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X-ray snapshots of the maturation of an antibody
response to a protein antigen
Yili Li1, Hongmin Li1,2, Feng Yang1, Sandra J Smith-Gill3 & Roy A Mariuzza1
The process whereby the immune system generates antibodies of higher affinities during a response to antigen (affinity
maturation) is a prototypical example of molecular evolution. Earlier studies have been confined to antibodies specific for small
molecules (haptens) rather than for proteins. We compare the structures of four antibodies bound to the same site on hen egg
white lysozyme (HEL) at different stages of affinity maturation. These X-ray snapshots reveal that binding is enhanced, not
through the formation of additional hydrogen bonds or van der Waals contacts or by an increase in total buried surface, but by
burial of increasing amounts of apolar surface at the expense of polar surface, accompanied by improved shape
complementarity. The increase in hydrophobic interactions results from highly correlated rearrangements in antibody residues at
the interface periphery, adjacent to the central energetic hot spot. This first visualization of the maturation of antibodies to
protein provides insights into the evolution of high affinity in other protein–protein interfaces.
The ability of the humoral immune system to produce high-affinity
receptors for virtually any antigen derives from its capacity to generate
a large repertoire of antibodies encompassing a vast range of specificities and to then select members of this repertoire with high affinity for
a given immunogen1–3. The extensive sequence diversity characteristic
of antibody molecules has several sources: (i) combinatorial diversification whereby three sets of heavy (H) chain gene segments, VH, D
and JH, and two sets of light (L) chain gene segments, VL and JL,
rearrange to produce functional variable (V) regions; (ii) imprecise
joining of these gene segments; and (iii) somatic hypermutation by
which base changes are introduced throughout the sequences encoding H and L chains4. Selection of high-affinity receptors is then
achieved by the expansion of B-cell clones on the basis of improved
binding to the immunogen1–3. Through this rapid evolutionary
process of mutation and selection, antibody affinity typically increases
10- to 100-fold over the course of an immune response, enhancing
host defense.
The few crystallographic studies of affinity maturation thus far have
used antibodies specific for haptens, such as phenyloxazolone and
nitrophenyl phosphonate5–8, rather than proteins, the major class of
biological antigens. These have shown that somatic mutations in
combining-site residues directly or indirectly involved in binding
hapten permit the formation of additional hydrogen bonding, electrostatic and van der Waals interactions. In one case, large changes in the
conformation of the combining site, mediated by mutations in framework region (FR) residues, were noted upon binding of hapten to a
germline antibody, whereas the free and hapten-bound forms of the
affinity-matured antibody showed few structural differences8. Such
conformational preorganization is supported by thermodynamic
evidence for the modulation of combining-site flexibility during the
maturation process9,10. Whether these mechanisms of affinity maturation apply to protein antigens is unknown, because the physicochemical properties of haptens are different from those of protein
epitopes. Moreover, whereas haptens bind in a cleft between the third
complementarity-determining regions (CDR3s) of the H and L
chains, burying only ∼400 Å2 of total surface area, protein antigens
occupy the entire antibody combining site, contacting all six CDRs
and burying 1,400−2,300 Å2 of surface11,12.
We earlier derived four independent monoclonal antibodies (H8,
H10, H26 and H63) from mice immunized with HEL that recognize
overlapping epitopes on the antigen with relative affinities H26 < H63
< H10 < H8 (refs. 13,14). All four antibodies use the same VL germline
gene (Igk-V23) and have identical VL-JL junctions. The L chains differ
at only three amino acid positions, with the differences attributable to
somatic hypermutations. The H26, H63, H10 and H8 VH genes differ
from the VHM460 germline sequence by 7, 7, 11 and 21 mutations,
respectively, with two of the mutations common to all four VH genes.
The antibodies therefore show an overall correlation between affinity
and number of somatic mutations that is characteristic of affinity maturation1–3. However, the diversity of the VH sequences hinders determination of whether the antibodies are the products of the same
maturation pathways or different ones. Nevertheless, the VH regions,
like their VL counterparts, show >90% sequence identity, with no
amino acid insertions or deletions in VHCDR3.
To obtain snapshots of the maturation of antibodies to protein, we
determined the crystal structures of H8 and H26 in complex with HEL
1Center for Advanced Research in Biotechnology, W.M. Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive,
Rockville, Maryland 20850, USA. 2Wadsworth Center, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, New York 12201, USA.
