From the Dana-Farber Cancer Institute and Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02115 and the ¶ Institut für
Organische Chemie und Biochemie, Technische Universität
München, Lichtenbergstrasse 4, Garching 85747, Germany
Received for publication, June 15, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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The Fab fragment of the murine monoclonal
antibody, MAK33, directed against human creatine kinase of the
muscle-type, was crystallized and the three-dimensional structure was
determined to 2.9Å. The antigen-binding surface of MAK33 shows a
convex overall shape typical for immunoglobulins binding large
antigens. The structure allows us to analyze the environment of
cis-prolyl-peptide bonds whose isomerization is of key
importance in the folding process. These residues seem to be involved
with not only domain stability but also seem to play a role in the
association of heavy and light chains, reinforcing the importance of
MAK33 is a murine monoclonal antibody of subclass During the biosynthesis of antibodies, the ER-specific hsp70 chaperone
family member, BiP, plays an important role in the assembly of the
mature antibody. BiP has been shown to bind to the variable domain of
certain light chains to assist in folding or as a means of removing
improperly folded chains from the secretory pathway (7). In addition,
binding to the newly synthesized heavy chain allows the polypeptide
chain to remain soluble until the appropriate light chain is translated
and available to form the complete antibody molecule (8, 9).
Based on the screening of a phage display peptide library for
BiP-binding sequences (10), an algorithm has been developed that allows
for the prediction of potential protein sequences, which might
constitute BiP-binding sites in proteins (11, 12). Analysis of the
primary sequence of MAK33 as well as that of a related antibody
indicated that binding does seem to require exposed hydrophobic
residues. Notably leucine, but also tryptophan, residues are
specifically enriched in BiP-binding sequences compared with the
overall protein sequence. As evidenced from the stimulation of the
ATPase activity of BiP upon heptapeptide binding in vitro, potential BiP-binding sites in vivo have been identified
(11, 12).
In the present study, we have determined the three-dimensional
structure of the MAK33 Fab fragment by x-ray crystallography. This
structure enables us to discuss the protein environment of the
cis-proline peptide bonds as well as the position of
BiP-binding sites identified biochemically using synthetic
heptapeptides. In this way one can begin to address the critical
questions of subunit recognition within the context of the complete
folding pathway of a multidomain protein.
Protein Preparation and Crystallization--
The proteolytically
derived Fab fragment of the murine monoclonal antibody MAK33 of
subclass
Crystals of MAK33 Fab were grown at 4 °C by vapor diffusion in
4-µl sitting drops made up of equal volumes of protein solution and a
reservoir solution containing 50 mM citrate, pH 5.6, 16% (w/v) polyethylene glycol 8000, 1.0 M NaCl, and 16%
glycerol. Large (0.25 × 0.20 × 0.80 mm3),
single crystals were obtained after 8-10 weeks. The crystallographic asymmetric unit contains two Fab molecules and ~53% solvent with a
Matthews coefficient of 2.64 Å3/dalton and a cell volume
of 264,105.8 Å3 (14).
Data Collection and Structure Determination--
Diffraction
data were collected at 4 °C from CuK
Nonhomologous residues in BiP ATPase Activity Assays--
The binding of MAK33 peptides by
BiP was monitored by the measurement of BiP ATPase activity as
described previously (11). BiP used in this assay was purified from
bovine pancreas as described (22), with the addition of gel filtration
chromatography as a final purification step. The MAK33 peptides were
synthesized using Fmoc
(N-(9-fluorenyl)methoxycarbonyl)-protected amino acids as
described previously (23). The standard assay contained 40 mM HEPES, pH 7.0, 2 mM MgCl2, 500 µM unlabeled ATP, 10 µCi of [ Overall Structure--
The three-dimensional structure of the
MAK33 Fab fragment was determined using the molecular replacement
method (see Table I). Two independent
molecules were identified related by a pseudo 2-fold rotation. The
final electron density maps were of high enough quality in most regions
such that we could identify several previous DNA sequencing errors
within the heavy chain (1). The MAK33 Fab fragment is comprised of four
typical immunoglobulin domains, two each from the light and heavy
chains (Fig. 1A). Each domain
is characterized by a
Surface loops extending from the N-terminal variable domains of both
heavy and light chains form the antigen-binding site. The conformations
of these loops can be classified according to the observations of
Chothia and Lesk (24). They are typical for
This antibody is distinct in that it recognizes only the native dimeric
form of its antigen, CK-MM, and in contrast to many other antibodies it
does not bind the denatured form of this protein. Therefore, antigen
recognition is expected to be a conformation-specific, as opposed to a
sequence-specific, interaction, i.e. requiring either a
complex epitope comprising residues from both subunits or one whose key
residues are influenced by the dimer formation. Comparison of the
antigen-binding surface of MAK33 with those described in the complex
structures of two sequence-specific antibodies directed against hen egg
lysozyme reveals certain subtle differences. The contact surface of
HyHel-10, an
In contract to these antibodies, the analogous region in the MAK33
structure is slightly convex with a shallow groove or channel running
the width of the molecule resulting in a bipartite binding surface.
