As a common theme in cytokine signal-ling, single-span receptor chains are assembled in the cell membrane by a ligand enabling cross-activation of the aligned cytoplasmic receptor domain
Trang 1Molecular basis of cytokine signalling – theme and
variations
Delivered on 8 July 2009 at the 34th FEBS Congress in Prague
Walter Sebald1, Joachim Nickel1, Jin-Li Zhang2and Thomas D Mueller3
1 Department of Physiological Chemistry II, Theodor-Boveri Institute for Life Sciences (Biocenter), University of Wuerzburg, Germany
2 Institute for Developmental Biology, University of Cologne, Germany
3 Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute, University of Wuerzburg, Germany
Introduction
In 1968 I attended the FEBS meeting in Prague as
Doktorand I have lasting memories of the opening
ceremony in the opera house, which was initiated by
the fanfare of Janacek’s Sinfonietta This is one of the reasons why when I think of Prague I also think of music In the late 1960s, Theodor Bu¨cher gave a
well-Keywords
bone morphogenetic proteins (BMP); drug
development; interleukins; molecular
recognition; receptor oligomers
Correspondence
W Sebald, Department of Physiological
Chemistry II, Theodor-Boveri Institute for
Life Sciences (Biocenter), University of
Wuerzburg, Am Hubland, 97074 Wuerzburg,
Germany
Fax: +49 931 8884113
Tel: +49 931 3188322
E-mail: sebald@biozentrum.uni-wuerzburg.de
(Received 27 July 2009, revised
28 September 2009, accepted 4 November
2009)
doi:10.1111/j.1742-4658.2009.07480.x
Cytokine receptors are crucial for the maintenance, regulation and growth
of cells in multicellular organisms As a common theme in cytokine signal-ling, single-span receptor chains are assembled in the cell membrane by a ligand enabling cross-activation of the aligned cytoplasmic receptor domains Nature has created many variations of how this general principle is realized in a cell Here we focus on cytokines of the four-helix bundle (inter-leukins) and cystine knot (transforming growth factor-b⁄ bone morphoge-netic proteins) families Upon activation, receptor chains can form duos, trios, quartets and even larger assemblies The structure of the extracellular ligand-binding domain of a number of these receptor complexes has now been elucidated, providing the molecular basis for understanding the func-tional relevance of mechanistic diversity in a cellular context Biochemical and structural data have revealed ligand recognition mechanisms Contact sites are usually large and rather flat A limited number of contact residues provide most of the binding free energy (hot spots) Leaks in hydrophobic seals appear to provide a mechanism for adjusting the affinity of a hot spot interaction (scalability) Bone morphogenetic protein ligands are often pro-miscuous and interact not only with receptors, but also with a multitude of modulator proteins, which inhibit or enhance bone morphogenetic protein signalling Cytokine receptor systems offer promising targets for drug devel-opment Information on the structure and the activation mechanism provides leads for developing biologicals, such as engineered cytokines, cyto-kine mutants acting as receptor antagonists and receptor extracellular ligand-binding domain–Fc fusion proteins Possible indications exist in the areas of haematology, immunology, inflammation, cancer and tissue regeneration
Abbreviations
BMP, bone morphogenetic proteins; CV-2, crossveinless-2; GDF, growth and differentiation factor; IL, interleukin; SMAD, homologs to the protein from Caenorhabditis elegans SMA and Drosophila mothers against decplentaplegic; STAT, signal transducers and activators of transcription; TGF, transforming growth factor; VWC, Von Willebrand factor type C; cc, common c chain.
Trang 2received traditional Christmas lecture for the medical
students about the storage and realization of genetic
information As a sounding illustration of this topic,
one of the Brandenburg Concertos was played and in
parallel the single pages of the partitur, the musical
score, were projected; I had to change the slides in
har-mony with the music So this is one of the reasons
why when I think of Theodor Bu¨cher I also think of
music Another reason, of course, is his close
associa-tion with the Munich Bach-Chor, which has been
catalysed by Ingrid Bu¨cher, who I would like to thank
for attending the Theodor Bu¨cher Lecture at the 34th
FEBS Congress
The present Theodor Bu¨cher Lecture on the
mole-cular basis of cytokine receptor signalling – theme and
variations – has three movements, like a sonata First,
we will look at the basic mechanism and the many
variations realized in diverse receptor systems Second,
we will discuss molecular recognition in these
recep-tors; this means the structural basis for affinity and
specificity And, third, we will see how the
accumu-lated data on structure and mechanism aid in the
development of drugs
Basic mechanism – receptor
oligomerization
For a long time it was a mystery how single-span
membrane proteins, like cytokine receptors, can signal
into a cell These receptors have an extracellular
bind-ing domain, which is connected to a cytosolic domain
by only a short peptide segment probably folded in the
membrane as a single a-helix It is difficult to conceive
how such a segment can transduce a signal from the
outside to the inside of a cell How, therefore, can an
extracellular signal initiated by ligand binding be
prop-agated across the membrane?