3National Cancer Institute, Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702-1201, USA. Correspondence should be
addressed to R.A.M. ([email protected]).
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Figure 1 Electron density (stereo views) in antibody
combining sites. (a) Density from the final 2Fo – Fc
map of the H26–HEL complex in the region of
VHCDR2 at a resolution of 2.1 Å. (b) Density from
the final 2Fo – Fc map of the H8–HEL complex in
the same region at a resolution of 1.9 Å. Contours
are at 1σ.
© 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology
a
to resolutions of 1.9 and 2.1 Å, respectively.
The high-resolution structures of antibodies
H10 and H63 bound to HEL15,16, as well as of
H63 in free form16, were reported previously.
By correlating structural changes in these
complexes with differences in free energies of
association, we identified the basis for
increased affinity in this protein–protein
recognition system.
b
RESULTS
Overview of the complex structures
The electron density maps for the complexes
are of high quality (Fig. 1). The overall structure of the H26–HEL complex (Fig. 2a) is representative of all four complexes, including
H8–HEL. The r.m.s. differences in Cα positions of the V domains and HEL are 0.66, 0.54
a
b
d
c
Figure 2 Structure, sequence differences and binding energetics of antibody–HEL complexes. (a) Ribbon diagram of the H26–HEL complex. HEL is yellow,
the L chain is blue and the H chain is green. Residues of HEL (red) in contact with residues of the L (blue) and H (green) chains across the antigen-antibody
interface are drawn. CDRs 1−3 of the VL and VH domains are numbered. (b) Superposition of the H26–HEL (red), H63–HEL (green), H10–HEL (light blue)
and H8–HEL (dark blue) complexes. (c) Amino acid sequence differences among HEL-specific antibodies H26, H63, H10 and H8. In red are those residues
that contact HEL in the corresponding antibody–HEL complexes. Complete VL and VH sequences are given elsewhere14,16. (d) Space-filling model of the
surface of H63 in contact with HEL. Residues are color-coded according to the loss of binding free energy upon alanine substitution18: red, >4 kcal mol–1;
yellow, 2−4 kcal mol–1; green, 1−2 kcal mol–1; blue, <1 kcal mol–1. In magenta are contacting residues in the H63–HEL interface that were not tested by
alanine-scanning mutagenesis. VL residues are labeled in white and VH residues in black.
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and 0.74 Å for comparisons of H26–HEL with Table 1 Characteristics of antibody–HEL complexes
H63–HEL, H10–HEL and H8–HEL, respecH26–HEL
H63–HEL
H10–HEL
H8–HEL
tively. The interacting surface of the antibodies
with HEL is mainly hydrophobic, as observed Binding parameters
in other antigen–antibody complexes11,12. In Ka (M–1)
1.4 (±0.1) × 108 3.6 (±0.1) × 108 3.2 (±0.1) × 109 5.0 (±0.2) × 109
contrast, hydrophilic residues predominate on ∆∆G (kcal mol–1)
0
0.5
1.8
2.1
b
the HEL side of the interfaces.
