J. Biol. Macromol., 4 (2) 45-56 (2004)

Review
Antibody flexibility observed in antigen binding and its subsequent signaling
Masayuki Oda
Dept. Cellular Macromolecule Chemistry, Graduate School of Agriculture, Kyoto Prefectural University,
1-5 Shimogamo Nakaragi-cho, Sakyo-ku, Kyoto 606-8522, Japan.
Received June 7, 2004; Accepted July 12, 2004
Antibodies are well-known proteins that defend against bacterial and viral infections. At themolecular level, antibodies in solution are flexible and exist in populations of different structureswith different energy levels which are important for antigen binding and immunological functions.
In the present post-genome era, although many three-dimensional structures of proteins, including
antibodies, have been determined mainly by X-ray structural analysis, quantitative analyses of the
dynamic properties of proteins are needed for correlation with the protein functions. Since protein
molecules always function via binding, we have investigated the binding mechanism of antibodies
and the conformational change of antibodies induced upon antigen binding, which is critical for
intracellular signaling. In this article, I review several reports, including our recent results ofdynamic structural analyses for antibodies. These reports strongly suggest the difficulty ininterpreting snapshot pictures from static structural analyses. To detect the flexible nature ofprotein, the development or improvement of several techniques is also in progress. Used together, theconventional methods, such as binding thermodynamics and kinetics, can provide new information
on protein flexibility. These novel findings can not only shed light on the general features of proteinfunctions but also can be put to practical use, such as the rational design of an antibody with highantigen-binding affinity.
Keywords: antibody, antigen, binding affinity, structural flexibility, structure-function relationshipIntroduction
Antibodies such as IgG and IgM have a basic
four-chain structure consisting of two heavy andtwo light chains with two antigen binding sites,
termed combining sites. The antibody structure isextensively flexible, permitting antibodies to adapt
to a vast array of antigen shapes and sizes,
including soluble proteins and microorganisms. It
is well known that the cooperation of multiple
combining sites, two for an IgG and 10 for an IgM
pentamer, to multivalent antigens enhances theapparent binding affinity, termed avidity,
compared with that achieved with monovalentbinding. This type of cooperation among different
types of antigens depends on the flexibility of the
Fab arms involved in the antigen binding.
Correspondence to Masayuki Oda.
Tel & Fax: +81-75-703-5673
E-mail: oda@kpu.ac.jp

Antibodies also act as antigen receptors on Bcells (BCRs) and transmit signals resulting fromantigen binding. The different mechanisms involved in the effector function induced upon antigen
binding have long been debated; one mechanism isthe cross-linking of adjacent BCRs by the bindingof a multivalent antigen, and another is the conforThe abbreviations used are: BCR, antigen receptor
on B-cell; SPR, surface plasmon resonance; ITC,
isothermal titration calorimetry; Ka, equilibrium
association constant: ∆G, Gibbs free energychange; kon, association rate constant; koff, dissociation rate constant; ∆H, enthalpy change; ∆S,
entropy change; NP, (4-hydroxy-3-nitrophenyl)
acetyl; HEL, hen egg lysozyme; OVA, ovalbumin;
BSA, bovine serum albumin; CGG, chicken γglobulin; NP-Cap, NP-ε-aminocaproic acid;
ELISA, enzyme-linked immunosorbent assay;
SpG, streptococcal protein G; SpA: staphylococcal
protein A; ∆Cp, heat capacity change; CDR,
complementarity-determining region

46

Antibody flexibility

mational change in the antibody. Although many
crystal structures of liganded and unliganded Fabor Fv fragments of antibodies have been
determined, the central question about therelationship between effector function andconformational change remains unanswered,mainly because of the difficulty in detecting thefine and dynamic conformational differences.
Not only a global conformational change but
also a site-specific change occurs in an antibody
upon antigen binding. We have recently shownthat kinetic and thermodynamic analyses are
powerful tools to detect subtle conforamationalchanges and reveal the flexible nature of an
antibody. In these investigations, we have mainly
used the Biacore system, which utilizes surface
plasmon resonance (SPR) as a means of detection,and isothermal titration calorimetry (ITC), which
can detect heat in the binding of biologicalinteractions with high sensitivity. Both SPR
biosensors and ITC have been widely used tomeasure biomolecular interactions, not only for
determining equilibrium association constant (Ka)and Gibbs free energy change (∆G) but also forbinding kinetic rate constants, association rate (kon)and dissociation rate (koff) constants, andthermodynamic parameters, binding enthalpy (∆H)
and entropy (∆S) changes, respectively [1].
In this review article, I show several
examples, including our recent results of dynamic
structural
analysesofantigen-antibody
interactions, which can provide unknown featuresof an antibody in comparison with or in addition tostatic structural analyses. These results can aid inunderstanding the flexible nature of antibodies inantigen recognition and the implication ofsubsequent signaling via antibodies. In our investigations, we used a series of mouse monoclonal
antibodies specific to a hapten, (4-hydroxy-3nitrophenyl)acetyl (NP), which has beencommonly used as a T-cell-dependent antigen.