It is clear now that single-span receptor chains
cannot signal alone They function as oligomers Binding
of the ligand leads to an oligomeric state of the
extra-cellular domains, which is transmitted to the cytosolic
domains inside the cell This general theme ‘signalling
by oligomerization’ has been the ‘Leitmotiv’ of
recep-tor research for many years It was called ‘horizontal
signalling’ in a 2004 review by Stroud & Wells [1] to
set it apart from the ‘vertical signalling’ of multi-span
membrane receptors, such as G-protein coupled
recep-tors, which employ a transmembrane conformational
change (Fig 1) In the most simplistic model, the
receptor chains diffuse freely in the membrane and are
bound together – oligomerized – in the presence of the
ligand Recently, evidence has accumulated that some
single-span receptor chains can form complexes by
themselves, so-called preformed complexes, which are inactive without a ligand [2–4] Here, ligand binding probably initiates a conformational change, which is transmitted across the membrane On the other hand, some G-protein coupled receptors have been found to oligomerize during signalling [5] Thus, it seems that there exist a variety of intermediate receptor states between pure ‘horizontal’ and pure ‘vertical’ signalling This simple and elegant horizontal signalling mecha-nism integrating the membrane as the organizing prin-ciple was very successful during the evolution of multicellular organisms It is therefore not surprising that the signalling receptor oligomers vary consider-ably, differing in stoichiometry and topology An olig-omerization mechanism was postulated for the first time by Schlessinger [6] for the epidermal growth factor receptor Here, the formation of homodimeric receptors is triggered by the binding of two ligands However, Cunningham et al [7] showed that a homod-imeric growth hormone receptor is formed by binding
to a single ligand Another renowned example for a
1 : 2 stoichiometry is the receptor for erythropoietin [8] In the growth hormone receptor, the two receptor chains differ; they are bound to different ligand epi-topes in a high- and a low-affinity mode It is therefore not unexpected that heterodimeric oligomers exist, where two different receptor chains are bound by one ligand, as in the interleukin-4 (IL-4) receptor [9,10] This division of labour between different chains opens
Fig 1 Horizontal versus vertical receptor signalling [1] Signalling across membranes requires either a conformational change in a receptor or a change in the oligomerization state of the receptor (A) Single-span transmembrane receptors are examples of so-called horizontal signalling Upon ligand binding to one receptor subunit a binary complex intermediate is formed, in the subsequent step a second (or further) receptor subunit is recruited into the complex, leading to the activation of the cytoplasmic receptor parts, e.g by transphosphorylation of inherent or receptor-associated kinases (B) Vertical receptor signalling is initiated in a single receptor (or pre-formed oligomer) by transducing a ligand-induced conformational change from the extracellular to the intracellular side.
Trang 3up a whole range of new possibilities for cellular
sig-nalling Even more complex oligomers are assembled
by dimeric ligands, such as the bone morphogenetic
proteins (BMPs) and other members of the
transform-ing growth factor-b (TGF-b) superfamily Here, twice
heterodimeric receptors are assembled by the dimeric
ligand [11,12] This can lead to avidity effects, where
ligand affinity is increased by binding simultaneously
to two receptor chains The formation of heterodimeric
ligands and⁄ or multiple receptor chains might allow
specific signalling modes, for instance during
develop-ment
Receptor structures
Here we will discuss a few receptor structures, and
focus on the extracellular domains only, in particular
on the binding domains for the ligand Although the
first structures were elucidated in the early 1990s, the
more complex ones have only recently been described
The homodimeric complex of the growth hormone
receptor represents the prototype and the reference
structure for many other systems [13] The growth
hor-mone ligand consists of a helix bundle Site 1
consti-tutes a high-affinity epitope and site 2 a low-affinity
epitope Both bind the same receptor species It is
unclear why this polarization into high- and
low-affin-ity sites originated However, as a consequence, the oligomerization is often considered an ordered sequen-tial process (Fig 2) Step 1 is the binding of the solute ligand at the high-affinity site and in step 2 the second chain is recruited in the membrane to form the signal-ling oligomer The cytosolic parts of the homodimer carry tyrosine kinases, which transphosphorylate and thus activate the twin chain This creates docking sites for signal transducers and activators of transcription (STAT) proteins, which initiate and propagate the signal within the cell For the intracellular part, the homodimer is symmetrical Each chain can function as
a trigger, which transactivates, or as a driver, which initiates intracellular signalling
This symmetry is broken in the heterodimeric recep-tors, as shown in Fig 2 for the IL-4 receptor [14–16] One chain, called the common c chain (cc), is the trigger, which can only transactivate The other chain, IL-4Ra, is the driver, which can only initiate the intra-cellular signal The division of labour is indicated by the cytosolic domains The trigger, cc, contains only a binding site for the tyrosine kinase Janus kinase 3 (JAK3), which transactivates The driver, IL-4Ra, contains a large cytosolic domain with binding motifs for Janus kinase 1 (JAK1), the intracellular signalling protein STAT6, the insulin-receptor-substrate 2, and others Again there exist a high-affinity chain, IL-4Ra,