The HEL epitope recognized by H26 consists Intermolecular interactions
of 18 residues from three separate polypeptide
Hydrogen bondsa
24
25
20
23
segments of the antigen that form a contiguous
144
134
153
van der Waals contactsb 159
patch on its surface (Fig. 2a). This epitope is
c
1
1
1
1
essentially identical to those recognized by H8, Salt bridges
H10 (ref. 15) and H63 (ref. 16), as shown by
superposing the complex structures (Fig. 2b). Buried surface areas
1,812
1,825
1,824
1,872
In each complex, all six CDRs of the V domains ∆SAS (Å2)
1,149
1,101
1,075
1,052
are involved in contacts with HEL. Eight (H8, ∆SASpolar (Å2)
H10) or 9 (H26, H63) L chain CDR residues, ∆SASapolar (Å2)
663
724
749
820
10 (H10, H63) or 11 (H10, H26) H chain CDR ∆SASapolar / ∆SAS (%)
37
40
41
44
residues, and 0 (H10) or 1 (H8, H26, H63) H ∆SASL (Å2)
655
690
661
661
chain FR residues contribute to the contacts: ∆SAS
2
452
481
462
443
L-polar (Å )
Ser30, Asn31, Asn32 (VLCDR1); Tyr50, Gln53 ∆SAS
2
203
209
199
218
L-apolar (Å )
(VLCDR2); Ser91, Asn92, Trp94, Tyr96 ∆SAS
31
30
30
33
L-apolar / ∆SASL (%)
(VLCDR3); Thr/Ile30 (VHFR1); Ser/Arg31,
∆SASH (Å2)
1,157
1,135
1,163
1,211
Asp32, Tyr33 (VHCDR1); Tyr50, Ser52,
697
620
613
609
∆SASH-polar (Å2)
Tyr/Phe53, Ser54, Ser/Asn56, Tyr/Phe58
∆SASH-apolar (Å2)
460
515
550
602
(VHCDR2); Trp98 (VHCDR3); and Glu/Asp99
45
47
50
(VHCDR3). The antibody V regions differ at ∆SASH-apolar / ∆SASH (%) 40
several positions, including HEL-contacting
residues VL30 and VH30, 31, 53, 56, 58 and 99 Shape complementarity
0.69
0.70
0.70
0.75
(Fig. 2c). Importantly, VLAsn32, VHTyr33, Sc
VHTyr50 and VHTrp98, which were identified ScL
0.66
0.66
0.66
0.68
as hot spots for HEL binding by alanine- ScH
0.69
0.72
0.72
0.78
scanning mutagenesis of H10 and H63 aHydrogen bond distance is ≤3.5 Å. bvan der Waals contacts are ≤4.0 Å. cSalt bridge distance is ≤3.5 Å.
(Fig. 2d)17,18, are conserved in all four antibodies, where they are encoded by germline
sequences.
This need not necessarily have been the case, because the surface of
Association constants (KA values) were determined by surface plas- HEL contacted by the antibodies is dominated by polar and charged
mon resonance (SPR) under equilibrium binding conditions (see residues, including Asp18, Arg21, Arg73, Lys96 and Asp101, that offer
Methods). The antibodies bound HEL in the following order of many potential targets for the formation of new hydrophilic interincreasing affinity: H26 (KA = 1.4 × 108 M–1), H63 (3.6 × 108 M–1), actions with mutant or repositioned antibody residues.
The total solvent-accessible surface buried in the four interfaces
H10 (3.2 × 109 M–1) and H8 (5.0 × 109 M–1) (Table 1). This 35-fold
improvement in KA is comparable to the affinity increases observed in (∆SAS) is very similar: 1,812 Å2, 1,825 Å2, 1,824 Å2 and 1,872 Å2 for the
anti-hapten responses1–3.
H26–HEL, H63–HEL, H10–HEL and H8–HEL complexes, respectively
(Table 1). These values fall in the middle of the range for
protein–protein recognition sites21. However, decomposition of the
Sources of increased affinity
In principle, increased affinity could arise from any combination of total buried surface areas into polar (∆SASpolar) and apolar (∆SASapolar)
several variables, including improved shape complementarity, addi- components reveals marked differences among the interfaces, such that
tional interfacial hydrogen bonds or van der Waals contacts, increased there is a direct correlation between complex stability and amount of
buried surface area, or entropic restriction of interacting residues19. As apolar buried surface (Table 1). Thus, the highest-affinity complex
shown in Table 1, there is no apparent correlation between increases in (H8–HEL) has the most apolar buried surface (820 Å2) and the least
binding free energy (∆∆Gb = ∆Gb(H26) − ∆Gb(H63,H10,H8)) and number polar buried surface (1,052 Å2), whereas the lowest-affinity complex
of hydrogen bonds, with the least (H26–HEL) and most (H8–HEL) (H26–HEL) buries the least apolar surface (663 Å2) and the most polar
stable complexes having 24 and 23, respectively, of which 17 are strictly surface (1,149 Å2). The difference of 157 Å2 in apolar buried surface
conserved. Likewise, no correlation is observed between ∆∆Gb and between H8–HEL and H26–HEL represents a 24% gain relative to the
number of van der Waals contacts. These vary from 134 to 159 per latter complex, such that ∆SASapolar constitutes 44% of total ∆SAS in
complex, with the lowest-affinity antibody (H26) making the most H8–HEL, but only 37% in H26–HEL. An extension of this analysis to
