High-resolution structures of the IgM Fc domains
reveal principles of its hexamer formation
Roger Müllera,1, Melissa A. Gräwerta,b,1, Thomas Kerna,c,1, Tobias Madla,c,d, Jirka Pescheka, Michael Sattlera,c,2,
Michael Grolla,2, and Johannes Buchnera,2
a
Munich Center for Integrated Protein Science at the Department Chemie, Technische Universität München, 85748 Garching, Germany; bDeutsches
Elektronen Synchrotron, European Molecular Biology Laboratory, 22603 Hamburg, Germany; cInstitute of Structural Biology, Helmholtz Zentrum München,
85764 Neuherberg, Germany; and dInstitute of Chemistry, Karl-Franzens Universität Graz, 8010 Graz, Austria
Edited by Robert Huber, Max Planck Institute of Biochemistry, Planegg-Martinsried, Germany, and approved May 6, 2013 (received for review
January 10, 2013)
IgM is the first antibody produced during the humoral immune
response. Despite its fundamental role in the immune system, IgM
is structurally only poorly described. In this work we used X-ray
crystallography and NMR spectroscopy to determine the atomic
structures of the constant IgM Fc domains (Cμ2, Cμ3, and Cμ4)
and to address their roles in IgM oligomerization. Although the
isolated domains share the typical Ig fold, they differ substantially
in dimerization properties and quaternary contacts. Unexpectedly,
the Cμ4 domain and its C-terminal tail piece are responsible and
sufficient for the specific polymerization of Cμ4 dimers into cova-
lently linked hexamers of dimers. Based on small angle X-ray scat-
tering data, we present a model of the ring-shaped Cμ4 structure,
which reveals the principles of IgM oligomerization.
antibody oligomerization | hybrid approach | dimer interfaces
A ntibodies, also referred to as Igs, defend against infection
by the inactivation of viruses or bacteria and by recruiting
downstream effectors such as the complement system or cells
specialized to kill invading microorganisms (1, 2). IgM is the first
antibody to be produced during the humoral immune response
(3). Early IgM antibodies are secreted before B cells have un-
dergone somatic hypermutations and therefore tend to be of low
affinity. To compensate for the reduced binding efficiency of the
monomers, IgM forms oligomers (Fig. 1) whose multiple antigen-
binding sites confer high overall avidity (4). Moreover, the com-
plex structure of IgM makes it especially effective in activating the
complement system (5).
Whereas the antigen-binding fragment (Fab) of different Ig
classes follow a common topology, the constant fragment (Fc)
parts differ substantially in domain composition and architec-
ture. In contrast to IgG, the Fc part of IgM is composed of three
Ig domains (Cμ2, Cμ3, and Cμ4) and an additional C-terminal
tail piece (tp). The IgM polymer is composed of subunits in
which two heavy chains (μ) are covalently paired with two light
chains (L). IgM is present either as pentamers (μ2L2)5J in the
presence of the J chain, or as hexamers (μ2L2)6 in the absence of
the J chain, a small protein that is involved in IgM assembly and
secretion (6–8). Upon assembly, the heavy chains are covalently
linked in the Cμ2, Cμ3, and Cμ4 (tp) domains by interchain
disulfide bridges (Fig. 1).
The Fc region of IgM is of outstanding interest because its
structure, oligomerization, and effector protein binding clearly
differs from other Ig Fc regions. Our current understanding of
the IgM structure largely originates from negative-stain EM (9),
which identified the pentamer as a planar, star-shaped oligomer
with the Fab fragments pointing away from the inner core com-
posed of the Fc regions. Early structural models of complement
activation were based on small angle X-ray scattering (SAXS)
data and modeling (10) and suggest a planar Fc disk containing a
central Cμ4 ring. The Cμ3 and Cμ2 domains are attached in a star-
shaped manner. The latest model makes use of the high similarity
of IgM and IgE. IgM shares the basic IgE domain architecture
www.pnas.org/cgi/doi/10.1073/pnas.1300547110
with three domains in the Fc part (11). Fluorescence data (12)
and the subsequently determined crystal structure (11) of IgE Fc
revealed that the IgE Fc is sharply bent. Interestingly, the latest
IgM model suggests a similar Fc region with the Cμ4 domains
protruding out of the plane defined by Cμ2, Cμ3, and the Fab
domains (13).
Owing to its flexibility, crystallization trials of the IgM oligomer
have not been successful to date. In this study, we applied X-ray
crystallography and NMR spectroscopy to determine the atomic
details of all individual mouse IgM Fc domains (Cμ2, Cμ3, and
Cμ4). Analysis of their quaternary arrangements in solution re-
vealed unexpectedly that Cμ4 forms defined ring-like hexamers of
dimers. SAXS data of the hexameric domain assembly together
with modeling suggest a model for the IgM Fc structure.
Results
Characterization of the IgM Fc Domains. To gain insight into the
elusive structure of the IgM Fc segment, we produced the in-
dividual Fc domains recombinantly in Escherichia coli and char-
acterized their properties after purification. Investigation of the
influence of intersubunit disulfide bonds on oligomerization was
addressed by serine mutations of the corresponding cysteine res-
idues in Cμ2 (C337), Cμ3 (C414), and Cμ4tp (C575). During
thermal denaturation, all domains unfold cooperatively with
transition midpoints ranging from 59–65 °C (Table 1 and Fig. S1).
The lack of thermodynamic stability parameters is caused by the
irreversibility of the transitions. However, guanidium chloride
(GdmCl)-induced equilibrium unfolding (Fig. S1) resulted in re-
versible transitions of all domains, and thermodynamic parame-
ters are given in Table 1. All domains behave like two-state folders,
not significantly populating any equilibrium intermediates.
To investigate the quaternary structure of the isolated domains,
we first performed size-exclusion chromatography (SEC) experi-
ments (Fig. S2). The Cμ2 domain was purified as a disulfide-linked
dimer, as determined by nonreducing SDS/PAGE, which is in
agreement with the elution profile of the SEC column (Fig. S2A,
dotted line). Moreover, our data demonstrate that dimer forma-
tion was independent of the protein concentration. However, when
BIOCHEMISTRY
Author contributions: R.M., M.S., M.G., and J.B. designed research; R.M., M.A.G., T.K., T.M.,
and J.P. performed research; R.M., M.A.G., T.K., T.M., J.P., M.S., M.G., and J.B. analyzed data;
and R.M., M.S., M.G., and J.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org [PDB ID codes 4JVU (Cμ2 domain), 4BA8 (Cμ3 domain),
4JVW (Cμ4 domain), and 4BLE (C-alpha coordinates of the Cμ4tp hexamer of dimers small
angle X-ray scattering model).
1
R.M., M.A.G., and T.K. contributed equally to this work.
2
To whom correspondence may be addressed. E-mail: sattler@helmholtz-muenchen.de,
michael.groll@ch.tum.de, or johannes.buchner@tum.de.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1300547110/-/DCSupplemental.
PNAS | June 18, 2013 | vol. 110 | no. 25 | 10183–10188
 interaction in the SEC and aUC experiments. Taken together,
these experiments conclude that the Cμ2 and Cμ4 domains form
dimers in solution, whereas the Cμ3 domain is monomeric. Next,
we performed structural analysis of the respective Cμ domains.

