Structural Biology and Functions

of Immunoglobulins

©Dr. Colin R.A. Hewitt

2005-2006
 Topic 1

Immunoglobulin Structure-Function Relationship

Outline of Lectures
•Signalling antigen receptors on B cells - bifunctional antigen-binding
secreted molecules
•Structural conservation and infinite variability - domain structure.
•The Immunoglobulin Gene Superfamily
•The immunoglobulin fold
•Framework and complementarity determining regions - hypervariable
loops
•Modes of interactions with antigens
•Effector mechanisms and isotype – role of the Fc.
•Multimeric antibodies and multimerisation
•Characteristics and properties of each Ig isotype
•Ig receptors and their functions
Immunoglobulin Structure-Function Relationship

•Cell surface antigen receptor on B cells

Allows B cells to sense their antigenic environment

Connects extracellular space with intracellular signalling

machinery

•Secreted antibody
Neutralisation

Arming/recruiting effector cells

Complement fixation
 Immunoglobulins are Bifunctional Proteins

•Immunoglobulins must interact with a small number of

specialised molecules -
Fc receptors on cells

Complement proteins

Intracellular cell signalling molecules

•- whilst simultaneously recognising an infinite array of

antigenic determinants.
 Immunoglobulin domains

•Structural conservation and a capacity for infinite variability in a

single molecule is provided by a DOMAIN structure.
•Ig domains are derived from a single ancestral gene that has
duplicated, diversified and been modified to endow an assortment
of functional qualities on a common basic structure.
•Ig domains are not restricted to immunoglobulins.

•The most striking characteristic of the Ig domain is a disulphide

bond - linked structure of 110 amino acids long.
Ig gene superfamily - IgSF

The genes encoding Ig domains are

not restricted to Ig genes.

Although first discovered in

immunoglobulins, they are found in a
superfamily of related genes,
particularly those encoding proteins
crucial to cell-cell interactions and
molecular recognition systems.

IgSF molecules are found in most

cell types and are present across
taxonomic boundaries
 Domain Structure of Immunoglobulins
Domains are folded, compact, protease resistant structures

Fc Fab

S Light chain C
S
L VL domains
C
κ or λ
S S
S S
3H
C

CH1
CH2 S VH
S
Heavy chain C
domains
α , δ , ε , γ , or µ F(ab)2

Pepsin cleavage sites - 1 x (Fab)2 & 1 x Fc

Papain cleavage sites - 2 x Fab 1 x Fc
CH3
CH2

CH3
CH1

CH2

CH3
VH1
CH1

CH2

CH3
VH1
CH1

VL

CH2

CH3
VH1
CH1

VL CL

CH2

CH3
VH1
CH1

VL CL

CH2

CH3
 VH1
CH1

CL VL

CH2
Elbow

Hinge

CH3
Flexibility and

Fv

Fv
motion of

Fv
immunoglobulins Elbow

Fb

Fb
Fv
Fb Fv
Hinge
CH2

CH2
3

3
H

H
C

C
 Fv

VH1
CH1 Fb

CL VL

Fab

CH2
Elbow

Hinge
Carbohydrate Fc

CH3
View structures
 The Immunoglobulin Fold
The characteristic structural motif of all Ig domains

A β barrel of 7 (CL) or 8 (VL)

polypeptide strands connected A barrel made of a sheet of
by loops and arranged to staves arranged in a folded
enclose a hydrophobic interior over sheet

Single VL domain Barrel under construction

 The Immunoglobulin Fold

COOH

S S

NH2

Unfolded VL region showing 8 antiparallel β -pleated

sheets connected by loops.
View structures
 Immunoglobulins are Bifunctional Proteins

•Immunoglobulins must interact with a finite number of

specialised molecules -
Easily explained by a common Fc region irrespective of specificity

•- whilst simultaneously recognising an infinite array of

antigenic determinants.

In immunoglobulins, what is the structural basis for the

infinite diversity needed to match the antigenic universe?
Variability of amino acids in related proteins
Wu & Kabat 1970
100
Variability
80

60 Cytochromes C
40

20

20 40 60 80 100 120

Amino acid No.