contacts with HEL. A single salt bridge, linking hot-spot residues the interfaces between individual VL and VH domains and HEL permits
VHAsp32 and Lys97HEL (ref. 18), is present in all four complexes. us to map the site of these changes. The VL–HEL interfaces display only
Therefore, increased affinity most probably cannot be attributed to minor differences in ∆SASpolar and ∆SASapolar that show no correlation
increased electrostatic or van der Waals interactions, which are major with improved binding (Table 1). For the VH–HEL interfaces, by concontributors to affinity maturation in anti-hapten responses5–8,20. trast, ∆SASapolar increases in tandem with affinity: 460 Å2 (40% of total
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a
b
c
d
an antigen-antibody interface23, which is consistent with estimates of 15−30 cal mol–1 Å–2
from hydrocarbon solubility models24, ∆∆Gb
values of 1.3, 1.8 and 3.3 kcal mol–1 may be calculated for the H63–HEL, H10–HEL and
H8–HEL complexes, respectively, based on
differences in ∆SASapolar compared with the
H26–HEL complex. These predicted ∆∆Gbs
exceed the actual ∆∆Gbs (Table 1) by 0–1.2
kcal mol–1, suggesting two conclusions. First,
increased hydrophobic interactions are sufficient to explain the more favorable binding
free energies of the affinity-matured antibodies, in agreement with the idea that the
hydrophobic effect drives protein-protein
association19,21,25. Second, at least for H63 and
H8, the expected contribution of hydrophobic
forces to complex stabilization is reduced by a
concomitant loss of other types of favorable
interactions (such as electrostatic, through
decreases in ∆SASpolar) or by the introduction
of unfavorable ones (such as steric clashes,
torsional strain).
Structural basis of affinity maturation
To identify structural differences that could
explain the observed affinity differences, we
superposed the H26–HEL, H63–HEL,
H10–HEL and H8–HEL complexes through
HEL. The CDR loops of the antibodies, with
the sole exception of VHCDR1 of H26, align
very closely overall, as do the regions of HEL
that constitute the epitope (Fig. 4a). In particular, hot-spot residues VLAsp32, VHTyr33, VHTyr50 and VHTrp98, as
well as all three VLCDR loops, show nearly identical conformations.
However, important differences are evident in the conformations of
the VHCDR1 and VHCDR2 loops that are directly correlated with
enhanced binding. Residues in these loops are located at the periphery
of the interface with HEL, adjacent to the central hot-spot residues
(Fig. 2d). Compared with its position in H26, VHCDR1 of H8, the
highest-affinity antibody, is displaced by 2.9 Å in the position of the
Ser/Arg31 Cα atom (Fig. 4a). The corresponding shifts in the
VHCDR1 loops of H10 and H63 are 2.0 Å and 1.7 Å, respectively, in
order of decreasing affinity. Similarly, VHCDR2 of H8 undergoes a
rigid-body displacement of 1.4 Å in the position of the Tyr/Phe53 Cα
atom relative to its position in H26; the corresponding shifts in
VHCDR2 of H10 and H63 are 0.8 Å and 0.7 Å, respectively. Snapshots
of VHCDR2 (Fig. 4b) illustrate the progressive movement of HELcontacting residues Tyr/Phe53 and Ser54. These loop rearrangements
are partially mediated by replacement of non-contacting residue
VHArg97 in H26 by Ser/Asn in the higher-affinity antibodies (Fig. 2c).
In H26, the bulky Arg97 side chain displaces VHCDR1 toward
VHCDR2 through contacts with VHCDR1 residue Asp32, resulting in
a concerted shift of both loops relative to their positions in the other
antibodies (Fig. 4a).
On the antigen side of the interface, VHCDR1 contacts mainly HEL
residues 63, 73−77 and 97, whereas VHCDR2 interacts with residues
21, 63 and 100−103. The segments encompassing residues 73−77 and
100−103 are the only regions of the epitope with r.m.s. deviations in
Cα positions of >0.4 Å upon superposition of the bound HEL structures (Fig. 4a). That these same segments show several conformations
Figure 3 Shape complementarity at antibody–HEL interfaces. (a) Molecular surface of H26 viewed at
the site that interacts with HEL in the H26–HEL complex drawn using GRASP37. Regions with higher
Sc values22, indicating closer topological match with HEL, are more blue; regions with topologically
uncorrelated surfaces (Sc = 0) are white. (b) The same view of H26 as described in a, showing the
location of VH residues in contact with HEL. (c) Molecular surface of H8 viewed at the binding site for
HEL in the H8–HEL complex. As in a, regions with better geometric fits to the antigen are more blue.