B
A
Fig 2 A two-step sequential binding mech-anism allows for a simple design of antago-nists [9] Signal transduction of single transmembrane receptors, e.g cytokine receptors, often follows a sequential binding mechanism (A) In the first step, the ligand binds to its high-affinity receptor subunit forming an intermediate binary complex In the second step, the low-affinity receptor subunit is recruited into a ternary complex (higher oligomeric states are also possible), leading to intracellular receptor activation (indicated by the star) (B) A mutated vari-ant, which is not capable of binding to the second receptor subunit but with unaltered binding to its first receptor subunit, will still form the binary complex, but cannot pro-ceed to the second step and thus is unable
to activate the receptor [58,59,61,67] This antagonist is most efficient in blocking receptor activation if binding affinity to the second receptor subunit does not contribute significantly to the overall ligand–receptor binding affinity.
Trang 4and a low-affinity chain, cc Therefore, the assembly
of the signalling receptor heterodimer proceeds in two
steps: First, solute IL-4 binds to IL-4Ra The solute
IL-4 is concentrated 100- to 1000-fold at the membrane
surface This concentration effect and also probably the
two-dimensional diffusion in the membrane, facilitate
the following recruitment of cc The assembly of the
ternary IL-4 receptor complex can be simulated at a
biosensor surface [17] The solute IL-4 at 1–10 nm
concentrations associates rapidly with the immobilized
IL-4Ra chain Buffer alone results in a very slow
dissociation with a half-life of 5 min When the
immobilized IL-4Ra has been first saturated with the
IL-4 ligand, more and more of the ternary complex can
be formed after the addition of increasing
concentra-tions of cc Dissociation of cc is fast and its affinity to
IL-4 corresponds to a dissociation constant (KD) of
3 lm This is more than 10 000-fold lower than the
affinity for IL-4Ra
The IL-4Ra chain is shared by three receptor–ligand
complexes: two IL-4 receptors containing either cc or
IL-13Ra1 as a second chain, and one IL-13 receptor
containing IL-13Ra1 [18] As a consequence, genetic
or pharmacological inactivation of the shared IL-4Ra
will abolish not only IL-4, but also IL-13 signalling
This will be discussed further below The cc family is
larger, with cc being shared by at least five receptors,
including the IL-2 receptor [14]
The receptor for IL-2 exists in two forms A
medium-affinity heterodimeric receptor exists in natural killer
cells Its architecture corresponds to the IL-4 receptor
The driver is IL-2Rb, and cc again functions as the
trigger A second high-affinity IL-2 receptor exists in
activated T-lymphocytes It also contains the coreceptor
IL-2Ra, also called Tac [19] This coreceptor enhances
affinity specifically for IL-2 In other cells, a different
coreceptor, IL-15Ra, co-operates with the same
hetero-dimer to provide enhanced affinity for IL-15 The
struc-ture of the tetrameric high-affinity IL-2 receptor shows
that the coreceptor IL-2Ra interacts only with the IL-2
ligand It has no contacts with the other two chains
This is a telling example of the importance of
concen-trating the ligand at the surface of the membrane A
soluble IL-2Ra without membrane anchor functions as
an inhibitor of IL-2 signalling
Finally, as a further variation of horizontal signalling
we will discuss the hexameric BMP receptors (Fig 3)
These complexes are not true hexamers, as the BMP
ligand is a disulfide-bonded homodimer [11,12] The
dimeric ligand assembles a heterodimeric receptor at
each end The extracellular domains are small and
linked to the membrane-spanning segment by a short
peptide segment This places the binding domains close
to the membrane The binding domains of the receptor chains have no contact with each other They are bound together solely by the BMP ligand The BMP receptors are set apart from the cytokine receptors described above by employing a serine⁄ threonine kinase (and not tyrosine kinases) in their cytoplasmic domains and homologs to the protein from Caenorhabditis elegans SMA and Drosophila mothers against decplentaplegic (SMAD) proteins (and not STAT proteins) as intracel-lular signalling proteins However, BMP receptors obey the general rule that one chain (type II) is the transacti-vating trigger and the other chain (type I) is the driver activating the SMAD proteins by phosphorylation [20] Several proteins have been identified that qualify as
A
B
Fig 3 The ternary complex of BMP-2 ⁄ BMPR-IA ⁄ Act-RIIB forms a heterohexameric complex (A) A side view of the ternary complex
of BMP-2 (UniProtKB P12643; the BMP-2 dimer is indicated in blue and yellow) bound to the extracellular domains of its type I receptor BMPR-IA (UniProtKB P36894; green) and its type II receptor ActR-IIB (UniProtKB Q13705; red) The membrane surface is indicated
by yellow spheres The membrane-proximal C-termini of the recep-tor ectodomains were missing in the crystal structure of the ternary complex (PDB entry 2H64 [11]) and were therefore not modelled (B) A top view of (A) showing the two-fold symmetry of the ligand– receptor complex imposed by the symmetrical ligand homodimer.