Crystal Structure of the Cμ2 Domain. Crystals of the Cμ2 domain

Fig. 1. Schematic view of hexameric IgM (Left) and a single subunit (Right).
Heavy chains are depicted in light blue and light chains in orange. Red lines
represent intersubunit disulfide bridges and the green hexagons show
glycosylation sites. The cysteine residues that form interdomain disulfide
bridges between the Cμ2 domains (C337) and covalently link the IgM hex-
amer in Cμ3 (Cys414) and the C-terminal tp (C575) are depicted.
the interchain disulfide bond was removed by mutating Cys337 to
Ser, the elution time became concentration-dependent. The single
peak is indicative of a fast monomer/dimer equilibrium (Fig. S2A).
Both the wild type and the C414S mutant of the Cμ3 domain eluted
predominantly as monomers and did not show any peak-shift (Fig.
S2 B and C). However, in the case of wild-type Cμ3, small amounts
of covalent dimers were observed. The Cμ4 domain behaved
similar to the Cμ2C337S mutant. It also exhibits a fast monomer/
dimer equilibrium, although with lower affinity as estimated from
the smaller shift at higher protein concentrations (Fig. S2D). When
the C-terminal tp with the penultimate C575 was attached to the
Cμ4 domain, at least one additional prominent species with sub-
stantially increased molecular weight appeared (Fig. S2E). This
peak could be minimized by mutating Cys575 to Ser (Fig. S2F),
indicating that this species seems to correspond to a covalently
linked Cμ4tp dimer or higher-order oligomers (discussed below).
To gain further insight into Cμ domain association, we per-
formed sedimentation equilibrium (SE) and sedimentation ve-
locity (SV) analytical ultracentrifugation (aUC). Data from SE
runs performed at different rotor speeds and protein concen-
trations were fitted with a model for self-association (14) to cal-
culate dissociation constants for the monomer/dimer equilibrium
and were confirmed by SV-aUC (Table 1 and Fig. S3). For the
Cμ2C337S mutant without the interdomain disulfide bridge, a
Kd of 2.1 ± 0.1 μM was assigned. The weaker dimerization of the
Cμ4 domain deduced from the SEC experiment was verified by
aUC, resulting in a Kd of 86 ± 3 μM. The presence of the C-
terminal tp (Cμ4tpC575S) decreased the affinity of the Cμ4 do-
main dimer to a Kd of 224 ± 7 μM. This finding might be the
consequence of a not-fully-unstructured tp that is interacting with
the Cμ4 domain. In contrast, the Cμ3C414S domain showed no
were obtained in the space group C2, with the unit cell dimensions
a = 92.4 Å, b = 44.8 Å, and c = 54.6 Å, and two molecules in
the asymmetric unit accounting for 37% solvent content. The phase
problem of the IgM Cμ2 domain was determined by Patterson
search calculations using the IgE Ce2 (1O0V) (11) as a starting
model and the structure was refined to an Rfree of 18.1% at
1.3 Å resolution (Table S1). Overall the model is well defined in
its electron density map, yet some loops (gap between chain-A
Glu-293 to Thr-298 and chain-B Phe-248 to Lys-255 and Thr-293
to Gln-300) were structurally disordered and have therefore been
omitted. The Cμ2 domain crystallized as a dimer, and the con-
formation of Cμ2 shows a typical Ig fold with a two-layer sandwich
of seven antiparallel β-strands arranged in two β-sheets with
a Greek key topology (Fig. 2A). Structural superposition of the
Cα-atoms of both chains yields a rms deviation of 0.6 Å. Both
chains possess an Ig fold typical internal disulfide bond between
Cys-260 and Cys-320. The electron density map shows high oc-
cupancy and low Debye–Waller factors in the disulfide bridges,
which are oriented perpendicular to the β-sheets. A common
feature of many members of the Ig superfamily is the presence
of at least one cis proline residue in the native structure. Both
domains harbor this cis peptide bond between Thr-266 and Pro-
267. However, in chain A there is an additional cis peptide bond
involving Pro-253, whereas the corresponding loop in chain B is
not resolved.
To analyze the biological assembly state of the structure, the
PISA server (15) was used and confirmed that Cμ2 forms a phys-
iological dimer comprising a buried area of ∼1,660 Å2. The Cμ2
dimer shares the mode of domain pairing seen in IgE Ce2 with
its less extensive interaction surface and interchain disulfide bonds
(11). However, unlike IgE Ce2, the interface is dominated by
a central hydrophobic core (Fig. 2A) as typically seen in other Ig
domain structures. The dimer is further stabilized by six hydrogen
bonds, one salt bridge (between chain A Glu-261 OE2 and chain
B Lys-257 NZ, 2.77 Å) as well as an additional disulfide bridge
between the C-terminal Cys-337 residues of chain A and B (Fig. 2A).
The hydrophobic contact interface also accounts for the observed
dimer formation in the absence of the C-terminal disulfide bridge.
Solution Structure of the Cμ3 Domain. Because crystallization trials
of the Cμ3C414S domain failed, this structure was elucidated
by heteronuclear NMR spectroscopy (Table S2). We determined
the overall tumbling correlation time of Cμ3C414S to be 6.0 ± 0.4 ns
from 15N NMR relaxation data (Fig. S4). Comparison with an
expected value of 7.5 ns for the monomer and 14.4 ns for the
dimer confirms that Cμ3C414S is monomeric, in agreement with
the sedimentation data described above. The solution structure of
Cμ3C414S consists of the typical Ig fold with a β-sheet sandwich

Table 1. Oligomeric state and conformational stability of the IgM Fc domains

Domain Mass, kDa Kd, μM ΔGunfolding, kJ·mol−1 meq, kJ·mol−1·M−1 Tmelt, °C

Cμ2 24.6 — 36.2 18.6 64.5

Cμ2C337S 23.6 2.1 ± 0.1 26.0 16.6 60.0
Cμ3C414S 11.3 — 15.8 10.2 57.9
Cμ4 17.8* 86 ± 3 15.8 14.4 58.9
Cμ4tpC575S 15.4* 224 ± 7 21.1 17.5 60.9

Molar masses and Kd values for domain dimerization were calculated from SV and SE aUC runs, respectively.
*For Cμ4 and Cμ4tpC575S, monomeric and dimeric species could not be separated due to fast exchange rates.
ΔGunfolding and the cooperativity parameter (meq) originate from GdmCl denaturation experiments and melting
temperature (Tmelt) from thermal denaturation experiments.

10184 | www.pnas.org/cgi/doi/10.1073/pnas.1300547110 Müller et al.