antibodies used were anti-NP IgG1s, F8, A6, B2,
and C6, obtained at different stages of
immunization (Table 2). In the binding
experiments using SPR, an antigen or antibody
was immobilized on the sensor chip surface, andanother molecule, either antibody or antigen, wasinjected into the flow (Fig. 1). The former
immobilization method is a model of bacteria orviruses interacting with soluble antibodies, and thelatter is a model of BCRs interacting with soluble
antigens [2].
In the method of antibody immobilization, theanti-NP antibody was captured through its Fc
region on a rabbit anti-mouse Fc antibody
chemically attached to the sensor chip surface.
This method enabled us to determine the binding
Table 1. Sizes of carrier proteins used in the NP
conjugates
protein
HEL
OVA
BSA
CGG
MMG

14
45
67
150
900

surface areaa (Å2)
6.5 × 103
1.6 × 104
2.7 × 104
6.2 × 104
3.1 × 105a

The estimation was based on the crystal
structures [61, 62]. The surface areas of BSA and
CGG were assumed to be the same as those ofhuman serum albumin and mouse IgG1 antibody[33, 63]. The surface are of MMG was assumed tobe 5-fold that of mouse IgG1 antibody.
Table 2. Antigen binding affinity and avidity ofanti-NP IgG1 and IgM antibodies
antibody time after immu- Ka, affinitya
nization (week)(M–1)

Ka, avidityb(M–1)

2.7 × 105
2.5 × 106
3.4 × 106
3.3 × 107
2.5 × 106

2.5 × 108
4.5 × 108
4.3 × 108
4.1 × 1088.8 × 108

F8
A6
B2
C6
A6-hCµ ca

Binding avidity and stoichiometry of IgG
In order to analyze the effects of antigen sizeand valence on antibody binding, we prepared
antigens with different numbers of NPs conjugatedto various proteins, such as hen egg lysozyme(HEL), ovalbumin (OVA), and bovine serum
albumin (BSA) (Table 1). The monoclonalmolecular weight (k)

1
1
2
12
―

The Ka, affinity values for monovalent binding toNP-ε-aminocaproic acid (NP-Cap) were determined by ITC at 37°C [25].
b
The Ka, avidity values were determined from thedivalent binding to higher valenced NP-BSA at
25°C.
c
Transfectoma produced IgM pentamer, which has
A6 and human Cµ at the V and C regions,
respectively.

Masayuki Oda

A

(a)

(b)

B

(c)

(d)

(e)

C

D

(f)

(g)

(h)

(i)

Figure 1. Schematic representation of interactions
between antigen and antibody. (A) Models ofinteractions between monovalent antigens andimmobilized antibodies. Only two antigenantibody complexes would form with monovalentantigens. (B) Models of interactions between
multivalent antigens and immobilized antibodies.
(C) Models of interactions between free antibodies
and immobilized monovalent antigens. (D) Modelsofinteractions
betweenantibodies
andimmobilized multivalent antigens. The closed
circles and open ellipses represent haptens andcarrier proteins, respectively.
stoichiometry of antigen to antibody since all of
the immobilized anti-NP monoclonal antibodies
could interact with antigens. It was found that thetwo combining sites of an antibody were able tosimultaneously accommodate two NP1-HELs as
well as NP2-HEL at a high antigen concentration,
resulting in a tri-molecular complex, Ag2Ab1(Figs. 1a and 1c). In contrast, NP conjugates of thelarger proteins, such as OVA and BSA, formed
only a di-molecular complex, Ag1Ab1, irrespectiveof the hapten valence (Figs. 1b and 1d). The
variation in the binding stoichiometry with antigen
size can be explained in terms of the restriction of47