(d) The same view of H8 as described in c, showing VH residues that interact with HEL.
∆SAS at the VH–HEL interface), 515 Å2 (45%), 550 Å2 (47%) and
602 Å2 (50%) in the H26–HEL, H63–HEL, H10–HEL and H8–HEL
complexes, respectively. These increases in ∆SASapolar are accompanied
by decreases in ∆SASpolar, from 697 Å2 to 609 Å2 for the VH–HEL interfaces of the H26–HEL and H8–HEL complexes, respectively.
Accordingly, the progressive shift from polar to apolar buried surface
area must originate from structural rearrangements in the VH portion
of the interface, as described later.
All four interfaces show relatively high degrees of shape complementarity, based on calculated shape correlation (Sc) statistics22 ranging from 0.69 to 0.75 (Sc = 1.0 for interfaces with geometrically perfect
fits). Notably, the highest-affinity interface is also the most complementary, whereas the lowest-affinity interface shows the poorest topological match (Table 1), suggesting that improved fit is an additional
influence contributing to affinity maturation. As in the case of apolar
buried surfaces, the increase in Sc arises from improved complementarity at the VH–HEL, rather than VL–HEL, interface, as visualized by
mapping Sc values onto the antibody combining sites (Fig. 3). Indeed,
the Sc index for the VH–HEL interface of the H8–HEL complex, 0.78,
is at the upper end of those for oligomeric proteins (Sc = 0.70−0.76),
whose interfaces have co-evolved to optimize the fit (and presumably
the affinity) between the interacting components.
Although the energetic contribution of improved shape complementarity is difficult to assess, theoretical and experimental estimates
of the hydrophobic effect in protein–protein interfaces allow us to ask
whether burial of increased amounts of apolar surface could account
for the measured differences in binding free energy19. Using an experimental value of 21 cal mol–1 for the burial of 1 Å2 of apolar surface in
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and above-average temperature factors (B) in a
different crystal forms of free HEL26–28 may
explain why affinity maturation is mediated
through structural changes in VH, rather than
VL, which contacts more rigid regions of the
antigen.
One effect of the concerted movements in
VHCDR1 and VHCDR2 is to improve shape
complementarity at the VH–HEL interface
(Fig. 3). However, a more important consequence of these shifts, particularly in VHCDR2
(Fig. 4b), is to increase the amount of apolar
surface buried in the interfaces, concomitant
with tighter binding and a reduction in polar
buried surface. Thus, the VHCDR2 loops of
c
b
H26, H63, H10 and H8 contribute 89 Å2,
2
2
2
102 Å , 117 Å and 147 Å , respectively, to
∆SASapolar, whereas the corresponding contributions to ∆SASpolar are 129 Å2, 125 Å2, 113 Å2
and 56 Å2. The residues mainly responsible for
these changes are Phe/Tyr53 and Tyr/Phe58,
both of which are situated at the periphery of
the interface (Fig. 2d). Tyr/Phe53 fills a sizable
and predominantly hydrophobic pocket on
d
the surface of HEL formed by Trp62, Trp63,
Leu75 and Asp101, whereas Tyr/Phe58 is
much less buried (Fig. 4d). The apolar buried
surface contributed by VHTyr/Phe53 rises in
parallel with affinity: 52 Å2 (H26), 72 Å2
(H63), 80 Å2 (H10) and 86 Å2 (H8). Double
mutant cycle analysis of pairwise interactions
in the H63–HEL interface revealed substantial
coupling energies (as much as 1.5 kcal mol–1)
between VHTyr53 and hydrophobic residues
Trp62, Trp63 and Leu75 of HEL18. By contrast,
no coupling was detected between VHTyr53
Figure 4 Conformational differences in antibody–HEL complexes. (a) Comparisons of the combining
and Asp101, despite the loss of 12 van der sites (left) of antibodies H26 (red), H63 (green), H10 (light blue) and H8 (dark blue) (left) and of the