Fig. 2. Structures of the individual IgM Fc domains.
(A) Cartoon representation of the crystal structure of
the Cμ2 domain (Upper, Protein Data Bank ID code
4JVU), the arrangement of the two molecules in the
crystallographic asymmetric unit forming a stable di-
mer. Inter- and intramolecular disulfide bonds are
shown in stick representation. The surface of one Cμ2
domain (chain A, yellow), showing the contact area
with the second Cμ2 domain (chain B, red), is repre-
sented as Cα-trace (Lower). (B) Solution structure of the
Cμ3 domain (Protein Data Bank ID code 4BA8). Super-
position of the 10 lowest-energy NMR structures are
shown as cartoon (Upper). The highly flexible DE loop is
labeled. Chemical shift perturbations in the Cμ3 do-
main, when bound to the Cμ4 domain, are shown
(Lower). The color scale is as follows: yellow, 0.025–0.05
ppm; orange, 0.05–0.1 ppm; and red, >0.1 ppm. The
positions for the interdomain disulfide bridge (Cys414)
and the glycosylation site (Asn402) are labeled and
shown in stick representation. (C) Cartoon represen-
tation of the crystal structure the Cμ4 domain (Upper,
Protein Data Bank ID code 4JVW). Intramolecular disul-
fide bonds are shown in stick representation. The surface of one Cμ4 domain (chain A, blue), showing the contact area with the second Cμ4 domain (chain B, red), is
represented as Cα-trace (Lower). Color coding for the interfaces: gray, hydrophilic contact residues; green, hydrophobic contact residues.
that includes an intramolecular disulfide bridge between Cys367
and Cys426 (Fig. 2B). NMR signals of residues in the loop between
strands D and E connecting the two β-sheets are line-broadened
owing to chemical exchange, further confirmed by comparison of
the transverse R2 15N relaxation rates to offset-corrected R1ρ rates
(Fig. S4). The increased dynamics of this loop had been already
observed in the corresponding IgG Cγ2 domain in the absence of
glycosylation in the DE loop (16), suggesting that the DE loop in
Cμ3C414S may be stabilized by glycosylation of Asn402. Chemical
shift perturbations (CSP) of Cμ3C414S and Cμ3C414S–Cμ4 do-
main constructs (Fig. S4) have been obtained by comparing the
chemical shifts of their 1HN, 15N resonance from the backbone
assignments. The CSPs have been calculated according to Eq. S2.
In the spectra of the tandem construct the region between
Phe354–Phe358 has been broadened out completely owing to the
interaction in the interface, and therefore no exact CSP could
be assigned. Residues Thr374–Leu378 and Val396–Ser399 have
been excluded from the analysis, because their 1HN, 15N resonances
could not be assigned owing to line broadening in the single domain
construct. This allowed mapping of the binding interface of the Cμ3
domain with Cμ4 (Fig. 2B). Interestingly, this interface on Cμ3
comprises predominantly the two helices at the C-terminal side of
Cμ3 and is very similar to the interactions seen between the cor-
responding domains in other Ig classes. The mapped interface on
Cμ3 was then used for modeling experiments, eventually yielding
the overall domain arrangements below.
Crystal Structure of the Cμ4 Domain. The Cμ4 domain crystallized
in the space group C2 with the cell dimensions a = 169.2 Å, b =
41.2 Å, and c = 67.1 Å. Four molecules are located in the asym-
metric unit leading to 40% solvent content. Using the structure of
the Cμ2 domain as a starting model, the IgM Cμ4 could be de-
termined by molecular replacement methods and refined to a Rfree
of 23.4% at 2.0 Å resolution (Table S1). The conformations of all
four IgM Cμ4 monomers show a typical Ig topology that is stabi-
lized by a disulfide bond between Cys474 and Cys536 (Fig. 2C) and
a cis peptide bond involving Pro481 is observed.
The analysis of the biological assembly using the Protein Inter-
faces, Surfaces and Assemblies (PISA) server predicts that the
monomers A and D as well as B and C form stable dimers. The
conformation of both dimers in the asymmetric unit match to
each other with an rmsd on the Cα-atoms of 0.3 Å and 0.4 Å,
respectively. Approximately 1,900 Å2, which corresponds to 17%
of the surface area, are buried in the interface that comprises
Müller et al.
hydrophobic protein interactions resulting in a stable dimer of
Cμ4 (Fig. 2C). Interestingly, compared with the dimerization
behavior of other Ig folds, the two molecules are arranged in
a parallel manner (Fig. 2C). Particularly, the N termini are
located on the same side of the dimer, and the C termini on the
opposite end, which is important for the oligomer formation. A
summary of the different homo–dimer association modes of
Ig Fc domains is shown in Fig. S5.
Cμ4 Oligomerization. As shown above, the Cμ4tp domain forms
weak noncovalent dimers in the absence of Cys575 in the tp. In
the presence of Cys575, we also observed noncovalently linked
dimers in dilute solutions. At higher concentrations, however,
those dimers form disulfide-linked oligomers. The analysis of the
native Cμ4tp by SEC revealed a concentration-dependent equi-
librium between dimers and oligomers with masses of 31 kDa
and 198 kDa applying multiangle light scattering (MALS) (Fig.
3A). Interestingly, the latter species corresponds well to a hex-
amer of Cμ4 dimers, as observed for native IgM in the absence of
the J chain (6, 17, 18). Thus, the isolated Cμ4tp domain is suf-
ficient for the assembly of defined hexamers of dimers.
Further support of oligomer formation was received by SV
aUC runs at different protein concentrations (1–20 μM) (Fig.
3B), revealing a major species that sediments with 8.8 S and a
minor one with 2.6 S. Both the sedimentation coefficients and
the fitted frictional coefficient f/f0 of 1.34 calculated the masses
for the two species to be 190 kDa and 30 kDa, respectively.
These values validate the dimer/hexamer of dimer equilibrium
of Cμ4tp dimers. Furthermore, also the relative ratios could
be shifted toward the hexamer of dimers in a concentration-
dependent manner (Fig. 3B, Inset).
BIOCHEMISTRY
Model for the Cμ4 Hexamer Structure. SAXS experiments were
used to further characterize the hexameric structure of Cμ4tp
dimers (Materials and Methods gives details). The molecular
mass of Cμ4tp of 162 ± 4 kDa, determined based on the I(0), fits
well to the expected molecular mass of 176 kDa for a hexamer.
The radius of gyration (Rg) of 41.6 ± 0.5 and a maximum dimen-
sion of 114 Å also support hexamer of dimer formation (Fig. S6A).
The SAXS data together with the crystal structure of the Cμ4
domain allowed us to model the hexameric structure of Cμ4tp
dimers. Note that the additional Cμ4–Cμ4 interface observed in
the crystal structure cannot be used to construct the hexamer
(Fig. S6E). Therefore, no additional restraints have been used
PNAS | June 18, 2013 | vol. 110 | no. 25 | 10185