elbow-bending flexibility within the Fab arms andhinge bending between two Fab arms. Thesefindings are supported by the results ofexperiments related to binding of protein antigens,such as HEL, OVA, and BSA, to monoclonal
antibodies specific to the respective proteins (Odaet al., unpublished results), and the recent analysis
using cryo-electron microscopy, as described
below [3]. At a low concentration of multivalentantigen, the Ka values were higher than those at ahigh antigen concentration, suggesting thatmultivalent binding had occurred (Figs. 1e and 1f).
The method of antigen immobilization isuseful for providing information on the binding
avidity of an antibody and its correlation withmonovalent binding affinity. Since the dextran
layers of the Biacore sensor chip surface aremobile, the divalent binding of IgG can beclassified into intermolecular and intramolecularbindings, depending on the types of immobilized
antigens.
When
NP-HEL
antigensareimmobilized, intermolecular divalent binding
occurs due to the small size of HEL, in addition tomonovalent binding (Figs. 1g and 1h). In this case,the binding of the lower affinity antibody, F8, was
not detected, while the binding of higher affinity
antibodies was. These results indicate that neither
the monovalent binding nor intermoleculardivalent binding of F8 is under the detection limitof Biacore, probably due to the upper limit for the
koff value [4]. The results of the binding
experiments using immobilized NP-HEL and NPBSA surfaces indicate that the difference in avidity
between intermolecular and intramoleculardivalent bindings is smaller for the antibody withhigher monovalent affinity. In intramoleculardivalent binding with maximum avidity (Fig. 1i),the apparent binding strengths of F8, B2, A6, andC6 appeared likely to increase by approximately
1,000-, 200-, 100-, and 10-fold relative to themonovalent affinity (Table 2). These results
suggest that antibodies with lower affinity canincrease their binding strength to a larger extent. In
addition, it should be noted that the Ka values forthe maximum avidity are similar in all antibodies,
which seems to be due to the ceiling avidity. It has
been reported that the physiological role ofsomatic hypermutation is to allow antibodies toattain an affinity ceiling of Ka ≈ 1010 M–1 [5]. The

48

Antibody flexibility

ceiling affinity does not seem to be responsible forthe limitation of the antibody architecture because
the yeast-surface-displayed antibody has beenshown to have 2.1 × 1013 M–1 monovalent affinity[6]. Based on these results, the ceiling affinity or
avidity in vivo maturation is considered to beenough for the immune system.
Binding avidity and stoichiometry of IgM
The secreted IgM molecule is a pentamer
made up of five four-chain subunits and one Jchain, and it occurs in body fluid in the form ofnatural antibodies. When mice were immunizedwith proteins or haptenated proteins, IgM wasproduced as the primary antibody, followed by the
production of IgG as the secondary antibody. It
has been considered that an IgM pentamer uses 10
Fab arms to bind to antigens with higher avidity,
which can compensate for the lower binding
affinity of each combining site [7]. However, little
is known about how IgM binds to smaller antigens
using multiple combining sites. In order toexamine how the binding avidity of IgM depends
on the antigen size and epitope density, we
analyzed the interactions of anti-NP IgM with
antigens of different numbers of NP conjugated tovarious proteins, such as BSA, chicken γ-globulin(CGG), and mouse anti-dansyl monoclonal IgM,
designated as MMG (Table 1) [8]. The antibodies
used were an anti-NP IgG1, A6, and a chimericanti-NP IgM pentamer, A6-hCµ, having A6 andhuman Cµ at the V and C regions, respectively.
The analysis of binding stoichiometry has
shown that A6-hCµ binds to NP-BSA and NPMMG with Ag5Ab1 and Ag1Ab1 complexes,
respectively. The model in which one NP-BSA
molecule binds to one monomer subunit of IgM isin accordance with the results for IgG, as describedabove. This finding suggests that IgM is unable toform a complex involved with multiple subunits,
further suggesting that IgM is not flexible enoughto hold a single antigen of small size, such as a“seaflower-like” configuration. In contrast, with an
antigen larger than IgM, five IgM subunits would
participate in binding to a single antigen molecule.
It is interesting to reconsider the previous reportsthat electron microscopy showed that an antigenbound IgM has a “crab-like” configuration. Such a