Trang 5coreceptors For instance, repulsive guidance molecule
proteins determine affinity and specificity for certain
members of the BMP family [21], or b-glycan functions
as a coreceptor for TGF-b2, which belongs to the same
family as the BMPs [22,23] However, no structures
comprising such coreceptors have been determined and
therefore we do not know in molecular detail how they
function The binding of two trigger and two driver
chains to a dimeric ligand has profound consequences
for BMP signalling Multiple interactions of the ligand
with membrane receptor chains provide new
opportuni-ties for a cell to determine and tune receptor affinity
and, therefore, specificity Combinatorial assemblies of
heterodimeric BMPs and mixed receptor chains are
possible [24]
Molecular recognition
The structures of the complexes provide a wealth of
information on the mechanism of cytokine receptor
signalling As Theodor Bu¨cher put it: ‘Function is
structure in action’ Of particular importance is the
structural definition of the interfaces between a
cyto-kine and a receptor In principle, these contact sites,
called structural epitopes, carry all the determinants
for the molecular recognition among these proteins,
i.e for the affinity and the specificity of their
interac-tion However, it is still a big challenge to understand
or even to predict how these structural epitopes create
binding free energy during association One problem is
that these epitopes are large and flat [25] They have
sizes between 800 and 1500 A˚2 and comprise 20–25
residues This is similar to the interfaces of antibody–
antigen complexes Often there exist no obvious knobs
or holes that could suggest geometric complementarity
and therefore binding
It was an influential new concept that contact residues
are not of equal importance for binding Clackson &
Wells [26] performed a mutational analysis of growth
hormone and receptor and could demonstrate that a few
contact residues contribute the major part of the binding
free energy They coined the term ‘hot spots’, which is
now regularly used in the field The functional binding
epitope defined by alanine mutations is smaller than the
structural epitope defined by the residues buried in
the contact In the functional epitopes of the growth
hormone and the receptor exists one hot spot created by
two tryptophan residues (104 and 169) interacting with
complementary hydrophobic residues of the hormone
The difference between a structural and a functional
epitope has now been established in numerous
cyto-kine–receptor contacts [27] However, epitopes can be
mosaic in comprising several independent hot spots
Also, there exist strong polar bonds As an example, the IL-4 receptor system will be discussed (Fig 4), in particular the interface between IL-4 and the high-affinity IL-4Ra chain [16,28,29] Two main binding determinants are identified in IL-4: the acidic residue Glu9 and the basic residue Arg88 Mutation of either residues to alanine leads to 1000-fold loss in recep-tor affinity The crystal structure of the complex shows that the Arg88 forms a perfect salt bond with receptor Asp72 and that the Glu9 forms a hydrogen bond
B A
Fig 4 The hot spot of binding determinants in the IL-4 ⁄ IL-4Ra complex are formed by a so-called ‘avocado cluster’ [16] Two polar bonds (a hydrogen bond or a salt bridge) comprise the main binding determinants of the IL-4 ⁄ IL-4Ra ligand–receptor interaction, contrib-uting more than 80% of the overall binding free energy (A) The side chain guanidinium group of Arg88 of IL-4 (UniProtKB P05112) forms a bidentate salt bridge with the carboxylate group of Asp72
of IL-4Ra (UniProtKB P24394) This salt bridge is shielded from sol-vent access due to the surrounding hydrophobic residues from the receptor (Leu39, Phe41, Leu43 and Val69) as well as the ligand (Y56 and K84) (B) The side chain of Glu9 of IL-4 forms several hydrogen bonds to the main and side chain groups of IL-4Ra (Tyr13
OH, Ser70 main chain amide, Tyr183 OH) Similar to the salt bridge formed by Arg88 of IL-4, the hydrogen bonds emanating from Glu9 are effectively shielded by the hydrophobic environment provided
by Ile5 (IL-4), Tyr13, Val69, Tyr127 and Tyr183 of IL-4Ra The shielding from solvent embeds the polar bonds into a vacuum-like environment, thereby dramatically increasing the contribution of these noncovalent bonds to the overall binding energy Because the embedding of a polar bond into a surrounding hydrophobic envi-ronment is reminiscent of the placement of seeds in a fruit, this setup was called the avocado cluster [16].