Fig. 3. Characterization of the Cμ4tp oligomer. (A) SEC MALS profile of the
Cμ4tp oligomer. UV absorbance is shown in black, and calculated masses in
red. (B) Cμ4tp oligomer characterized by aUC SV runs at 20 μM (black), 5 μM
(red), and 1 μM (cyan). The histogram shows the percentages of hexamer of
dimers (dark gray) and dimers (light gray) of the total protein in solution.
in the SAXS modeling. All structures share a characteristic to-
roidal assembly (Fig. 4B). The generated structures fit well to the
experimental data (χ = 1.98–3.34) (Fig. S6G). Interestingly, in
the oligomer the Cμ4tp dimers contact each other involving the
surface of the β-sheets and several loop regions with a surface
area of ∼1,000 Å2 (Dataset S1). The N termini of Cμ4tp are
pointing outward of the hexameric ring, whereas the C termini,
which are structurally constrained by the disulfide bond, are
facing its interior (Fig. 4B).
In conclusion, the various structural and functional data
described above allowed us to generate a model of the entire
Cμ2–Cμ3–Cμ4tp hexamer (Fig. 4A) using (i) the structures of the
individual subunits, (ii) the SAXS-derived structure of the hex-
americ Cμ4tp dimers, (iii) the distances for the inter-Cμ3 disulfide
bonds, and (iv) NMR chemical shift mapping of the Cμ3–Cμ4
binding interface (Fig. 2C and Fig. S4). We conclude that the
Cμ2–Cμ3 subunits in IgM point apart from the hexameric ring
formed by Cμ4tp dimers (Fig. 4C). The domain interactions, in-
cluding the short linkers between the Cμ2, Cμ3, and Cμ4 domains,
sterically restrict the accessible conformational space (Fig. 4C).
These findings demonstrate that the Cμ4 domain orchestrates the
assembly of the oligomeric IgM, whereas the covalent linkages in
the tail piece and the Cμ3 domain stabilize this complex.
Discussion
Whereas the structure and assembly of IgG is well-studied (19),
for IgM we largely lack detailed structural information. Here
10186 | www.pnas.org/cgi/doi/10.1073/pnas.1300547110
we present a hybrid approach using X-ray crystallography, NMR,
and SAXS to solve the structures of the individual domains
of the mouse IgM Fc (Cμ2, Cμ3, and Cμ4) and to reconstruct the
Fc oligomer. Aside from the expected domain topologies with
their characteristic β-sheet sandwich and two short α-helices
(20), we observed unexpected domain associations that were not
considered in previous models. Based on electron microscopy,
X-ray scattering, and mutagenesis studies, we know that the IgM
polymer is assembled through interactions of identical domains,
that is, Cμ2–Cμ2, Cμ3–Cμ3, and Cμ4–Cμ4, and that the corre-
sponding cysteine residues (C337, C414, and C575) form the
interchain disulfide bonds (21–23). A detailed mutagenesis study
showed that the cysteine at position 575 is essential for efficient
assembly, whereas C337 and C414 are not needed for polymer
formation (24). The latest IgM model is based on the IgE
structure (13).
To obtain a better understanding of the elusive structure of
the IgM Fc, we first characterized the isolated Fc domains. The
Cμ2 domain, the most N-terminal domain of the IgM Fc, replaces
the hinge region found in IgG (25). It forms a disulfide-linked
dimer with a unique interface dominated by hydrophobic inter-
actions. In the absence of the disulfide bridge, the Kd is around
2 μM. This weak interaction needs to be further stabilized by the
covalent linkage (Cys337). The IgE Ce2 domain is functionally
equivalent to Cμ2 and it also bears some structural resemblance.
Both have a unique association mode with a small interface
area, compared with other Ig constant domains, and a distinct
rotation angle (110° for Cμ2 and 105° for Ce2) between the
domains (Fig. S5A). However, there are important differ-
ences. These include the arrangement of the disulfide bridges
in Ce2 (11) and the interface that is polar and less pronounced
in Ce2, leading to a monomeric protein in the absence of the
disulfide bonds (26).
The Cμ3 domain does not make any stable dimer contacts. This
is similar to the corresponding IgG Cγ2 and IgE Ce3 domains,
which do not have any direct contacts either. In contrast to IgG and
IgE, IgM forms larger oligomers. During assembly, the Cμ3 do-
mains come into close proximity and the weak Cμ3–Cμ3 inter-
actions are stabilized via disulfide bridges (Cys414) only in the
complete IgM oligomer. The position of the Cys414 at the edge of
the Cμ3 domain and its mainly polar environment supports such
a weak contact that must be stabilized by a disulfide bridge.
The mode of dimerization elucidated here for Cμ4 has so far
not been observed among Igs (Fig. 2 and Fig. S5A): Whereas all
other C-terminal antibody domain dimers such as IgG Cγ3, IgA
Cα3, and IgE Ce4 are arranged in an antiparallel fashion, with
the N and C termini on opposing sides, the IgM Cμ4 topology
results in the location of the N termini and C termini on the
same side. This is explained by the rotation angles between the
domains. Whereas IgG Cγ3, IgA Cα3, and IgE Ce4 have rota-
tion angles of 132°, 138°, and 142°, respectively, the Cμ4 of IgM
harbors a rotation angle of 70°, which is important for the self-
assembly of the dimers into pentamers or hexamers of dimers.
Interestingly, IgA Cα3 and IgE Ce4 share up to 50% sequence
identity with Cμ4. Whereas the dimer interfaces of IgM Cμ4, IgA
Cα3, and IgE Ce4 share a conserved, mostly hydrophobic, core,
most residues lying at the outer part of the Cμ4 interface show
little identity or homology to IgA or IgE (Fig. S5B). These dif-
ferences must lead to the different dimer association of Cμ4.
Hence, their different oligomeric states are caused by the unique
amino acid composition at the interface that defines the con-
figuration of the dimer.
A hallmark of the current work is the identification of the
minimum requirements for IgM oligomerization. Attaching the
C-terminal tp to the Cμ4 domain is already sufficient for oligomer
formation, including the intermolecular disulfide bridge involving
Cys575, the penultimate amino acid in the tp. Our results therefore
show that the Cμ4 domain possesses two dimerization interfaces
Müller et al.
Fig. 4. SAXS analysis of Cμ4tp and hexameric IgM Fc modeling. (A) Scheme of the IgM Fc hexamer with the information used for model building is in-
dicated. (B) Structural model of the Cμ4tp hexamer of dimers (Protein Data Bank ID code 4BLE). The hexameric subunits and N/C termini of the Cμ4 dimer
structure are annotated. One single Cμ4tp dimer is shown in blue. (C ) Cartoon representation of the IgM Fc hexamer model (Left). A side view of the Fc
hexamer is given (Right), showing that the inner core (Cμ4) is protruding out of the plane defined by the Cμ2 and Cμ3 domains. Cμ2 dimers are shown in
yellow, Cμ3 in gray, and the Cμ4 core ring in blue. The termini and linker residues are represented as Cα dummy atoms by the program CORAL and are
shown as spheres.

consisting of the four β-strands as well as a weak interaction site
distinct from the first one that is stabilized by the interdimer
disulfide bridge. Thus, our data reveal the existence of a non-
covalently associated Cμ4 dimer that is covalently surrounded by
two adjacent Cμ4 dimers. The noncovalent Cμ4 oligomer does not
seem especially stable (Fig. 3) at protein concentrations usually
found in plasma (ca. 1 μM). However, the oligomer is further
stabilized by the Cμ2 dimer interface and the inter-HC disulfide
bridge at the C terminus of the Cμ2 domain (23).
Based on the ring-like Cμ4 oligomer and the data from the
single domains, this study describes a model for the hexameric
IgM Fc, composed of the Cμ4 inner core and the Cμ3 and Cμ2
domains that build a flexible star-shaped arrangement around
the inner core. This is similar to previous models based on
electron microscopy (9) and SAXS (10) data. However, the
model presented here is not planar and the central region (Cμ4)
is projecting out of the plane defined by the Cμ2 and Cμ3
domains (Fig. 4C). The dimensions of this structure are in good
agreement with previous estimations based on cryo-atomic force
microscopy (AFM) data (13). In addition to the nonplanar
shape, also the size of the central circular region, composed of
the Cμ3 and Cμ4 domains, with a diameter of ∼180 Å, fits well to
of six or more subunits are secreted (30, 31). Furthermore, the J
chain plays an important role in the selective assembly and it is
expected that its major function is a place holder for one IgM
subunit in the pentamer compared with the hexamer (32). This is
supported by the hexameric ERp44/ERGIC53 assembly platform
that is involved in the formation of pentameric and hexameric
IgM assemblies. Therefore, the hexameric Cμ4 ring presented here
might be identical to a pentameric Cμ4/J-chain ring with one Cμ4
dimer subunit exchanged by one J-chain protein. Notably, hex-
americ IgM that lacks the J chain activates the complement system
15- to 20-fold more efficiently than pentameric IgM (33, 34).
In summary, our study provides a comprehensive structural
analysis of the IgM Fc domains as well as of the oligomerization
of IgM subparticles. Despite the high structural similarity of in-
dividual Ig domains from various classes, interactions between
the domains are different and adapted for the desired function.
Notably, it is the Cμ4 domain of IgM together with its C-terminal
extension that, apart from any antibody domain characterized
to date, confers the intrinsic ability to oligomerize into defined
hexamers of dimer. Because the sequences of mouse and human
IgM Fc are conserved (68% identity), we assume that the model
BIOCHEMISTRY

the core region dimension of 190 ± 20 Å seen in the cryo-
AFM experiments.
In conclusion, the Cμ4tp domain is responsible and sufficient
for the specific IgM polymerization. In vivo the situation seems
more complicated. In the crowded environment of an antibody-
producing cell, a sophisticated quality control system is present
(27). In the case of IgM, the formation of pentamer/hexamer
complexes is stringently controlled by the ERp44/ERGIC53
assembly platform including a thiol retention mechanism via
Cys575 (28, 29). In addition, it was shown that carbohydrates
have an influence on oligomerization (30), particularly the glycan
linked to Asn563 in the tp of Cμ4 that interacts with ERp44. In
its absence, pentamers cannot be formed in vivo and polymers
presented here is also true for human IgM Fc.
Materials and Methods
Protein Production and Purification. Proteins were expressed as inclusion
bodies in E. coli, oxidatively refolded, and purified as described in SI Mate-
rials and Methods.
Analytical Gel Filtration. Analytical SEC measurements were performed in PBS
as outlined in SI Materials and Methods. For the Cμ4tp domain detection and
mass calculations of the observed species a Dawn Heleos MALS detector
was used.
Analytical Ultracentrifugation. Domain association and determination of
dissociation constants was assessed with analytical ultracentrifugation as