These structural differences suggest a possible alternate recognition
mode for MAK33 consistent with its requirement for the dimeric form of
the antigen. The longer CDR H3 loop contains 10 residues and, as seen
in the lysozyme antigen/antibody complexes, would be expected to affect
the surface presented for antigen binding. Although the corresponding
electron density for this loop is weak in our structure, thus
prohibiting the determination of its specific conformation, this
disorder is suggestive of a possible direct interaction with the
antigen such that upon binding this loop might assume a stable conformation.
The molecular surfaces presented for complex formation between the
light and heavy chain of any given antibody molecule comprises symmetrical arrangements of the Proline Environments: Implications for Folding
Studies--
Antibody molecules in general have a high degree of
primary sequence conservation. In particular, they contain a number of conserved proline residues in both the light and heavy chain domains. These residues appear to play important structural roles by stabilizing the common scaffolding motifs that maintain the characteristic Ig
domain secondary structure and also factor in the determination of the
orientations of the hinge regions between individual domains. As
observed in previous antibody structures, within the MAK33 Fab several
of these conserved proline residues adopt the cis conformation. In general, the locations of the cis prolines
can be broadly divided into those that are located near chain termini appearing on the molecular surface and those that are buried within the
domain interfaces. The final assembly of the native Ig molecule must
include steps to facilitate the isomerization process to the
thermodynamically less stable cis form. Therefore,
examination of the environment of these cis prolines within
the context of the entire antibody molecule helps to differentiate them
in terms of any potential roles they might play in protein folding or
assembly, such as reactivity with respect to PPIases, the FK506 binding protein, or cyclophilin (2, 30, 31).
In the MAK33 structure a conserved cis proline residue is
found at position Pro-L9.2
This proline is in the N-terminal
The only other cis proline within the light chain is found
at position Pro-L142. This conserved residue is located in the loop
connecting
The two final cis proline residues, His-149 and His-151, are
located in close proximity within the heavy chain. It has been previously suggested that the trans to cis
isomerization of Pro-H1513 is
the rate-limiting step in the overall folding process of the MAK33 Fab
(2). The present structure demonstrates that this cis isomer
is stabilized by the structural constraints of its immediate
neighborhood (Fig. 3D). This residue is located in the upper
edge of the constant domain of the heavy chain, where it protrudes into
the elbow region and helps define the intrastrand angle between the
VH and VCH1 domains. It is in an analogous
location to that of Pro-L142 with respect to the secondary structure
arrangement of the individual domains of each protein chain. However,
the pairings of heavy and light chains occur in such a way that
Pro-L142 is not directly involved in the interstrand constant domain
contact site but is within ~4-5 Å of the terminal residues (His-111
through His-113) of the heavy chain variable domain. The Pro-H151
residue, in the context of the heavy chain alone, has a large solvent
accessible surface area of 23Å2, but in the intact Fab it
is positioned within the constant domain subunit interface. These
structural observations provide an explanation for the differential
interplay of isomerization and association reactions of individual
domains versus longer fragments (6).
However, it is clear from this structure that a critical aspect of the
stabilization of Pro-H151 comes from the presence of the other
cis proline just 2 residues removed, at position H-149. This
proline residue has a very small accessible surface area, 3 Å2, and is involved in numerous contacts. These include,
stacking on Phe-H148 and the direct involvement in an intrachain
contact with the His-H2O1 side chain (Fig. 3D). Pro-H149 is
also buried within a hydrophobic pocket formed by Leu-H8, Ala-H2O3,
Thr-H123, and Pro-H151. This stacking pattern is observed in the
closely related antibody, 3D6 (25), as well as where, despite changes in individual residues, the overall structural interactions of this
immediate region are conserved.