Trang 6network with three tyrosines of the receptor These
bonds represent the hot spots in the receptor epitope
A more thorough analysis by a double mutant cycle
indicated that the two hot spots bind independently of
each other and that each of them is surrounded by a
shell of hydrophobic side chains, which co-operate
with the polar core in binding This motif has been
called an ‘avocado cluster’ in order to suggest that the
polar bond of the hot spot has to be shielded from the
bulk solvent by a hydrophobic shell It has also been
called the ‘O-ring model’ by Bogan & Thorn [30] or
‘core⁄ rim patches’ by Conte et al [25]
The IL-4 contact with the IL-4Ra chain contains an
additional third element, which is positively charged at
IL-4 and negatively charged at the receptor [31]
Molecular dynamics calculations suggest that the very
highly charged interfaces of IL-4 and IL-4Ra – not the
avocado nature of the site – lead to electrostatic
steer-ing dursteer-ing the association of the two proteins and,
thus, to an 10-fold increase in the association rate
constant This unusually fast association can be
mea-sured by Biacore interaction analysis, as described
above, and contributes to the high affinity of the IL-4
receptor corresponding to a very low dissociation
constant KDof 100 pm
Sharing receptor chains is common among cytokines
[14,32] cc functions with IL-2, IL-4 and several other
ILs, as discussed above Other receptor families employ
the common b chain or the common gp130
Promiscu-ity and sharing receptor chains also exist in the
BMP⁄ growth and differentiation factor (GDF) ⁄
acti-vin⁄ TGF-b superfamily [33] Of particular interest are
the type II activin receptor chains IIA and IIB They
bind with high affinity to activins and certain GDFs
and with low affinity to BMPs The structural epitopes
at the interfaces are largely hydrophobic with a single
serine at the core [11] According to the structure, this
serine establishes a hydrogen bond with the receptor
Leu61 main chain However, mutational analyses
indi-cate that this bond does not contribute to the binding
affinity of BMP-2 It does not represent a hot spot, not
even a minor determinant Surprisingly, this hydrogen
bond is conserved in the receptor complexes with
acti-vin A and BMP-7 In the complex with BMP-2 and
BMP-7 it does not contribute to binding affinity
How-ever, in the activin complex it is a hot spot of binding
energy, and it is responsible for the high-affinity
inter-action with this ligand What makes this bond binding?
When the residues surrounding Ser88 are compared
in BMP-2 and activin A, a few differences are found
Fortunately, swapping two activin residues, an aspartic
acid and a lysine, yielded a BMP-2 with activin-like
affinity We know the structure of the complex
between the aspartic acid⁄ lysine mutant of BMP-2 and ActR-IIB The structure does not indicate any new bonds in trans between the ligand and the receptor The swapped side chains form an ion pair in cis, which fixes the hydrophobic parts of the lysine in such a way that it seals the Ser88 from the bulk solvent Evidence
is accumulating that the sealing effect in an avocado cluster is used by some receptors to scale affinity according to the signalling requirements [18]
Inherited diseases demonstrate that small changes in receptor affinity can be crucial for in vivo function (Fig 5) Human BMP-2 and human GDF-5 bind with high affinity to the BMP receptor IB BMP-2 has an even slightly higher affinity for the IA subtype, whereas GDF-5 affinity for IA is nearly 20 times lower Nickel et al [34] identified the determinant for this specificity as Arg57 occurring in a loop region of GDF-5 A mutation of this large basic residue to an alanine in GDF-5 causes a 20-fold gain in IA affinity
A substitution of Arg57 by a leucine residue produces
an intermediate effect In Berlin, Seemann et al [35] studied a family with inherited symphalangism They identified the very same Arg57Leu substitution in the GDF-5 of the afflicted individuals These observations suggest that the gain of affinity in the GDF-5 mutant leads to an inappropriate high signalling by the IA subtype The outcome is a hyperproliferation of chon-drocytes and, as a consequence, a loss of certain joints The recently established structure of GDF-5 in com-plex with the IB receptor [36] reveals the molecular
Fig 5 (A) Familial symphalangism caused by a gain-of-function mutation in GDF-5 (UniProtKB P43026) [35] Joints are replaced by bone in finger V and defective in finger IV (see arrows) The R438L mutation is located in the wrist epitope of GDF-5 (R57L in the mature protein) The mutant GDF-5 has a several-fold increased affinity for the BMPR-IA receptor (B) A similar phenotype is pro-duced by loss-of-function mutations in the NOG gene coding for the BMP and GDF-5 inhibitor Noggin (UniProtKB Q13253) (Repro-duced with kind permission of The Journal of Clinical Investigation via the Copyright Clearance Center.)