Müller et al. PNAS | June 18, 2013 | vol. 110 | no. 25 | 10187
described in SI Materials and Methods. For dimerizing domains, data were
fitted to a self-association model (14).
Optical Spectroscopy. Fluorescence and CD measurements were carried out in
PBS as described in SI Materials and Methods. To obtain thermodynamic
parameters from the GdmCl-induced unfolding and refolding transitions, a
two-state model was applied (35).
Crystallization and Structure Determination. Crystals of Cμ2 and Cμ4 were
grown at 20 °C using the sitting drop vapor diffusion method. Protein so-
lution (10 mg/mL) in 10 mM Tris·HCl, pH 7.5, was mixed with an equal vol-
ume of buffer C2 [1.0 M lithium chloride, 0.1 M MES (pH 5.4), and 23% PEG
6000] for the Cμ2 domain and buffer C4 [15% PEG 8000 and 0.1M Hepes (pH
7.2)] for the Cμ4 domain, respectively. Diffraction datasets were collected
using synchrotron radiation at the X06SA beamline at the Swiss Lightsource
(Cμ2) or on a Bruker Microstar/X8 Proteum (Bruker AXS Inc.) with a Cu ro-
tating anode (λ = 1.54 Å; Cμ4 domain). For structure determination, mo-
lecular replacement was performed in Phaser (36) using the coordinates of
the Ce2 domain of human IgE (1O0V) for Cμ2 and the coordinates of the
here determined structure of the Cμ2 domain for Cμ4, respectively. Details
are provided in SI Materials and Methods.
NMR Spectroscopy. NMR data were acquired at 25 °C on a Bruker Avance III
600 MHz spectrometer with a 300-μM uniformly 15N, 13C-labeled Cμ3C414S
sample except for the stereospecific assignment of valine and leucine
side chains, which was based on a 1H-13C heteronuclear multiple quantum
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10188 | www.pnas.org/cgi/doi/10.1073/pnas.1300547110
correlation acquired on a 10% 13C-labeled sample as previously described
(37, 38). Details are provided in SI Materials and Methods.
SAXS Measurements and Modeling. SAXS data for solutions of Cμ4tp were
measured for several solute concentrations in the range from 1 to 10 mg/
mL. The structure of the Cμ4tp hexamer was modeled using the program
CORAL (39) as described in SI Materials and Methods. The model of the IgM
Fc hexamer was generated in CORAL using the structures of (i) Cμ2, (ii) Cμ3,
(iii) the Cμ4 hexameric ring as determined based on SAXS data, (iv) the
disulfide linkages (C414 and C575), and (v) the Cμ3–Cμ4 interface derived
from NMR chemical shift titrations as input. Details are provided in SI Materials
and Methods.
ACKNOWLEDGMENTS. The mouse IgM cDNA was a kind gift from Roberto
Sitia (San Raffaele Scientific Institute, Milan). We thank the staff of the Swiss
Lightsource for their assistance in X-ray data collection. We are grateful to
Matthias Feige and Moritz Marcinowski for helpful discussion and techni-
cal support and Thomas Kriehuber for assistance during MALS measure-
ments. We thank the Bavarian NMR Centre for NMR measurement time and
Anton Paar GmbH for access to the SAXSess mc2 in-house SAXS instrument.
This work was supported by grants from the Swiss National Foundation (to
R.M.), DFG (to M.S., M.G., and J.B.), the Emmy Noether Program Grant MA
5703/1-1 (to T.M.), the Helmholtz Association, the Bavarian Ministry of Sci-
ences, Research and the Arts, Bavarian Molecular Biosystems Research Net-
work (to T.M.), and the Austrian Academy of Sciences (Austrian Programme
for Advanced Research and Technology Fellowship, to T.M.). M.A.G. was
supported by the Peter und Traudl Engelhornstiftung and J.P. by the Stud-
ienstiftung des deutschen Volkes.
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32. Randall TD, Brewer JW, Corley RB (1992) Direct evidence that J chain regulates
the polymeric structure of IgM in antibody-secreting B cells. J Biol Chem 267(25):
18002–18007.
33. Davis AC, Roux KH, Shulman MJ (1988) On the structure of polymeric IgM. Eur J Im-
munol 18(7):1001–1008.
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tion. Eur J Immunol 20(9):1971–1979.
35. Bolen DW, Santoro MM (1988) Unfolding free energy changes determined by the
linear extrapolation method. 2. Incorporation of delta G degrees N-U values in a
thermodynamic cycle. Biochemistry 27(21):8069–8074.
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Müller et al.
Supporting Information
Müller et al. 10.1073/pnas.1300547110
SI Materials and Methods
Cloning, Mutagenesis, Expression, and Purification of the Constant
Fragment Domains. The Cμ2 (Ile225–Ala339), Cμ3 (Asp344–
Lys443), Cμ4 (Glu446–Lys558), and Cμ4tp (Glu446–Tyr576) genes
were obtained by PCR amplification using the mouse IgM heavy-
chain cDNA as template. Amino acid numbering is according to
the UniProt entry P01872 (IGHM_MOUSE) with the VH (122
amino acids) added. The genes were cloned into the pET28a ex-
pression vector (Merck/Novagene) via the NcoI and HindIII re-
striction sites. The C337S, C414S, and C575S mutations were
introduced using the QuikChange site-directed mutagenesis strat-
egy (Stratagene/Agilent). Expression was performed in Escherichia
coli BL21 cells at 37 °C. At an OD600 of 0.6–0.8, expression was
induced with 1 mM isopropylthio-β-galactoside. Cells were har-
vested after overnight growth and inclusion bodies were prepared
as previously described (1). Inclusion bodies were solubilized in
50 mM Tris·HCl (pH 7.5), 10 mM EDTA, 8 M urea, and 2 mM
β-mercaptoethanol at 4 °C for 2 h. Insoluble components were
removed by centrifugation (46,000 × g, 25 min, 4 °C). The su-
pernatant was applied to a Q-Sepharose column equilibrated with
50 mM Tris (pH 7.5), 10 mM EDTA, and 5 M urea. The protein
was present in the flow-through, and refolding was carried out
via dialysis into 250 mM Tris·HCl (pH 8.0), 100 mM L-arginine,
10 mM EDTA, 1 mM oxidized glutathione, and 0.5 mM reduced
glutathione at 4 °C overnight. To remove misfolded protein and
remaining impurities, the protein was applied to a HiLoad Su-
perdex75 26/60-pg gel-filtration column (GE Healthcare) equil-
ibrated in PBS buffer. For NMR (chemical shift perturbation
analysis) and SAXS measurements, a Cμ3C414S–Cμ4 construct
was cloned, which included the cysteine at position 337 (Cys337–
Lys558) and the protein was expressed, refolded, and purified as
disulfide linked dimer. Isotope-labeled proteins were expressed
in M9 minimal medium containing 15N ammonium chloride as the
only nitrogen source and in addition13C glucose as the only carbon
source. Partially deuterated protein was expressed in 70% D2O-
containing medium. All constructs were sequenced and the mass
of the purified proteins was verified using matrix-assisted laser
desorption ionization time-of-flight mass spectrometry.
Analytical Gel Filtration. The oligomeric state of the domains were
determined by analytical size-exclusion chromatography (SEC)
measurements using a Shimadzu HPLC System and an analytical
Superdex75 10/300 GL column (GE Healthcare) equilibrated
with PBS (8.09 mM Na2HPO4, 1.76 mM KH2PO4, 137 mM NaCl,
and 2.65 mM KCl, pH 7.4) at 20 °C. All experiments were per-
formed with a flow rate of 0.5 mL/min. Fifty-microliter samples
were injected at concentrations of 1, 10, and 100 μM, and the
elution profiles were detected by absorbance at 280 nm or by
fluorescence emission at 350 nm. For the Cμ4tp domain, two
Superdex200 5/150 GL columns (GE Healthcare) in series and a
Dawn Heleos multiangle light scattering detector (Wyatt) were
used in addition.
Analytical Ultracentrifugation. Analytical ultracentrifugation was
carried out with a ProteomLab XL-I (Beckman-Coulter) equip-
ped with absorbance optics. All experiments were performed
using PBS at 20 °C. For the determination of the masses of the
individual domains and mutants, sedimentation velocity experi-
ments were performed at a rotor speed of 50,000 rpm. Sedi-
mentation was detected at 230, 280, or 300 nm, depending on
protein concentration. For the Cμ4tp oligomer, sedimentation
velocity runs were performed with protein concentrations of
Müller et al. www.pnas.org/cgi/content/short/1300547110
1–20 μM at 35,000 rpm. Data analysis was carried out by the C(s)
method with TI (time invariant) and RI (radial invariant) noise
fitting using SEDFIT version 14.1 software (2).
For Kd measurements, sedimentation equilibrium runs with
protein samples of different concentrations were performed.
Sedimentation was detected at 230, 280, or 300 nm, depending
on protein concentration. Data from various concentrations and
rotor speeds were fitted globally to a self-association model (3)
in Origin (OriginLab) according to Eq. S1:


ω2  2 2 
aðrÞ = c1 ðr0 Þ«1 d exp Mb r − r0
2RT

 [S1]
ω2  2 2 
+ K12 c1 ðr0 Þ2 2«1 d exp 2Mb r − r0 :
2RT
The c1(r0) value denotes the molar concentration at a reference
position r0; Mb, e1, and d designate the buoyant mass, molar ex-
tinction coefficient, and the optical path length, respectively. K12
is the association constant for the monomer/dimer equilibrium.
Fluorescence and CD Measurements. CD measurements were carried
out at 25 °C in a Jasco J-720 spectropolarimeter. Far UV (FUV)-CD
spectra were recorded from 195–260 nm at a protein concentra-
tion of 10 μM in 1 mm quartz cuvettes. Near UV (NUV)-CD
spectra were collected form 260–340 nm at a protein concentration
of 40 μM in 2-mm quartz cuvettes. All spectra were accumulated
16 times and buffer-corrected. For temperature-induced equi-
librium unfolding, the change in ellipticity at 205 nm was recorded
and the rates for heating and cooling were set to 10 °C h−1.
Fluorescence measurements were performed in a Spex Fluo-
roMax-4 spectrofluorimeter (Instruments SA). To determine the
maximum difference between native and guanidium chloride
(GdmCl)-induced unfolded protein, intrinsic tryptophan fluo-
rescence spectra were obtained from 310–450 nm, with exitation
at 280 nm and excitation and emission slit width set to 1 nm and
6 nm, respectively. Measurements were carried out using a pro-
tein concentration of 1 μM in 1-cm quartz cuvettes.
GdmCl-induced equilibrium unfolding was monitored by fluo-
rescence and CD spectroscopy. For fluorescence measurements,
the excitation wavelength was set to 280 nm and the emission was
determined at 360 nm with slit width of 1.5 nm and 6 nm, re-
spectively. For CD measurements, the change in ellipticity at 212
nm (Cμ2), 213 nm (Cμ3), 220 nm (Cμ4), or 221 nm (Cμ4tp) was
monitored. All measurements were performed at a protein con-
centration of 10 μM. For GdmCl-induced unfolding transitions,
the samples were incubated over night at 25 °C at different
GdmHCl concentrations before measurements.
Data were evaluated using Origin (OriginLab); for GdmCl-
induced unfolding transitions, a two-state model was applied (4).
Crystallization and Structure Determination. Crystallization of the
Cμ2 and Cμ4 domains of IgM was performed by the sitting drop
vapor diffusion method at 20 °C. Ten milligrams per milliliter
(800 μM) of protein solution in 10 mM Tris·HCl (pH 7.5) were
mixed with an equal volume of buffer C2 [1.0 M lithium chloride,
0.1 M MES (pH 5.4), and 23% PEG 6000] for the Cμ2 domain
and buffer C4 [15% PEG 8000 and 0.1M Hepes (pH 7.2)] for the
Cμ4 domain. In both cases, crystals grew to a final size of ∼50 ×
50 × 25 μm3 within 48 h. Crystals were either soaked in 2 M
lithium sulfate at pH 5.4 (Cμ2 domain) or buffer C4 with addi-
tional 25% PEG 200 (Cμ4 domain) before being cooled in a
1 of 11
stream of liquid nitrogen at 100 K. Native diffraction datasets were
collected using synchrotron radiation at the X06SA-beamline
(Swiss Lightsource) (Cμ2 domain at 1.3 Å resolution, Table S1)
or on a Bruker Microstar/X8 Proteum (Bruker AXS Inc.) with
a Cu rotating anode (λ = 1.54 Å; Cμ4 domain at 1.9 Å reso-
lution, Table S1).
For structure determination of the Cμ2 domain, data were
processed using the program package XDS (5). The self-rotation
function, calculated with MOLREP (6), revealed noncrystallo-
graphic twofold symmetries indicating two Cμ2 domain mole-
cules in the asymmetric unit cell. Determination of the crystal
structure was performed by molecular replacement using the
program PHASER (7). Coordinates of the corresponding do-
main of the human IgE (1O0V, residues 228–328) were applied
as starting model for the ligand structure. After rigid body
and preliminary positional refinement with REFMAC (8) as
well as noncrystallographic symmetry averaging using the soft-
ware package MAIN (9), both Cμ2 domains were traced. The
refined model resulted in improved density and allowed final-
ization of the last missing secondary structures and loop con-
nections. The structures were completed in successive rounds
using the interactive 3D graphic programs COOT (10) and an-
isotropically refined with REFMAC5 (8). Water molecules were
located either by using ARP/wARP (11) within the CCP4i GUI
(12) or by using COOT (10). All solvent molecules were verified
manually in COOT (10). Temperature factors were corrected with
restraints between bonded atoms and between noncrystallo-
graphic symmetry related atoms using translation/libration/
screw (TLS) parameters with current crystallographic values of
Rwork = 0.138 and Rfree = 0.168 (13). Data collection and re-
finement statistics are summarized in Table S1. Coordinates
were confirmed to have good stereochemistry from the Ram-
achandran plot using PROCHECK (14), which displays 99.0% of
residues in the most favored region and 1.0% of residues in the
additionally allowed regions. The asymmetric unit cell contains
two Cμ2 domains (with the N-terminal Met and the last two Ala
residues at the C terminus being structurally disordered) and 291
water molecules (Table S1). The data of the Cμ2 domains have
been deposited in the Protein Data bank (PDB ID code 4JVU).
For structure determination of the Cμ4 domain, data were
processed with the Proteum 2 software suite (version 2006;
Bruker AXS Inc.). MOLREP revealed twofold symmetries sug-
gesting four Cμ4 domains in the asymmetric unit cell. Initial
phases were received by Patterson search calculations applying
PHASER and using the coordinates of the previously determined
model structure of the Cμ2 domain as starting model. Similarly
as described for the structure elucidation of the Cμ2 domain,
cycles of model building, fourfold noncrystallographic symmetry
averaging for electron density map improvement and TLS
refinement yielded the final Cμ4 model. Data collection and
refinement statistics are summarized in Table S1. The stereo-
chemical quality of the Cμ4 domains were assessed with PRO-
CHECK, displaying in total 97.9% of residues in the most
favored region and 2.1% of residues in the additionally allowed
regions. The asymmetric unit cell contains four Cμ4 structures
(with the N-terminal five amino acids being structurally disor-
dered) and 428 water molecules (Table S1). The data of the Cμ4
domains have been deposited in the Protein Data bank (PDB
ID code 4JVW). Molecular illustrations were prepared using
the PyMOL Molecular Graphics System (DeLano Scientific).
NMR Spectroscopy and Structure Calculation. NMR data were ac-
quired at 298 K on a Bruker Avance III 600-MHz spectrometer
equipped with a TCI CryoProbe head. Processing was done with
NMRPipe (15) and analysis was carried out using Sparky (16).
The backbone resonances were assigned manually based on
HNCACB, HN(CO)CACB, HNCO, and HN(CA)CO experi-
ments (17). Side chains were assigned using an (H)CCH-TOCSY
Müller et al. www.pnas.org/cgi/content/short/1300547110
experiment. Distance restraints were extracted from 15N and 13C
edited NOESY spectra with mixing times of 70 ms (17, 18). Fi-
nally, 15N R1 and R2 relaxation rates, as well as {1H}-15N het-
eronuclear NOE values, were measured at 600 MHz proton
Larmor frequency at 298 K (19). All data were acquired with a
300-μM uniformly 15N,13C-labeled Cμ3C414S sample, except for
the stereospecific assignment of valine and leucine side chains,
which was based on a 1H-13C heteronuclear multiple quantum
correlation acquired on a 10% 13C-labeled sample as previously
described (20, 21).
NOE cross-peaks were assigned automatically by the software
CYANA 3.0 (22) and then inspected manually for assignment and
completeness. The distance restraints extracted from the CYANA
calculation as well as torsion angles derived by TALOS+ (23) were
used in a water refinement calculation in Aria 2.2 (24). Finally, the
quality of the structure ensemble was validated using WHATIF
(25) and PROCHECK (26). The data of the Cμ3 domain have been
deposited in the Protein Data bank (ID code 4BA8).
To map the Cμ3/Cμ4 interface, chemical shift perturbation
analysis was performed, following chemical shifts in the HNCA
spectra of the 15N-labeled dimeric Cμ3C414S–Cμ4 construct
compared with the labeled Cμ3C414S and plotted for each res-
idue in Cμ3C414S. The chemical shift perturbations have been
calculated according to Eq. S2:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
 