conformational change would be necessary for
activation of the complement cascade [10].
The Ka values of A6-hCµ were always higher
than those of A6 on comparing their binding with
NP-MMG of the same NP valence, although thedifference became smaller for the higher valenced
NP-MMG. These results indicate that IgM issuperior to IgG in interactions with antigens of alarger size and lower hapten density. The highest
Ka value of A6-hCµ for NP-conjugates was 8.8 ×
108 M–1 (Table 2). Again, this should be theceiling avidity. Assuming that the combining sitesof IgM are freely accessible to epitopes on theantigen molecule without any steric hindrance, theapparent Ka value can be expressed as Ka = (K1)10,
where K1 is an intrinsic association constant ofeach combining site. However, in practice, themaximum Ka value of IgM is much lower than thetheoretically predicted value, which is the same asthat of IgG, suggesting that the respective
combining sites can not act independently eachother, and the binding of one Fab arm provides anegative influence on the binding of other.
A similar analysis was recently reported
which shed light on the correlation between theantigen binding ability and segmental flexibility ofan antibody [11]. In this article, the binding
properties of the IgM monomer and IgD to theantigens of different epitope densities arecompared, and correlated with the respective
flexibilities determined by electron microscopy.
IgD is the major component of BCR, coexpressedwith IgM on the surface of naïve B-cells, and has
long and Cys-free hinge regions which give IgD a
more flexible nature than IgM. It has been shownthat an IgM monomer forms a more stablecomplex with an antigen of high epitope density
due to the small loss of entropy, while IgD has anadvantage for the binding to an antigen of low
epitope density due to its flexibility. The samegroup also reported the rank orders of the mean
Fab-Fab angles and the hinge-fold flexibility
function of human subclasses; the former rankorder is IgD, IgE > IgG3 > IgG4 > IgG2 > IgG1 >IgA2 > IgM, and the latter rank order is IgD > IgM> IgG3 > IgG1 > IgG4 > IgA2, IgE > IgG2 [1113].

Masayuki Oda

Application for the detection of different
affinities of antibodies
We have developed simple methods using
enzyme-linked immunosorbent assay (ELISA) andflow cytometry to estimate the relative affinities ofantibodies and BCRs based on antibody affinity
and avidity, as described above [14]. In thedetection system, the binding of NP-specific BCRs
to NP-BSA conjugated with biotin molecules isobserved using peroxidase-labeled goat anti-mouse
antibodies in ELISA and streptavidin-R-phycoerythrin by flow cytometry. At a concentration of
100 ng/ml of antibodies in ELISA, the binding
ratio of lower valenced NP-BSA per higher
valenced NP-BSA was less than 0.1 for lower
affinity anti-NP antibodies, such as N1G9 and A6,
while it was close to 1 for higher affinity
antibodies, such as B2 and C6 (Table 3). Similarly,
at a concentration of 10–8 M of antigens in flow
cytometry analysis, the binding of lower valenced
NP-BSA to lower affinity BCRs on hybridomas
and transfectants was observed to a small extent
only, while that to higher affinity BCRs was
detected with a mean fluorescence intensity similar
to the binding of higher valenced NP-BSA. These
results are in good agreement with those obtainedby SPR and indicate the possibility for practicalmeasurements of the antibody affinity, includingthat of BCR on the cell surface. Furthermore, ourreagent and method can be useful to identify thememory B-cell population at different stages ofaffinity maturation.
Table 3. Comparisons of the relative affinities
analyzed by different methods
antibody

ELISA

FACS

SPR

N1G9
A6
B2
C6

0.02
0.12
1.17
1.48

0.02
0.07
0.73
1.16

0.12
0.14
0.64
0.72

The relative affinity is shown as the ratio of NP0.5BSA-bio21 to NP7.4-BSA-bio21.
Conformational change induced by antigen
binding
We have addressed the question of whether
antigen binding induces a conformational change
in the constant domain of an antibody using