Trang 7basis of receptor specificity and discrimination A rigid
disulfide-stabilized loop has different orientations in
the subtypes In the IA receptor, the loop occludes the
binding site and allows the binding of only a small
ala-nine side chain In the BMP receptor IB, the loop is
oriented away and gives room for the bulky arginine
of GDF-5 In summary, small structural variations
leading to small and selective changes in affinity can
be of high functional importance and result, in
the case of GDF-5, in profound chondrodysplasias of
skeletal elements in vivo
BMPs not only interact with receptors A large
vari-ety of proteins occur in the extracellular compartment
that bind BMPs and regulate their activity [37,38]
These proteins provide fascinating paradigms for
molecular recognition, as they often interact with the
same epitope Well-known representatives are Noggin,
follistatin and the members of the differential
screen-ing-selected gene aberative in neuroblastoma (DAN)
family Numerous proteins belong to the Chordin
family, which typically contain one or multiple Von
Willebrand factor type C domains (VWC domains)
[39] Members are Chordin itself, the Chordin-like
pro-teins 1 and 2, crossveinless-2 (CV-2), connective tissue
growth factor and others These proteins are essential
during gastrulation for dorsal–ventral patterning and
neural induction [40] They occur in the Spemann
orga-nizer (Chordin) and in the ventral centre (CV-2, twisted
gastrulation) Later in development they regulate organ
formation; in the adult they regulate the regeneration
of organs and tissues The VWC domain is a versatile
protein module that occurs in many forms Some of
them can bind BMPs or other proteins; some seem to
exert a purely structural role Of particular interest is
VWC1 of CV-2 Zhang et al [41,42] demonstrated that,
with zebrafish CV-2, out of the five modules present,
only VWC1 binds BMP-2 The affinity is high,
compa-rable with the BMP receptor IA Two CV-2 proteins
can bind one BMP molecule
The complex of VWC1 and BMP-2 has been
iso-lated The crystal structure revealed how VWC1
inhib-its BMP signalling [43] (Fig 6) The small module of
only 66 residues is tripartite A short N-terminal
seg-ment of eight residues occupies the binding epitope for
the IA receptor; a subdomain SD1 of 34 residues binds
to the epitope for the type II receptor; the C-terminal
subdomain SD2 points away from the complex and
has no contacts with BMP-2 Most of the binding
energy is provided by the SD1 part This hydrophobic
interaction alone has a micromolar KD The
N-termi-nal segment extends across the small ridge, like a paper
clip, onto the other side of BMP-2 and provides a
1000-fold increase in affinity The SD1 and the clip
together compete efficiently for receptor binding and therefore prevent BMP-2 signalling The BMP inhibi-tor Noggin uses a similar trick for the generation of high-affinity binding [44] This beautiful structure has been elucidated by Groppe et al [44] It shows that Noggin also uses an N-terminal extension to block the binding epitope of BMP-7 for the type I BMP recep-tors Thus, a clip-like extension to generate an addi-tional binding epitope might represent a more general mechanism to increase affinity
Drug design and development
When working in the Bu¨cher Institute, I experienced not only the atmosphere of competitive and ambitious basic research, but there was also always a readiness
to improve or to invent something A major stimulus,
of course, was the invention and the design of the Eppendorff system The Eppendorff caps, pipettes, centrifuges, incubators and photometers have estab-lished a worldwide standard for equipment in aca-demic, industrial and clinical laboratories A keen sense for industrial applications is also a hallmark of cytokine research Cytokine signalling is vital for the growth, maintenance and repair of cells and tissues in our body Dysregulation of cytokine function can result in serious and widespread diseases Not surpris-ingly, therefore, cytokines and cytokine receptors are promising targets for drug design and development Basic research has generated a remarkable spin-off of new drugs Several of them are already very successful