δ15N 2
CSPðppmÞ = + δ1H 2 : [S2]
10
The chemical shift perturbations of the different nuclei have been
normalized using their gyromagnetic ratio.
Small Angle X-Ray Scattering Measurements. Small angle X-ray
scattering (SAXS) data for solutions of Cμ4tp were recorded
on an in-house SAXS instrument (SAXSess mc2; Anton Paar
GmbH) equipped with a Kratky camera, a sealed X-ray tube
source, and a one-dimensional reverse-biased silicon diode array
detector (Mythen 1K; Dectris). The scattering patterns were
measured with a 30-min exposure time (three frames, each 10 min)
for several solute concentrations in the range from 188 to 751 μM.
Radiation damage was excluded based on a comparison of in-
dividual frames of the 30 min exposures, where no changes were
detected. A range of momentum transfer of 0.01 < s < 0.7 Å−1
was covered (s = 4π sin(θ)/λ, where 2θ is the scattering angle and
λ = 1.5 Å is the X-ray wavelength).
SAXS data for solutions of Cμ4tp and Cμ3C414S–Cμ4 in the
presence of 5 mM DTT were recorded at the X33 beamline of
the European Molecular Biology Laboratory at Deutsches Elek-
tronen Synchrotron using a MAR345 image plate detector. The
scattering patterns were measured with a 2-min exposure time
(eight frames, each 15 s) for several solute concentrations in the
range from 110 to 578 μM. Radiation damage was excluded based
on a comparison of individual frames of the 2-min exposures,
where no changes were detected. Using the sample–detector
distance of 2.7 m, a range of momentum transfer of 0.01 < s < 0.6
Å-1 was covered (s = 4π sin(θ)/λ, where 2θ is the scattering angle
and λ = 1.5 Å is the X-ray wavelength).
All SAXS data were analyzed with the package ATSAS. The
data were processed with the SAXSQuant software (version 3.9)
and desmeared using the program GNOM (27). The forward
scattering, I(0), the radius of gyration, Rg, the maximum di-
mension, Dmax, and the interatomic distance distribution func-
tions, (P(R)), were computed with the program GNOM (27).
The masses of the solutes were evaluated by comparison of the
forward scattering intensity with that of a human serum albumin
reference solution (molecular mass 69 kDa). The structure of the
Cμ4tp hexamer was modeled using the program CORAL (28).
The crystal structure of Cμ4 was taken as input, a C6 symmetry
2 of 11
was defined, and the flexible N- and C-terminal regions (residues
E446–H448 and Gly557–Tyr576) were randomized. No con-
nections between the Cμ4 dimers have been defined owing to
lack of experimental data defining the interface. A total of 50
structures were calculated, and the best scoring structure (based
on the solvation free energy gain upon formation of the in-
terface, ΔiG) was selected to model the flexible C terminus. To
test whether the additional Cμ4 interface observed in the crystal
structures can be used to construct the structure of the hexamer
we calculated an ensemble of structures using the crystal struc-
ture of the Cμ4 dimer of dimers as input. The calculations were
carried out using the program CORAL (28) with the same set-
tings as described above, but defining C3 symmetry and distance
restraints between the dimers of dimers according to the addi-
tional interface observed in the crystal structure. Because the
Cμ4 dimers of dimers might adopt orientations different from
those in the hexamer, we used all possible combinations of dis-
tance restraints (i.e., contacts of chain A with chain B/C of the
next Cμ4tp dimer of dimers, chain A–chain C/B, chain D–chain
B/C, and chain D–chain C/B). The resulting structures are in
worse agreement with the experimental data compared with the
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Müller et al. www.pnas.org/cgi/content/short/1300547110
calculation in which none of the Cμ4 interfaces in the hexamer
was restrained (Fig. S6). C-alpha coordinates of the Cμ4tp hex-
amer of dimers SAXS model have been deposited in the Protein
Data Bank (ID code 4BLE).
To model the structure of the entire IgM, the Cμ4tp hexamer
modeled based on SAXS data was taken as input for its exten-
sion with Cμ2, Cμ3, and the flexible linkers/termini (Cμ2–Cμ3,
Cμ3–Cμ4, and the tail piece extending Cμ4). The following input
and data were used in the rigid body docking program CORAL:
the atomic resolution structures of (i) Cμ2, (ii) Cμ3, (iii) the
SAXS model of the Cμ4 hexameric ring, (iv) the disulfide link-
ages (C414 and C575), and (v) the Cμ3–Cμ4 interface derived
from NMR data as input. The connections of Cμ4 to the Cμ3
domains has been made based on the NMR chemical shift per-
turbations observed for the free Cμ3C414S domain compared
with the Cμ3C414S–Cμ4 dimer (Fig. S4). Based on that, am-
biguous distance restraints defining that any proton in Cμ3 res-
idues F354 and F358 has to be within a distance closer than 6 Å
to any proton of Cμ4 have been defined. A total of 50 structures
were calculated, and the structure with the best fit to the ex-
perimental data was selected to prepare Fig. 4C.
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and PROCHECK-NMR: Programs for checking the quality of protein structures solved
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27. Svergun DI (1992) Determination of the regularization parameter in indirect-
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Fig. S1. Stability against thermal and chemical unfolding. (A) Temperature denaturation of the Cμ2 (black), Cμ3C414S (red), Cμ4 (blue), and Cμ4tpC575S
(green) domains, followed by the far-UV CD signal at 205 nm at a heating rate of 20 °C/h. Data were fitted by a Boltzmann function. GdmCl-induced equi-
librium unfolding (filled circles) and refolding (open circles) of the (B) Cμ2, (C) Cμ3C414S, (D) Cμ4, and (E) Cμ4tpC575S domains. GdmCl-induced unfolding/
refolding transitions were monitored by intrinsic tryptophan fluorescence. Data were fitted by a two-state unfolding model.

