6 and TIR domains of both TLR2 and TLR6
activates the NF- B signaling. This active signaling complex further recruits other intracellular
adapter molecules such as MyD88 and TIRAP. This active signaling results in the expression
of pro- and anti-inflammatory cytokines IL-1, IL-6, IL-12 and TNF-α. IL-12 plays a pivotal
role in TH1 cell differentiation to stimulate immune responses.
54
Chapter 5
CONCLUSION
TLRs not only recognize pathogens but also, upon ligand binding, initiate a cascade of cellular
signaling that direct the subsequent immune responses. We conclude that heterodimerization of
TLR2 with TLR6 is an evolutionary process which enhance the ligand recognition capacity to
enable the innate immune system. We found that the amino acid residues ASN248, LEU250
and LYS338 of TLR2 are interacting with residues ASP233, ASN231 and LEU318 of TLR6
respectively in three regions while ILE745 and GLN747 residues of TIR domain of TLR2 are
interacting with PRO680 and PHE678 residues of TIR domain of TLR6. These amino acid
residues play an important role in the formation of heterodimer. After the formation of
heterodimer, TLR2/TLR6 complex can recognize the numerous structures of LP present in
various pathogens, thus, providing a sort of basic level specificity to the innate immune system
in humans.
In our project work we have studied about the heterodimerization of TLR2/TLR6 which is
essential for the activation of pro- and anti-inflammatory cytokine cascade and T cell
proliferation. In future this heterodimer can act as a drug target to stimulate immune responses.
55
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Web links used
http://biocarta.com/
http://blast.ncbi.nlm.nih.gov/Blast.cgi
http://en.wikipedia.org/wiki/
http://ncbi.nlm.nih.gov/
http://nihserver.mbi.ucla.edu/SAVES_3
http://swissmodel.expasy.org//SWISS-MODEL.html
http://www.biochem.vt.edu/modeling/homology.html
http://www.cryst.bbk.ac.uk/PPS2/course/section3/rama.html
http://www.csd.abdn.ac.uk/hex/
http://www.genome.jp/kegg/
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