100
Variability
80 Human
60 Ig heavy
40 chains
20

20 40 60 80 100 120

Amino acid No.

 Framework and Hypervariable regions
•Distinct regions of high variability and conservation led to the
concept of a FRAMEWORK (FR), on which hypervariable regions were
suspended.
•Most hypervariable regions coincided with antigen contact points -
the COMPLEMENTARITY DETERMINING REGIONS (CDRs)

FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4

100
Variability
80

60

40

20

20 40 60 80 100 120

Amino acid No.

Hypervariable CDRs are located
on loops at the end of the Fv regions

Hypervariable regions
Space-filling model of (Fab)2, viewed from above,
illustrating the surface location of CDR loops

Light chains Green and brown

Heavy chains Cyan and blue
CDRs Yellow
Hypervariable loops and framework: Summary

•The framework supports the hypervariable loops

•The framework forms a compact β barrel/sandwich with a

hydrophobic core
•The hypervariable loops join, and are more flexible than, the
β strands
•The sequences of the hypervariable loops are highly variable
amongst antibodies of different specificities
•The variable sequences of the hypervariable loops influences
the shape, hydrophobicity and charge at the tip of the antibody
•Variable amino acid sequence in the hypervariable loops
accounts for the diversity of antigens that can be recognised by
a repertoire of antibodies
 Antigens vary in size and complexity

Protein: Hapten:
Influenza haemagglutinin 5-(para-nitrophenyl
phosphonate)-pentanoic acid.
 Antibodies interact with
antigens in a variety of ways

Antigen inserts into a

pocket in the antibody

Antigen interacts
with an
extended
antibody surface
or a groove in
the antibody
surface
View structures
Flexibility and

Fv

Fv
motion of

Fv
immunoglobulins Elbow

Fb

Fb
Fv
Fb Fv
Hinge
CH2

CH2
3

3
H

H
C

C
Models of
Human 30nm
Rhinovirus 14
neutralised by
monoclonal
antibodies Human Rhinovirus 14
- a common cold virus 30 strongly neutralising McAb

60 strongly neutralising McAb Fab regions 60 weakly neutralising McAb Fab regions
 Electron micrographs of Antibodies
and complement opsonising
Epstein Barr Virus (EBV)

Negatively stained EBV

EBV coated with a corona of EBV coated with antibodies and

anti-EBV antibodies activated complement components
Electron micrographs of the effect of antibodies and
complement upon bacteria

Healthy E. coli Antibody + complement- mediated

damage to E. coli
 Non-covalent forces in
antibody - antigen interactions

Electrostatic forces Attraction between opposite charges

Hydrogen bonds Hydrogens shared between electronegative atoms
Van der Waal’s forces Fluctuations in electron clouds around molecules
oppositely polarise neighbouring atoms
Hydrophobic forces Hydrophobic groups pack together to exclude
water (involves Van der Waal’s forces)
 Why do antibodies need an Fc region?

The (Fab)2 fragment can -

•Detect antigen
•Precipitate antigen
•Block the active sites of toxins or pathogen-associated
molecules
•Block interactions between host and pathogen-associated
molecules
but can not activate
•Inflammatory and effector functions associated with cells
•Inflammatory and effector functions of complement
•The trafficking of antigens into the antigen processing
pathways
 Structure and function of the Fc region

IgA IgD IgG

3
H
C

C H2

IgE IgM
The hinge region is
4
H

C H2
C

replaced by an additional Ig
C H3 domain

Fc structure is common to all specificities of antibody within an ISOTYPE

(although there are allotypes)
The structure acts as a receptor for complement proteins and a ligand
for cellular binding sites
 Monomeric IgM