49

streptococcal protein G (SpG) or staphylococcal
protein A (SpA) as probes since these proteins are
known to bind to IgG domains such as CH1 and theCH2-CH3 interface [15, 16]. The results of binding
experiments using SPR and ITC between theprobes and anti-NP monoclonal antibodies, IgG1
and IgG2b subclasses, in the presence or absenceof an antigen show that antigen binding brought
about a 30-60% decrease in affinity with theprobes as shown in Table 4 [17]. In addition, theantigen binding accelerated the dissociation of theantibody from the complexes with the probes.
Similar results were also observed with anti-HEL
monoclonal antibodies, suggesting that ourobservations are not specific to anti-NP
monoclonal antibodies. Since small NP antigens,such as NP-ε-aminocaproic acid (NP-Cap) and
NP-Gly, were used in this experiment, the antigens
could cause no steric hindrance of the interaction
between antibodies and the probes. In addition, thepresence of an antigen resulted in no aggregationof antibodies. These results confirm that thedecreased binding affinity for the probes in thepresence of an antigen is due to the conformationalchange in binding sites for the probes, which are
distant from the combining sites.
A controversy has long existed over whether
antigen binding induces a conformational change
in CH domains and results in subsequent signalingby an allosteric mechanism, such as the activationof the complement cascade or IgG transport
through the membrane [10, 18, 19]. Nanosecond
fluorescence polarization studies of anti-dansyl
monoclonal antibodies have shown that the rankorders of mean rotational correlation times ofmouse subclasses are IgG2b > IgG2a > IgG1 >IgE, and they are correlated with the ability tocomplement fixation [20]. These results indicate
the transmission of conformational change fromthe Fab to the Fc region, which changes to thefavorable conformation for complement fixation.
In addition, it has been shown that B-cell lines ortransfectants can present monovalent antigens to
T-cells, indicating that the cross-linking of BCRs
is not required for antigen presentation by B-cells
and is still necessary for signal 1 in the
differentiation of naïve B-cells to antibodyforming cells [21, 22]. We have succeeded in
detecting the fine conformational change in CH do-Antibody flexibility

50

Table 4. Ka values of anti-NP IgG1 antibodies for the interactions with the immobilized SpG-C2 and SpAB in the absence or presence of NP-Cap
antibody
F8 (IgG1)
B2 (IgG1)
C6 (IgG1)
6L1 (IgG2b)
9L2 (IgG2b)

SpG-C2 a–antigen (M–1) +antigen (M–1)
8.3 × 106
5.4 × 106
3.3 × 106
2.9 × 107
2.5 × 107

3.8 × 106
1.7 × 106
1.7 × 106
1.7 × 106
1.6 × 107

ratio
0.46
0.31
0.52
0.59
0.64

b

SpA-B a–antigen (M–1) +antigen (M–1)
7.2 × 106
7.4 × 106
4.3 × 106
1.5 × 107
1.4 × 107

4.8 × 106
2.1 × 106
1.4 × 106
6.0 × 106
6.3 × 106

ratio b
0.67
0.28
0.33
0.40
0.45

Biacore experiments were carried out in PBS containing 0.005% Tween 20 at 25°C.
a
SpG-C2 and SpA-B are the C2 domain of SpG and the B domain of SpA, respectively.
b
The ratio was calculated by dividing the Ka value obtained in the presence of NP-Cap by that obtained in
the absence of NP-Cap.
mains [17], and have further shown that a B-celltransfectant is activated by NP-protein conjugates
which cannot cross-link antibodies (Oda et al.,unpublished results). In addition, we haveanalyzed the interactions of IgG2a or its enzymatic
fragments with SpA in the presence or absence ofthe hapten and shown novel features of thecomplex formation to support the conformationalchanges in the antibody constant domains uponantigen binding (Sagawa et al., submitted). It
should be noted that Fab and F(ab’)2 fragments ofanti-NP IgG2a antibodies bind to not only SpG but
also SpA and the hapten binding weakens theinteractions of F(ab’)2 fragments. Based on theseresults, we propose a new model of the antibodySpA complex which can explain the conformational change induced by antigen binding.
Alteration of antigen recognition during affinity
maturation
Affinity maturation refers to an increase in
antibodies,
generallyobserved
following
immunization with T-cell-dependent antigen
which consists of the ongoing generation of Bcells with a mutant BCR due to the somatic
hypermutation and selection of these cells on thebasis of affinity. The amino acid substitution of
antibodies, caused by somatic hypermutation,
creates variability for antigen recognition. It has
been shown that affinity of anti-NP antibodies to
NP increases more than 8,000-fold with time after
immunization [23].
In order to understand the mechanism ofaffinity maturation, thermodynamic analysis wasappliedtoantigen-antibody
interactions.