on the pharmaceutical market Most of these therapeu-tics are, however, biologicals; this means they are recombinant proteins The development of synthetic drugs is made difficult by the architecture of the binding epitopes and the activation mechanism, in par-ticular of heteromeric receptors, as discussed above Recombinant erythropoietin [45] and granulocyte colony-stimulating factor (Neupogen) [46] are now well-established therapeutics New players in tissue engineering and regenerative medicine are the BMPs [47], which induce the formation of new bone at criti-cal size defects that otherwise would not heal Recom-binant BMP-2 is a powerful protein that allowed the repair of a 5 cm defect in the mandible of a Go¨ttingen minipig [48] (Fig 7) A functional, mechanically stable and vascularized new bone formed in situ within 8–12 weeks Spinal fusion, bone augmentations and the treatment of nonhealing fractures represent major clinical applications of BMPs In the USA alone, more than 100 000 patients with unstable or collapsed verte-bral bodies were treated last year Mechanical load during healing is essential After ectopic application of
Trang 8BMP, for instance in a muscle pouch, the induced
bone is resorbed at later stages when transplanted in a
functional site under mechanical stress Thus, the
culti-vation of artificial bone with a certain desired shape is
science fiction at the present state of the art
Soluble receptor ectodomains are specific inhibitors
of their genuine cytokine ligands Fusion proteins
con-sisting of the constant Fc part of an immunoglobulin
and two receptor domains are even more potent, as
the cytokine can be bound at two sites They function
as efficient ligand traps The Fc-fusion protein with
the ectodomain of the activin receptor IIA is a
power-ful inhibitor of its high-affinity ligands, in particular
activin A ActRIIA–Fc induces an increase in bone
mass in ovariectomized mice [49] A clinical study has
recently shown that the human fusion protein provides
an effective treatment of osteoporotic bone loss in
postmenopausal women [50] Most importantly, the
inhibition of ActR-IIA ligands stimulates bone forma-tion by osteoblasts and therefore increases bone mass Treatment with, for instance, biphosphonates inhibits bone degradation by osteoclasts and thus at best preserves the status quo
Following the same approach, an Fc-fusion protein with the ectodomain of the activin receptor IIB was developed The IIB receptor subtype has two ligands: GDF-8 and the very similar GDF-11 These GDFs are bound with even higher affinities than the activins The signalling of GDF-8 and -11 is inhibited by the fusion protein ActR-RIIB⁄ Fc at the very low IC50 of
100 pm [51] GDF-8 has also been called myostatin This protein became well known because disruption of the myostatin gene in mice [52], cattle [53] and man [54] leads to a dramatic increase in muscle mass, the so-called double-muscling phenotype The ActR-IIB fusion protein when injected into mice produces an
A
B
C
Fig 6 Clip-like structures gain binding strength by co-operative interactions (A) A schematic representation of the binding mechanism of the BMP modulator proteins ⁄ domains Noggin and CV-2 (UniProtKB Q5D734) VWC1 to BMPs An N-terminal extension (clip) binds into the epitope for the type I receptor of the ligand, whereas the main core structure binds into the epitope for the type II receptor of the BMP ligand Therefore, the binding of the receptors of both subtypes is blocked and BMP activity is effectively suppressed Because of the strong co-operativity of both interfaces (clip and core structure) the contribution of the individual binding interfaces can be small (B) The binding of two N-terminal VWC domains of CV-2 (grey, left in surface representation) to the dimeric BMP-2 (blue and yellow) resembles the stacking
of a paperclip (VWC1 of CV-2) to a sheet of paper (BMP-2) (PDB entry 3BK3 [43]) (C) The binding of Noggin to BMP-7 (PDB entry 1M4U [44]) follows a similar mechanism as in (B) An N-terminal clip folds into the type I receptor-binding site of BMP-7, whereas the core structure blocks the type II receptor binding The much higher binding affinity of Noggin for BMP ligands can possibly be explained by the homodimeric nature resulting in four binding interfaces for a single Noggin molecule.