Binding of MAK33 Fab Peptides to BiP--
BiP has for some time
been an appealing target of studies to understand how chaperones
identify denatured proteins. Antibodies are the best-studied natural
substrate of this chaperone (7, 10, 12, 32-35). Potential binding
sites identified within the primary sequence of MAK33 Fab by a
computerized scoring model (11) were found to be distributed in small
clusters within the sequences of both the heavy and light chains of
MAK33 (Fig. 4). Although we, and others,
have used the results of this program, i.e. the "BiP
score," for assessing the relative potential binding of hydrophobic
amino acid sequences to BiP, there are ambiguities in selecting among
sequences with mid-range-positive scores. However, a true strength of
this particular scoring algorithm lies in its ability to definitively
identify those heptapeptide sequences that have no binding affinity for
BiP. Therefore, in the present study we used this algorithm to
eliminate from our initial list those sequences with no binding
potential. This allowed us to preselect a small subset of potential
binding sites. These sequences were in turn analyzed biochemically,
using synthetic heptapeptides, to assay binding directly.
The binding of peptide substrates to hsp70 class members is accompanied
by a stimulation of ATP hydrolysis thought to be required for
conformational changes within the binding domain and/or a re-orientation of that domain with respect to the N-terminal portion of
the protein. Therefore, an increase in ATPase activity of BiP in the
presence of a particular peptide is indicative of its interaction with
BiP. To this end, we have tested the eight peptides corresponding to
potential binding sites within the light chain of MAK33, designated LM1-176, (scores of +10 and higher) and another eight peptides corresponding to potential binding sites within the heavy chain of
MAK33, HM14-166, (scores ranging from +6 to +20) for their ability to
bind to BiP and stimulate the ATPase activity of BiP in
vitro. One additional peptide (HM164) was tested despite its strongly negative BiP score, because of its particular location within
the constant domain of the heavy chain (as discussed further below).
The sequences and BiP scores of these peptides are presented together
with the results of the ATPase stimulation in Table II. Of the 17 peptides tested, two
peptides, one each within the variable and constant domains of the
light chain, and three peptides within the heavy chain stimulated the
ATPase activity by factors ranging from 2.0 to 3.0, similar to values
reported previously for other peptides (11). An additional three
peptides displayed consistent, but lower stimulatory effects of 1.4 to
1.7. The remaining nine peptides caused no significant stimulation of
the ATPase activity and, therefore, were not further considered as
likely BiP-binding sites.
Location of the Possible Binding Motifs on the Three-dimensional
Structure of MAK33--
The BiP score based on primary structure
analysis indicates possible BiP binding motifs based on the residue
characteristics within short peptide segments. Our structure allows
detailed topological analysis of binding motifs within the context of
the overall antibody structure. We observe a strong correlation of high
BiP scores with the structural criteria of lying buried within the
antibody molecule (Fig. 4). The majority of identified peptides involve residues that participate in interdomain contact interactions within
one subunit or between the light and heavy chains, suggesting that
these generally hydrophobic sequences would only be exposed during
folding and assembly or under denaturing conditions. A more detailed
analysis of the location of specific peptides provides additional
information on the exact nature of possible interactions.
The conserved CH1 domain is known to be an essential target
zone in the binding of antibodies by BiP in vivo. Sites were
identified within the sequence of MAK33 that are found at the interface
of the constant domains. In particular, HM137 and LM134 are located at
analogous positions within strand
One additional heavy chain peptide displayed a moderate BiP score as
well as a significant ATPase stimulation factor, indicating that it may
be a favored binding site. This peptide, HM14, is located on the outer
surface of the protein very near the N terminus of the heavy chain. The
most likely explanation for binding in this region would be to
stabilize the growing protein chain and promote the proper folding of
the protein as it is being produced in the ER. The presence of a bound
chaperone might be expected to affect positively the ratio of folded to
aggregated heavy chain and promote antibody production.
Two sequential light chain peptides, LM91 and LM93, are located on the
The results reported here provide an additional perspective from
which to consider questions, previously addressed by solution studies,
relating to protein folding and antibody assembly. The detailed
structural view of any particular residue(s) of interest, e.g. Pro-H151, whose isomerization was implicated as the
likely rate-limiting step in the folding of the entire Fab, emphasizes not only its own contribution but also the context within which these
effects are implemented. From the MAK33 structure it is difficult to
discern why the isomerization of His-151 is a more likely candidate for
the rate-limiting step than His-149. However, it is clear that the
combined effects of these two highly constrained residues make this
overall site structurally unique as compared with the immediate
environment of the other cis prolines. Perhaps it is the
combination of the isomerization of both residues that contribute to
the rate-limiting step in the folding pathway as opposed to the
isomerization of either of these in particular.