Previously, using three anti-NP monoclonal
antibodies; N1G9, a germline antibody; and 3B44
and 3B62, affinity matured antibodies; themagnitude of ∆H, ∆S, and heat capacity change(∆Cp) for the binding of NP-Cap was shown toincrease the corresponding binding affinity almost
linearly [24]. These results suggest that a bettersurface complementarity was attained in thespecific complex in order to obtain a higher
affinity. Our recent thermodynamic analyses using
more than 30 anti-NP monoclonal antibodies haveshown that the ∆H and ∆S values of antibodies
with higher affinity do not always increase,although the correlation between ∆H and ∆S
values is almost linear, as shown in part in Table 5[25]. This finding indicates that the architecture of
the antibody, including not only the combining
sites but also the framework regions, totallycontributes to the increased binding affinity. The
effects of protein flexibility and salvation can also
make the thermodynamic origin complex. In thecourse of our studies, we obtained a group of antiNP monoclonal antibodies, 9T7, 9T8, 9T10, and
9T13, 9 weeks after the immunization of C57BL/6
mice, and found them to originate from a common
ancestor, judging from the nucleotide sequences[26]. It should be noted that their increased
binding affinities (9T7 < 9T8 < 9T10 < 9T13)
were accompanied by increased ∆S values without
any significant change in enthalpy (Table 5). In
addition, the kinetic measurements show that boththe kon and koff values decreased during affinity
maturation (Table 6). These results suggest that the
antigen-binding mechanism of these antibodies
shifts from a “induced-fit” type to a “lock-and-Masayuki Odakey” type during antibody evolution [27]. The
decreased kon values of more mature antibodies
provide a negative effect on affinity and are incontrast to kinetic maturation [28]. In the case of
the NP-hapten system at least, antibodies undergo
an increase in the Ka value by a change in both the
kon and koff values (Table 6), and at the late stage ofaffinity
maturation,theantigen-antibody
interaction should shift from a “induced-fit” typeto a “lock-and-key” type of mechanism. The
affinity-maturation-dependent decreases in koff
values have been interpreted in terms of rigidifying
mutations that stabilize favorable conformations ofcombining sites, while increases in kon values havebeen interpreted in terms of kinetic selection for
rapid antigen association [28-31].
Table 5. Thermodynamic parameters of theinteractions between NP-Cap and anti-NP IgG1
antibodies
antibody∆G˚∆H(kcal/mol)

T∆S˚

F8
A6
B2
C6
9T7
9T8
9T10
9T13

–10.2
–11.5
–11.7
–13.1
–13.2
–13.5
–13.7
–13.9

–13.4
–25.6
–18.1
–24.2
–18.9
–19.1
–18.8
–19.1

–3.2
–14.1
–6.4
–11.1
–5.7
–5.6
–5.1
–5.2

ITC experiments were carried out in PBS at 37°C.
Table 6. Kinetic rate constants of the interactions
between NP-HEL and anti-NP IgG1 antibodies
antibodykon (M–1s–1) koff (s–1)

Ka,kina (M–1)

B2
C6
9T7
9T8
9T10
9T13

8.7 × 104
1.7 × 105
5.5 × 105
5.4 × 105
1.8 × 105
3.9 × 105

1.1 × 106
1.0 × 107
4.2 × 107
7.2 × 107
7.8 × 107
1.7 × 108

8.1 × 10–2
1.7 × 10–2
1.3 × 10–2
7.5 × 10–3
2.3 × 10–3
2.5 × 10–3

Biacore experiments were carried out in PBS
containing 0.005% Tween 20 at 25°C.
a
The Ka,kin values were calculated from the two
kinetic rate constants, Ka,kin = kon/koff.
Comparison with crystal structural analyses
Two intact mouse monoclonal antibodies,
IgG1 and IgG2a, were crystallized and their51

molecular structures determined [32, 33]. The
results show that IgG1 has an angle of 115°
between two Fab arms so that it exhibits a“distorted Y shape”, while IgG2a has an angle of
172° so that it exhibits a “distorted T shape”.
Because proteins are flexible and exist in
populations of different structures in general, it isnatural to consider that these crystal structures arelike “snapshots” within the wide spectrum ofconformers that can be attained by the antibody
molecule. Our method, using various antigens ofdifferent sizes and epitope densities, is a novel
approach to analyze the antibody flexibility inantigen-antibody interactions [2, 8, 14].
The crystal structures of the Fab fragments ofan anti-NP monoclonal antibody, N1G9, in
unliganded and liganded states, are available andshow major structural differences induced byantigen binding, especially in the complementarity-determining regions (CDRs) [34]. This finding
suggests that an adaptation occurs in CDRs uponantigen binding, similar to other antigen-antibody
complexes [35, 36]. In addition to the changes in
CDRs, relatively large differences upon complex
formation can also be observed in the VH and CH1
domains of N1G9. Although it is not yet certain
whether the changes observed in the crystal
structures are related to the conformational change
induced by antigen binding in solution, theobserved change in the CH1 domain is of particularinterest since this region was shown to be involvedin SpG binding and affected by antigen binding in
our investigations [17]. With respect to the
influence on SpA binding in the CH2-CH3 region,
there is no direct structural evidence that theconformational change is transmitted to the CH2CH3 region through the flexible-hinge region.
In terms of affinity maturation, somestructural analyses are available in which the
crystal structures of germline antibodies arecompared with those of the corresponding affinitymatured antibodies. Nitrophenyl phosphonate, ahapten antigen, binds to the germline antibody
with significant changes in the configuration of the
combining site, while the hapten binds to thematured antibody by a “lock-and-key” mechanism[37]. In the matured antibody, many of the somatic
mutations reconfigure the combining sites byreorganizing networks of hydrogen-bonding,Antibody flexibility