Waals contacts. Therefore, repositioning of the HEL epitope (right) recognized by these antibodies in the corresponding complexes, after least-squares
VHTyr/Phe53 side chain during affinity matu- superposition of their common HEL component. Each HEL structure is the same color as the particular
ration (Fig. 4b) most probably serves to aug- antibody to which it is bound. The antibodies and antigen are oriented such that they can be docked by
ment hydrophobic interactions with HEL folding the page along a vertical axis between them. (b) Close-up view of the VHCDR2 loops (residues
residues Trp62, Trp63 and Leu75. The VH50−58) in a, showing the progressive shift of the Tyr/Phe53 side chain from its position in the
lowest-affinity (red) to the highest-affinity complex (dark blue). (c) Conformational changes in VHCDR2
VHTyr/Phe58 side chain is rotated by 25° in H8
associated with antigen binding. The V domains of complexed H26 (red), H63 (green), H10 (light blue)
relative to its orientation in the lower-affinity and H8 (dark blue) were superposed onto three crystallographically independent structures of free H63
antibodies (Fig. 4b), resulting in the burial of (yellow)16. (d) Stereo diagram of the H26–HEL interface showing interactions of VHCDR1 and VHCDR2
42 Å2 of apolar antibody surface, compared with HEL. HEL is yellow and VH is green. Nitrogen and oxygen atoms are colored blue and red,
with only 10–20 Å2 for the other complexes. respectively. Hydrogen bonds are represented as dotted black lines.
Together, movements in VHTyr/Phe53 and
VHTyr/Phe58 account for most of the difference in ∆SASapolar between the H26–HEL and H8–HEL complexes, lowest-affinity antibody (H26) distorts the combining site most from
with VHThr/Ile30 and VHSer/Arg31 contributing much of the remain- its conformation in the free H63 structures, whereas binding to the
der.
highest-affinity antibody (H8) distorts it least (Fig. 4c). Thus,
The structure of free H63 in different crystal forms16 allows an VHCDR2 of bound H26 is displaced by 2.6 Å in the position of the
assessment of conformational changes in the antibodies upon com- Tyr53 Cα atom relative to its average position in the free H63 strucplex formation. In the free H63 structures, the side chains of hot-spot tures; the corresponding shifts in VHCDR2 of bound H63, H10 and
residues VHTyr33, VHTry50 and VHTrp98, as well as the main chain of H8 are 1.9 Å, 1.8 Å and 1.3 Å, respectively, in order of increasing affinVHCDR2 (Fig. 4c), show several conformations, indicating flexibility. ity. These conformational differences are similar in magnitude to those
Moreover, the combining sites of the bound antibodies align more observed in other complexes involving protein antigens11,12,21. On the
closely with one another than with that of free H63, particularly with assumption that the free H26 and H8 structures resemble that of H63
respect to the VHCDR loops, resulting in convergence toward a com- in terms of the disposition and flexibility of their VHCDR loops,
mon conformation upon binding antigen. The binding of HEL to the another important effect of somatic mutation seems to be to permit
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Table 2 Crystallographic data statistics
antibodies is unique in being the only evolutionary mechanism known to operate on a
H26–HEL
H8–HEL
molecule in an organism’s own body1–3.
In the present study, affinity maturation was
Data collection
found to improve the binding of proteinP42212
Space group
P21212
Unit cells (Å)
a = 90.68, b = 164.02, c = 40.00
a = b = 89.87, c = 148.96 specific antibodies through burial of increased
amounts of hydrophobic surface at the periphAsymmetric unit
1 Fab H26–HEL
1 Fab H8–HEL
ery of the interface with antigen, as the result
Resolution (Å)
2.1
1.9
of rearrangements in non–hot spot residues.