IgM only exists as a monomer on the surface of B cells

Monomeric IgM has a very low affinity for antigen

N.B. Only constant

heavy chain
4

Cµ 2 domains are shown

µ

3 Cµ
C

Cµ 1

Cµ 4 contains the transmembrane and cytoplasmic regions. These are

removed by RNA splicing to produce secreted IgM
 Polymeric IgM

IgM forms pentamers and hexamers

N.B. Only constant

heavy chain
4

Cµ 2 domains are shown

µ

3 Cµ
C

Cµ 1

Cµ 3 binds C1q to initiate activation of the classical

complement pathway

Cµ 1 binds C3b to facilitate uptake of opsonised antigens

by macrophages

Cµ 4 mediates multimerisation (Cµ 3 may also be involved)

 Multimerisation of IgM
1. Two IgM monomers in the ER

Cµ 2
(Fc regions only shown)
2. Cysteines in the J chain
form disulphide bonds

3
Cµ
with cysteines from each 2
monomer to form a dimer Cµ
Cµ 3

4
µ
3. A J chain detaches

C
2
ss Cµ

C C

sC
leaving the dimer 4
Cµ

s
C

3
disulphide bonded. C

Cµ
Cµ 4
C

C
C
4. A J chain captures C
another IgM monomer

ss
CC C

CC
C

C
and joins it to the dimer. C

Cµ 4
ss

C
Cµ 4
5. The cycle is repeated
Cµ

twice more
3

Cµ
3
6. The J chain remains
C
µ

C
attached to the IgM
2

µ
2
pentamer.
 Antigen-induced conformational changes in IgM

Planar or ‘Starfish’ conformation found in Staple or ‘crab’ conformation of IgM

solution. Conformation change induced by
Does not fix complement binding to antigen.
Efficient at fixing complement
 IgM facts and figures

Heavy chain: µ - Mu
Half-life: 5 to 10 days
% of Ig in serum: 10
Serum level (mgml-1 ): 0.25 - 3.1
Complement activation: ++++ by classical pathway
Interactions with cells: Phagocytes via C3b receptors
Epithelial cells via polymeric Ig receptor
Transplacental transfer: No
Affinity for antigen: Monomeric IgM - low affinity - valency of 2
Pentameric IgM - high avidity - valency of 10
 IgD facts and figures
Heavy chain: δ - Delta
Half-life: 2 to 8 days
% of Ig in serum: 0.2
Serum level (mgml-1 ): 0.03 - 0.4
Complement activation: No
Interactions with cells: T cells via lectin like IgD receptor
Transplacental transfer: No

IgD is co-expressed with IgM on B cells due to differential RNA splicing

Level of expression exceeds IgM on naïve B cells
IgD plasma cells are found in the nasal mucosa - however the function of IgD in
host defence is unknown - knockout mice inconclusive
Ligation of IgD with antigen can activate, delete or anergise B cells
Extended hinge region confers susceptibility to proteolytic degradation
 IgA dimerisation and secretion
IgA is the major isotype of antibody secreted at mucosal sufaces
Exists in serum as a monomer, but more usually as a J chain-
linked dimer, that is formed in a similar manner to IgM pentamers.

S S
S S
C J C
C C
C ss C
S S
S S

IgA exists in two subclasses

IgA1 is mostly found in serum and made by bone marrow B cells
IgA2 is mostly found in mucosal secretions, colostrum and milk and is made
by B cells located in the mucosae
 Secretory IgA and transcytosis
‘Stalk’ of the pIgR is degraded to release IgA
containing part of the pIgR - the secretory SS SS
C ss C
C

component
C
S C J C S
S S

SS SS SS SS
C ss C C ss C
C C C C
S C J C S S C J C S
S S S S

IgA and pIgR

are transported Epithelial
SS SS
to the apical CJC
C C
CssC cell
surface in S
S S
S

vesicles

pIgR & IgA are Polymeric Ig receptors

S
S C J C
C C
S
S
internalised are expressed on the
C ss C
SS SS
basolateral surface of
epithelial cells to
capture IgA produced