electrostatic, and van der Waals interactions overdistances of 15 Å. The improved complementarity
to hapten is accompanied by the entropic
restriction of residues in the combining site, which
contributes to the 30,000-fold increased binding
affinity. The recent structural analysis of germline
and the mature antibodies specific to bothpolyether jeffamine and its cognate hapten Nmethylmesoporphyrin provide an interestingfinding of the structural plasticity of the germline
antibody and the structural effects of the somatic
mutations that result in increased affinity for thehapten and decreased affinity for jeffamine [38].
Significant conformational changes upon thebinding of these two structurally distinct antigens
are observed in this germline antibody. The effects
of somatic mutations are not only the fixing of the
optimal combining site for the hapten but also theintroduction of the interactions that interfere withthe binding of jeffamine, resulting in the antigen
specificity of the matured antibody. The reorganization of the combining site in the affinitymatured antibody is also supported by the resultsof molecular dynamics simulations [39]. Based onthese results, together with our investigations for
antigen-antibody interactions in solution, at least
three mechanisms can be proposed for increasing
affinity by somatic mutations; 1) direct interaction
with the bound antigen, 2) decreased structural
flexibility of a germline antibody, and 3)
reorganization of the antigen-antibody interface[40]. Through general mechanisms by the immune
response, the germline antibody repertoire isexpanded to multiple combining-site configurations, while somatic mutations can stabilize theconfiguration to interact with specific antigens,resulting in high affinity and specificity.
Protein flexibility in correlation with the energy
landscapeAlthough the crystal structure at atomic
resolution is important to understand the protein
structure and the recognition mechanism between
the biological molecules, there remain someproblems to be considered. The obtained structure
is like a snapshot, as described above, and it can beaffected by unpredictable errors originating fromthe special packing environment within the
crystals. A collection of these snapshotscorresponds to the equilibrium conformation of aprotein, which is critical for understanding thebiological function. The determination or
prediction of the functions of the encoded protein
sequences, based on the three-dimensional
structures, will be a major challenge in the postgenome era. The ensemble nature of protein
conformations is depicted on the energy landscapeof protein folding, namely the folding funnel,
which covers the entire conformational space
allowed for a protein [41]. This concept has been
widely accepted for protein folding and isconsidered to apply for protein binding [42]. In
solution, proteins frequently coexist in a wide
range of conformations (Fig. 2). In the “lock-andkey” binding, the funnel bottom is an associationof two proteins in their most populated native
conformations. In “induced-fit” binding, thecomplex is generated by the minor population(s) ofprotein(s) with conformational changes, which aresometimes coupled with the local folding of aprotein [43, 44].
Although the higher energy conformers aswell as the lower ones are required in order to und-Entropy

Energy52

Native structure
Conformational coordinate
Figure 2. Schematic illustration of the energy
landscape. When the energy landscape is smooth,the native protein would have small fluctuations.
When the landscape is rugged, the ensemble ofprotein structures would include various
conformations. In protein binding, “lock-and-key”
and “induced-fit” types can be explained using theenergy landscape, as described in the text. The
landscape in “lock-and-key” binding would havefew energy minima and the lowest energy bottom,
separated clearly from others. In contrast, thelandscape in “induced-fit” binding would have arugged bottom with rather low barriers separatingthem.