Observations
84,092
403,172
Several considerations suggest that this same
Unique reflections
31,484
46,465
basic strategy may be widely used in the evolua
90.0 (82.3)
95.0 (80.7)
Completeness (%)
tion of protein-protein interfaces. As in the
Mean I / σ(I)a
10.8 (3.1)
21.5 (3.2)
case of the HEL-specific antibodies described
8.6 (20.8)
5.3 (30.0)
Rsym (%)a,b
here, most hot spots may already be optimized
for ligand binding, such that any substitutions
Refinement
or rearrangements at these sites would lead to a
net loss of binding free energy. The periphery,
Resolution range (Å)
100–2.1
100–1.9
on the other hand, may offer more suitable
Rwork (%)b
20.7
21.5
sites for optimization, because these regions
Rfree (%)b
26.2
25.4
are typically more flexible and tolerant to
Non-hydrogen protein atoms
4,245
4,252
mutations25. Indeed, somatic hypermutation
Water molecules
597
547
has been found to spread structural diversity
Average B-factor (Å2)c
generated by V-D-J recombination from
Overall
17.0 (21.6)
22.6 (27.3)
central to peripheral regions of the antibody
V domains
14.9 (17.0)
20.2 (23.1)
binding site30. Additionally, double mutant
C domains
17.1 (19.0)
23.4 (25.8)
cycle analysis of hydrogen bonds between
Lysozyme
20.3 (23.1)
25.7 (28.4)
residues located at the periphery of proteinWater
29.6
34.7
protein interfaces has shown that they usually
R.m.s. deviations from ideal
make little or no net contribution to complex
Bonds (Å)
0.006
0.005
stabilization, presumably because the strength
Angles (°)
1.4
1.4
of these solvated interactions is comparable to
those of the water–protein hydrogen bonds
Dihedrals (°)
26.4
26.5
they replace19,25,31. Similar results were
Improper dihedrals (°)
0.79
0.76
obtained for peripheral residues making only
Ramachandran plot outliers
VLAla30, VLSer51
VLAla51
van der Waals contacts, where the loss of
aValues in parentheses correspond to the highest-resolution shell (2.0–2.1 Å for H26–HEL; 1.8–1.9 Å for H8–HEL).
protein–protein contacts at solvent-accessible
bR
sym= Σ|(Ihkl – I<hkl>)| / (ΣIhkl), where I<hkl> is the mean intensity of all reflections equivalent to reflection hkl by symmetry; Rwork (Rfree) = Σ||Fo| – |Fc|| / Σ|Fo|; 5% of data were used for Rfree. cValues not in parentheses are for main
sites is largely compensated by rearrangements
chains; values in parentheses are for side chains.
in solvent structure18,19,25,31. By contrast, we
have shown that increasing hydrophobic interantibody binding with the least distortion from the ground state, actions and improving the fit at peripheral sites that have not been
which may offset the entropic penalty associated with quenching the optimized for binding, and whose plasticity and ability to accommodate mutations render them permissive to such optimization, constimobility of these loops upon complex formation.
tute effective strategies for evolving higher affinity in protein-protein
interfaces.
DISCUSSION
Evolution of other protein–protein interfaces
Mutagenesis and binding studies of diverse protein-protein complexes
METHODS
have shown that only a small subset of contact residues on both pro- Production of Fab fragments. DNA fragments encoding the V C and V C 1
L L
H H
tein surfaces generally dominate the energetics of the association reac- chains of H8 and H26 were generated by PCR and inserted into expression vec21,25,29
tion
. Moreover, these hot-spot residues nearly always cluster at tor pET-22b (Novagen). Escherichia coli BL21 (DE3) cells were separately transthe center of interfaces, shielded from bulk solvent by peripheral formed with plasmids pET-22b-VLCL or pET22-VHCH1. Bacteria were grown
residues that contribute considerably less to the binding free energy, as at 37 °C in LB medium containing 80 µg ml–1 ampicillin to an absorbance of
in the H63–HEL complex (Fig. 2d). To engineer increased affinity in 0.8 at 600 nm, and isopropyl β-D-thiogalactoside was added to a concentration
such interfaces, Darwinian evolution may, at least theoretically, use of 1 mM. After incubation for 5 h, the bacteria were harvested by centriin 50 mM Tris-HCl (pH 8.0) conany of several strategies. The interfaces may be remodeled at central or fugation. The cell pellets were resuspended
–1
peripheral locations through substitutions in contacting residues, or taining 2 mM EDTA, 100 µg ml lysozyme, and 0.5% (v/v) Triton X-100; cells
were lysed in a French press. The inclusion bodies were washed three times with
in noncontacting residues that influence the conformations of con50 mM Tris-HCl (pH 8.0) containing 0.5% (v/v) Triton X-100 and 2 mM
tacting ones. More favorable binding free energies may then be EDTA and once with the same buffer without Triton X-100, and solubilized in
achieved through increased electrostatic or hydrophobic interactions, 50 mM Tris-HCl (pH 8.0), 6 M guanidine-HCl, 2 mM EDTA and 10 mM DTT.