Trang 9even more pronounced muscle phenotype, possibly
because it neutralizes both GDF-8 and GDF-11 [53]
The fusion protein also increases muscle mass in an
mdx mouse, an animal model of muscular dystrophy
Thus, it represents a promising drug candidate for the treatment of diseases associated with muscle loss or wasting
Another type of inhibitor has been generated by mutating cytokine ligands An IL-4 mutein, Aerovant,
is now in clinical phase IIB trials as a drug candidate for the treatment of allergic asthma [55]; a growth hormone mutein, Pegvisomant, is already in clinical use for the treatment of acromegaly [56]
Allergies and asthma represent a nuisance in the case
of seasonal rhinitis or conjunctivitis and a life-threat-ening condition in anaphylactic shock and asthma IL-4 and IL-13 are the hormones that make us allergic During the sensitization phase, IL-4 triggers the forma-tion of type 2 T helper lymphocytes Type 2 T helper cells then secrete cytokines that initiate the formation
of IgE in B cells, which finally leads to the symptoms
of a delayed hypersensitivity reaction In the effector phase, IL-4 co-operates with IL-13
A rational drug design is straightforward on the basis of the activation mechanism (see Fig 2) and of the functional epitopes [57] (Fig 8) As discussed above, there exist two IL-4 receptors and one IL-13 receptor, all of which use the IL-4 receptor a chain as the essential driver An inhibition of the a chain will therefore inhibit IL-4 as well as IL-13 signalling Two mutations of IL-4 are necessary to disrupt the interac-tion with the low-affinity chains cc and IL-13Ra1 [58] The double mutein binds with nearly unchanged affin-ity to the cellular IL-4 receptor, as the low-affinaffin-ity chains contribute only marginally to the affinity The double mutein, Aerovant, is therefore a potent antago-nist of IL-4 and IL-13 Animal studies have shown that the IL-4 mutein effectively inhibits an anaphylac-tic shock in mice when applied during the sensitization phase [59] Recently, clinical trials have shown that Aerovant can also ameliorate allergic asthma in human patients [55]
Following the same rationale, an antagonist of growth hormone has been designed and developed [60] Increased growth hormone production by, for instance, a pituitary adenoma, leads to a phenotype called acromegaly, which is typically associated with large body size and, among other symptoms, a promi-nent supraorbital ridge and a large nose and jaw In the homodimeric growth hormone receptor, the second chain is bound with low affinity to the ligand, as described above This interaction can be abolished by introducing a mutation in the functional epitope, substituting a small glycine with a large arginine This mutein has efficiently inhibited growth hormone action
in an animal model However, large amounts had to
be applied, as the affinity of the mutein for the cellular
A
B
C
Fig 7 Direct reconstitution of the mandible bone of a minipig [48].
(A) X-ray control taken immediately postoperative (B) A critical size
5 cm defect in the mandible was treated with carrier material plus
recombinant BMP-2 Full regeneration of the mandible with a
mechanically stable bone is visible in the X-ray taken after 8 weeks.
The control defect treated with carrier alone formed a
pseudar-those and the defect was filled with connective tissue (C)
Explant-ed mandible bone shown in (B) (12 weeks postoperative)
demonstrates complete reconstitution of the bone (Reproduced
with kind permission of Springer Science+Business Media.)
Trang 10receptor was severely reduced compared with normal
growth hormone Therefore, six additional mutations
were introduced, which increased affinity of the mutein
to wild-type levels In addition, the mutein was
pegy-lated (i.e covalently modified with polyethyleneglycol),
in order to prolong the half-life of the protein in the
body This engineered and modified growth hormone
antagonist (pegvisomant) is in clinical use for the
treat-ment of acromegaly
Cytokine signalling still provides a fertile ground
for the development of biologicals – protein drugs
However, it is still a big challenge to find chemical
compounds that bind to functional epitopes of
cyto-kines or their receptors It appears that small
peptides can function as agonists in homodimeric
receptors, such as in the receptor for erythropoietin
[61] Chemicals have been found that inhibit IL-2,
but, surprisingly, they bind outside the functional
epitope The compound Ro26-4550 distorts the conformation of IL-2 and therefore destroys the receptor-binding epitope [62] An elegant method called ‘fragment tethering’ has been invented by Erlanson et al [63] to screen for ligands with very low affinities The future will show whether such ligands may be used as lead structures for further drug development Other approaches involve large synthetic chemicals, such as dendromers or foldamers [64,65], which can expose large surfaces similar to the binding epitopes of cytokine receptors So, the quest continues to reach high-hanging fruit [66]
Acknowledgement
W Sebald wishes to thank the organizers of the 34th FEBS Congress It was a great privilege to present the Theodor Bu¨cher Lecture
D
Fig 8 An electrostatic mismatch is the basis of the antagonistic property of the IL-4 variant Y124D [67] (A) The first step of IL-4 receptor activation is the binding of IL-4 (green) to its high-affinity receptor IL-4Ra (cyan) (B) The binary complex then recruits the low-affinity receptor subunit cc (orange surface representation) into a heterotrimeric complex (C) (PDB entry 3BPL [15]) In the case of the IL-4 antagonist variant Y124D the formation of the ternary complex is blocked (D) Circles mark the interaction of the tyrosine residue
of IL-4 with residues of cc (E) Closer inspection of this area reveals that the side chain of Tyr124 of IL-4 is embedded in a hydro-phobic cleft formed by the residues His159, Cys160, Leu208 and Cys209 of cc, with both cysteine residues forming a disulfide bond (F) A model of this interaction with IL-4Y124D instead of wild-type IL-4 shows that the negatively charged carboxylate group of Asp124 would be placed in the centre of the hydrophobic interface, thereby causing electrostatic repulsion, which explains the loss of binding
of IL-4Y124D to cc [67].