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Fig. S2. Characterization of domain oligomerization. SEC profiles of (A) Cμ2 (dotted line) and Cμ2C337S domain, (B) Cμ3 domain, (C) Cμ3C414S domain, (D)
Cμ4, (E) Cμ4tp domain, and (F) Cμ4tpC575S domain. The proteins were loaded with 1 μM (black line), 10 μM (red line), and 100 μM (blue line). The fluorescence
signal was normalized to the highest peak intensity.

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Fig. S3. Sedimentation velocity analytical ultracentrifugation runs of (A) Cμ2, (B) Cμ2C337S, (C) Cμ3C337S, (D) Cμ4, and (E) Cμ4tpC575S. Runs were performed
at 50,000 rpm in PBS buffer. Representative analytical ultracentrifugation sedimentation equilibrium runs of (F) Cμ2C337S (10 μM), (G) Cμ3C414S (25 μM), (H)
Cμ4 (5 μM), and (I) Cμ4tpC575S (20 μM). Runs were performed at 34,000 rpm (black triangles), 38,000 rpm (red circles), and 42,000 rpm (blue squares) in PBS
buffer. Data were fitted globally to a self-association monomer–dimer equilibrium model (solid lines).

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Fig. S4. Experimental 15N backbone amide relaxation parameters and chemical shift perturbation (CSP) of the free Cμ3C414S domain compared with the
Cμ3C414S–Cμ4 dimer. (A) hetNOE values, (B) longitudinal (R1) rates, and (C) comparison of transverse relaxation rates R2, measured directly (black) and derived
from R1ρ (red). All experiments have been recorded at a Larmor frequency of 600 MHz at 298 K. Correlation times values were determined from 15N relaxation
data as described in SI Materials and Methods. (D) Chemical shifts in free form (Cμ3C414S) and the tandem construct (Cμ3C414S–CH4) have been assigned using
3D HNCA spectra. The secondary structure is pictured above (α-helixes in red, β-sheets in blue).

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Fig. S5. Comparison and alignments of Ig constant domains. (A) Comparison of the various Ig dimerization modes (cartoon representation). (Top) Display of
the respective chain A. (Middle) Display of the dimer form. (Bottom) Surface representation of the dimers. (B) Structure and sequence alignments of IgM Cμ4
(pink), IgA Cα3 (blue), and IgE Ce4 (green). The amino acids involved in the dimer interface are colored in green. Interface residues that are not conserved are
shown in the alignment in red and highlighted in the structure by red arrows. The following coordinates were used (IgA, 1OW0; IgE,1O0V; IgG, 3HKF).

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Fig. S6. Summary of the SAXS data and modeling. (A) Guinier plots of unmerged Cμ4tp scattering data collected at different concentrations. The Rgs and
calculated molecular weights (based on BSA standard) are shown. (B–D) SAXS data showing a comparison of the experimental radial density distributions of
Cμ4tp (B) and Cμ4tp (C) and Cμ3C414S–Cμ4 (D) in the presence of 5 mM DTT at different concentrations. (E) Modeling of the hexamer structure using the
program CORAL and defining C3 symmetry. Different combinations of the additional Cμ4 interface observed in the crystal structures were used as restraints
(SI Materials and Methods gives details). The fit (χ) between the experimental and back-calculated data are shown. (F) SAXS data showing a comparison of the
experimental radial density distribution of Cμ4tp (blue) and the Cμ4 dimer (red) [back-calculated from the crystal structure determined here, using the program
Crysol (1)]. (G) Comparison of experimental Cμ4tp SAXS data with SAXS data back-calculated from the CORAL model of the Cμ4tp hexamer. Both the s and I(s)
axes are shown in a logarithmic representation. The angular ranges from 0.0014–0.6 nm−1 are compared.

1. Svergun DI, Barberato C, Koch MHJ (1995) CRYSOL – A program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Cryst 28:768–773.

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 Table S1. Crystallographic statistics
Parameters Cμ2 domain Cμ4 domain

Crystal parameters
Space group C2 C2
Cell constants a = 92.44 Å, b = 44.78 Å, c = 54.57 Å a = 169.16 Å, b = 41.21 Å, c = 67.1 Å
α = γ = 90°; β =118° α = γ = 90°; β = 92.3°
Data collection
Beamline X06SA, SLS CuKα, rotating anode
Wavelength, Å 1.0 1.5418
Resolution range, Å* 40–1.30 (1.4–1.30) 40–2.0 (2.1–2.0)
No. of observations 15,5963 15,4481
No. of unique reflections† 48,207 31,721
Completeness, %* 99.3 (99.6) 100 (100)
Rmerge, %*,‡ 3.8 (30.9) 4.6 (35.4)
I/σ (I)* 16.1 (5.2) 19.0 (3.1)
Refinement (REFMAC5)
Resolution range, Å 15–1.3 15–2.0
No. of reflections working set 45,796 28,566
No. of reflections test set 2,414 1,537
No. of nonhydrogen 1,615 3,356
No. of solvent water 291 428
Rwork/Rfree, %§ 13.8/16.8 19.7/23.4
Rmsd bond, Å{/angles, ° 0.019/1.857 0.007/1.256
Average B-factor, Å2 20.0 26.1
Ramachandran plot, %k 99.0/1.0/0.0 97.9/2.1/0.0
PDB ID code 4JVU 4JVW

*The values in parentheses of resolution range, completeness, Rmerge, and I/σ (I) correspond to the last resolution shell.
†
Friedel pairs were treated as different reflections.
‡ P P P
Rmerge ðIÞ = hkl j ½IðhklÞj -IðhklÞ =½ hkl Ihkl , where I(hkl)j is the jth measurement of the intensity of reflection hkl and <I(hkl)> is the
average intensity.
P P
§
R = hkl kFobs j − jFcalc k= hkl jFobs j; where Rfree is calculated without a sigma cutoff for a randomly chosen 5% of reflections, which were
not used for structure refinement, and Rwork is calculated for the remaining reflections.
{
Deviations from ideal bond lengths/angles.
k
Number of residues in favored region/allowed region/outlier region.

Table S2. NMR and refinement statistics for Cμ3C414S domain

Parameters For all residues For residues 3-29,38-51,66-100

NOE-based distance restraints

Intraresidual, sequential 772
Medium range (2 ≤ ji-jj ≤ 4) 158
Medium range (ji-jj ≥ 5) 563
Total 1,493
Violated 0
Other restraints
ϕ + ψ dihedral restraints 100
Coordinate precision
Backbone, A 0.49 ± 0.10
Heavy atom, A 0.94 ± 0.11
Common Interface for NMR structure Generation
Red, % 16 11
Orange, % 18 14
Green, % 67 67
Whatcheck
First-generation packing quality 2.918 ± 1.251
Second-generation packing quality 4.617 ± 1.785
Ramachandran plot appearance 0.886 ± 0.227
χ1/χ2 rotamer normality −3.471 ± 0.486
Backbone conformation 1.647 ± 0.186
Ramachandran plot, %
Most favored regions 91.9
Allowed regions 7.0
Generously allowed regions 0.9
Disallowed regions 0.3
PDB ID code 4BA8

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Other Supporting Information Files

Dataset S1 (TXT)

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