reduced flexibility or improved complementarity. Unfortunately,
For in vitro folding, the solubilized VLCL and VHCH1 chains were mixed in
archaeological records for tracing the evolutionary pathway of specific an equimolar ratio and diluted into 50 mM Tris-HCl (pH 8.0), 0.4 M Lprotein–protein interfaces are unavailable. Affinity maturation of arginine, 2 mM EDTA, 3 mM reduced glutathione and 0.9 mM oxidized glu-
NATURE STRUCTURAL BIOLOGY VOLUME 10 NUMBER 6 JUNE 2003
487
ARTICLES
© 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology
tathione at a concentration of 60 µg ml–1. The folding reaction was allowed to
proceed at 4 °C for 96 h. The mixture was applied to an HEL affinity column,
and bound Fab was eluted with 50 mM glycine-HCl (pH 2.5). Further purification was carried out on a Mono Q FPLC column (Amersham Pharmacia) equilibrated with 50 mM Tris-HCl (pH 8.5); Fab was eluted using a linear NaCl
gradient.
Crystallization and data collection. The Fab H8–HEL and H26–HEL complexes were crystallized at room temperature in hanging drops from mixtures
containing a 1.2:1 molar ratio of antibody to antigen at total protein concentrations of 5−10 mg ml–1. The H8–HEL complex crystallized in 20% (w/v) PEG
8000, 50 mM KH2PO4 and 100 mM sodium acetate (pH 4.6), whereas crystals
of the H26–HEL complex grew in 14% (w/v) PEG 4000, 100 mM ammonium
acetate and 50 mM sodium acetate (pH 5.0).
X-ray diffraction data for the Fab H8–HEL complex were collected at 100 K
on beamline X12B of the Brookhaven National Synchrotron Laboratory using
an ADSC Quantum-4 charge-coupled device detector. Diffraction data for the
Fab H26–HEL complex were measured at 100 K using an in-house 345-mm
MarResearch Image Plate detector. Crystals of both complexes were transferred
to a cryoprotectant solution (mother liquor containing 25% (v/v) glycerol) and
flash-cooled in a nitrogen stream. Diffraction data were processed and scaled
using DENZO and SCALEPACK32, and data were reduced using programs
from the CCP4 suite33. Data collection statistics are summarized in Table 2.
Structure determination and refinement. The H8–HEL complex crystallized
isomorphously with the H63–HEL complex (PBD accession code 1DQM)16 in
space group P42212 (Table 1). After rigid-body refinement of the H8–HEL
structure starting from the H63–HEL complex with all water molecules deleted,
further refinement was carried out using CNS34, including interactive cycles of
simulated annealing, positional refinement, torsion angle refinement and
B-factor refinement, interspersed with model rebuilding into σA-weighted Fo –
Fc and 2Fo – Fc electron density maps using TURBO-FRODO35. The structure
of the H26–HEL complex was solved by molecular replacement using AMoRe36,
with the H63–HEL complex as a search model; refinement was carried out as for
the H8–HEL complex. Refinement statistics are summarized in Table 2.
Calculation of accessible surface areas and shape correlation statistics.
Changes in polar, apolar and aggregate-accessible surface areas were calculated
using AREAIMOL and DIFFAREA from the CCP4 suite33 with a probe radius
of 1.4 Å. Shape correlation statistics22 were calculated using Sc (version 2.0)
from CCP4.
Affinity measurements. The interaction of soluble Fab H8, H10, H26 and H63
with immobilized HEL was assessed by SPR using a BIAcore 1000 biosensor as
described18. Association constants were determined from Scatchard analysis,
after correction for nonspecific binding, by measuring the concentration of free
reactants and the complex at equilibrium. Standard deviations for two or more
independent KA determinations were <10% (Table 1).
Coordinates. Atomic coordinates and structure factors for the H26–HEL and
H8–HEL complexes have been deposited in the PDB (accession codes 1NDM
and 1NDG, respectively).
ACKNOWLEDGMENTS
We thank M.K. Gilson, E.J. Sundberg and C.P. Swaminathan for critical reading of
the manuscript. This work was supported by grants from the National Institutes of
Health.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 13 December 2002; accepted 16 April 2003
Published online 12 May 2003; doi: 10.1038/nsb930
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