Masayuki Oda

erstand the mechanisms of protein folding andfunction, the minor population of the higher
energy conformers is normally hidden behind theoverwhelming population of the lower energy
conformers [45]. It is difficult to detect the minor
population using spectroscopic methods since evenconventional NMR can only provide an averaged
and single three-dimensional structure close to the
crystal structure. In contrast, it should be notedthat thermodynamic analysis has an advantage fordetecting the total energy of all populations insolution, including not only the intra- or intermolecular interactions but also the protein
flexibility and the solvation effect. For example,
our thermodynamic analyses of major histocompatibility complex class II molecules reveal themechanisms of peptide binding and peptide
exchange at acidic and neutral pHs, which arecorrelated with the dynamic conformational
property of the protein [46-48]. These findings
provide new insight into the current structuralmodel. At the next stage, our understanding will beadvanced if the thermodynamics can be classified
according to each contribution of its origin andcorrelated with the atomic-resolution structure
more quantitatively. With respect to this challenge,
NMR relaxation methods have been applied forthe interpretation in terms of heterogeneouspartitioning between enthalpy and entropy [49,50]. In the thermodynamics of protein folding,molecular dynamics calculations of both nativeand denatured proteins have shown that about 25%of the total ∆S is reflected by changes in simulated
values based on the NMR-derived orderparameters [51].
Future development
Cryo-electron microscopy will be one of theuseful methods to detect the ensemble nature of aprotein since the sample in solution is quenched tothe temperature of liquid nitrogen. The structure ofa neutralizing monoclonal antibody bounddivalently to the human rhinovirus has revealed
that the spanning distance for divalent binding iswithin a range of 60-140 Å [52, 53]. Recent cryoelectron microscopic tomography experiments
using single-particle analysis have shown that the
average value of the Fab-Fab angle of a
monoclonal IgG2a antibody is approximately 110°53

and is distributed with a standard deviation of
approximately 30° [3]. This finding is quite
different from the crystal structure of the same
subclass antibody, which has a “distorted T shape”[32], and demonstrates the difficulty ininterpreting protein structures.
The development or improvement of othertechniques to detect the flexible nature of protein
is underway. One technique is multidimensional
NMR spectroscopy in conjunction with variablepressure perturbation [54]. The idea is based on
the fact that the conformational order of globular
protein normally decreases in parallel with its
partial molar volume and the population shifts to aless-ordered conformer with increasing pressure.
The same group has applied this technique tovarious proteins, such as HEL, prion, and the Rasbinding domain of RalGDS, and shown a goodcorrelation with the respective protein functions.
The traditional method using stopped-flow
measurement is also applicable. Pre-steady-statekinetics and crystal structural analysis revealed anequilibrium between different conformers of an
antibody, which generates multispecificity todifferent antigens [55]. It has been shown thatphoton-echo spectroscopy can measure theresponse to the electronic excitation of an antigen,fluorescein, bound to an antibody [31, 56]. Thismethod can determine the time scale of the
motions of combining sites in a range from tens offemtoseconds to nanoseconds. These flexibility
analyses in comparison with germline and affinitymatured antibodies demonstrate that the antibody
evolved from a flexible germline antibody into
more rigid mature antibody.
With respect to the practical application of
antibodies, such as humanized antibodies as drugs,
many questions remain unanswered. For instance,
how can antigen binding affinity, specificity, andantibody stability rationally be increased?
Although the present methods of rational designs
for not only antibodies but also other target
molecules seem to focus only on the improvementof the binding-surface complementarity, the knowledge of global architecture is needed. It has beenshown that substitution outside the combining site
can increase antigen-binding affinity and alter theconformation of the combining site [26, 30, 37]. In
general, conformational changes due to the protein

54

Antibody flexibility

flexibility can cause the failure of computermodels to properly identify inhibitory compounds[57]. As described above, the residues at a distancefrom the combining site are mutated in the
antibody with femtomolar antigen-binding affinity[6]. Similarly, the residues at the joint between the
VH and CH domains of a humanized antibody are
critical for potent neutralizing activity against
human IFN-γ, suggesting the importance of theantibody flexibility in an active state [58]. In
addition, several reports have shown the criticalresidues at the VH and VL interface for antigen
binding and antibody stability [59, 60]. In order tosolve these problems, we should learn much morefrom nature and apply the knowledge to rational
design. For example, because affinity maturation
results from the repetitive generation and selectionof B-cells, the structural analysis of antibodies in
the process should provide new information which
is applicable to the design of antibodies. With thedevelopment and improvement of techniques todetect dynamic structural and functional propertiesof antibodies, a new stage in the post-genome era
will begin.
Acknowledgements
The author thanks to graduate students at
Tokyo University of Science, Toranosuke Tobita
and Takuma Sagawa, for the collaboration, andProf. Takachika Azuma as a supervisor of ourinvestigations presented in this review article.
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Communicated by Hiroshi Ueno