B
B cells located in the submucosa in the mucosa
produce dimeric IgA
 IgA facts and figures
Heavy chains: α 1 or α 2 - Alpha 1 or 2
Half-life: IgA1 5 - 7 days
IgA2 4 - 6 days
Serum levels (mgml-1 ): IgA1 1.4 - 4.2
IgA2 0.2 - 0.5
% of Ig in serum: IgA1 11 - 14
IgA2 1 - 4
Complement activation: IgA1 - by alternative and lectin pathway
IgA2 - No
Interactions with cells: Epithelial cells by pIgR
Phagocytes by IgA receptor
Transplacental transfer: No
To reduce vulnerability to microbial proteases the hinge region of IgA2 is truncated,
and in IgA1 the hinge is heavily glycosylated.
IgA is inefficient at causing inflammation and elicits protection by excluding, binding,
cross-linking microorganisms and facilitating phagocytosis
 IgE facts and figures
Heavy chain: ε - Epsilon
Half-life: 1 - 5 days
Serum level (mgml-1 ): 0.0001 - 0.0002
% of Ig in serum: 0.004
Complement activation: No
Interactions with cells: Via high affinity IgE receptors expressed
by mast cells, eosinophils, basophils
and Langerhans cells
Via low affinity IgE receptor on B cells
and monocytes
Transplacental transfer: No

IgE appears late in evolution in accordance with its role in protecting against
parasite infections
Most IgE is absorbed onto the high affinity IgE receptors of effector cells
IgE is also closely linked with allergic diseases
 The high affinity IgE receptor
(Fcε RI)
The IgE - Fcε RI interaction
1 is the highest affinity of any
C ε
Fc receptor with an
22 ε 1
ε extremely low dissociation
33CCε C
ε rate.
44CCε
CCεε Cε
Binding of IgE to Fcε RI
C C1ε
Cεε 4C 2 1 increases the half life of IgE
4 C ε 3 Cεε 2
ε 3 C Cε 3 of IgE interacts with
α chain
S S
S S
the α chain of Fcε RI
causing a conformational
γ 2 β chain change.
S S
 IgG facts and figures
Heavy chains: γ 1 γ 2 γ 3 γ 4 - Gamma 1 - 4
Half-life: IgG1 21 - 24 days IgG2 21 - 24 days

IgG3 7 - 8 days IgG4 21 - 24 days

Serum level (mgml-1 ): IgG1 5 - 12 IgG2 2-6
IgG3 0.5 - 1 IgG4 0.2 - 1
% of Ig in serum: IgG1 45 - 53 IgG2 11 - 15
IgG3 3-6 IgG4 1-4
Complement activation: IgG1 +++ IgG2 +
IgG3 ++++ IgG4 No
Interactions with cells: All subclasses via IgG receptors on macrophages
and phagocytes
Transplacental transfer: IgG1 ++ IgG2 +
IgG3 ++ IgG4 ++
 C1q binding motif is
located on the Cγ 2
domain

Carbohydrate is essential for

complement activation

Subtly different hinge regions

between subclasses accounts
for differing abilities to activate
complement
 Fcγ receptors
High affinity Fcγ receptors from the Ig superfamily:

Receptor Cell type Effect of ligation

Fcγ RI Macrophages Neutrophils,
Eosinophils, Dendritic cells Uptake, Respiratory burst
Fcγ RIIA Macrophages Neutrophils,
Eosinophils, Platelets
Langerhans cells Uptake, Granule release
Fcγ RIIB1 B cells, Mast Cells No Uptake, Inhibition of stimulation
Fcγ RIIB2 Macrophages Neutrophils,
Eosinophils Uptake, Inhibition of stimulation
Fcγ RIII NK cells, Eosinophils,
Macrophages, Neutrophils
Mast cells Induction of killing (NK cells)
 The neonatal Fcγ receptor

Human Fcγ Rn Human MHC

Class I

The Fcγ Rn is structurally related to MHC class I

In cows Fcγ Rn binds maternal IgG in the colostrum at pH 6.5 in the gut.
The IgG receptor complex is trancytosed across the gut epithelium and
the IgG is released into the foetal blood by the sharp change in pH to 7.4
Some evidence that this may also happen in the human placenta,
however the mechanism is not straightforward.
Molecular Genetics of Immunoglobulins