As has been suggested previously, it is plausible that being
temporarily slowed by cis/trans proline
isomerizations is not necessarily a disadvantage among refolding
procedures but could possibly provide the time needed to allow
previously trapped refolding steps to correct or complete themselves
(36, 37). From biochemical studies it is known that individual light
chains are able to be completely reversibly denatured (3), whereas
similar treatment of heavy chains results in significant losses due to
aggregation.4 This might be
related to the differential reactivity of prolines in these two
components, as the ease at which these prolines assume their final
conformation might be related to the degree to which exposed B-strands
are involved in nonspecific aggregate formation. The surface
characteristics of individual domains would affect interchain contacts
as well as possible interactions with any other proteins necessary for
proper antibody folding, such as prolyl isomerases and chaperones.
In certain myeloma cell lines it is the variable domain of the light
chain that is specifically associated with BiP (38). This increased
binding has been attributed to the fact that the variable domains
exhibit a greater degree of sequence variability, especially relating
to their function in antigen recognition, and hence might be harder to
fold than constant regions. As a result improperly exposed hydrophobic
surfaces would become likely binding sites for chaperone interaction.
The primary sequence of a peptide segment does influence its general
propensity to form fundamental secondary structure elements. The
alternating hydrophobic nature of those segments identified by the BiP
score suggests that better binders are involved in Taken together, these findings suggest that, as an ER-specific hsp70
class chaperone, BiP might have a dual function. Interactions with many
misfolded proteins could take place through binding of short
hydrophobic sequences in a manner much like that described for DnaK.
The apparent ambiguity in the scores for antibody peptides, i.e. that not all high scoring potential binders are located
at the domain interfaces, is consistent with BiP's purely primary sequence-specific mode of interaction. A more specific functional role
of BiP, however, might utilize an interaction analogous to that seen in
the complexes of bacterial immunoglobulin type chaperones involved in
pili assembly with their specific cellular targets. In contrast to the
sequence-based binding of exposed residues, the crystal structures of
these complexes reveal another mode of interaction that is based on the
swapping of a The overall folding pathway of antibodies involves a series of
interactions not only between the protein subunits but also with
folding factors such as isomerases or chaperones. It is therefore likely that there is a kinetic competition for binding between these
proteins that must dictate the rules of this complicated interplay.
This is also consistent with the findings that, despite the highly
conserved secondary structure, similar residues within different
individual domains can have quite diverse influences on the formation
of the native folded structure. It seems that antibodies have evolved
to modulate a highly conserved structural motif (the immunoglobulin
fold) according to different functions (antigen binding, complement
binding, stability, association) through intrinsic signals for -strand recognition in antibody assembly. The structure also allows
the localization of segments of primary sequence postulated to
represent binding sites for the ER-specific chaperone BiP within the
context of the entire Fab fragment. These sequences are found primarily
in
-strands that are necessary for interactions between the
individual domains.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/IgG1
specific for muscle-type creatine kinase
(CK-MM),1 a key enzyme
involved in the formation of creatine-phosphate in muscle cells. This
antibody-antigen interaction is specific for the native dimeric form of
the antigen only and results in the inhibition of the enzymatic
activity, thus making MAK33 a valuable tool in the diagnosis of
myocardial infarction (1). In recent years, the Fab fragment of MAK33,
consisting of the entire light chain and the two N-terminal domains of
the heavy chain, has been used as a model for studies of protein
folding for multidomain proteins (2-5). Slow steps in the folding
process of the MAK33 Fab were found to be due to isomerizations of
prolyl-peptide bonds, however, the structural basis for these phenomena
remained unknown. Folding catalysts of the peptidyl prolyl isomerase
(PPIase) family have been shown to catalyze these slow folding
processes in vitro (2). The resultant isomeric state in turn
has been shown to influence subunit association. Kinetic analyses of
the folding pathway have demonstrated that the relative position of polypeptide chain association within the folding pathway leading to the
native molecule is different for the Fab fragment and the CH3 dimer of the Fc fragment (4, 6). Although the
isomerization of key proline residues could occur at various stages
relative to subunit association for the Fab (4), dimer prolyl
isomerization had to take place before association in the case of the
CH3 (6). These results indicate a complex relationship
between prolyl isomerization and intermolecular association in the
folding of the antibody molecule.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
/IgG1 directed against dimeric muscle-specific human
creatine kinase (CK-MM, E.C. 2.7.3.2) was obtained from Roche Molecular
Biochemicals GmbH. The lyophilized Fab was resuspended to ~6 mg/ml in
150 mM NaCl by gently rocking at 4 °C for 1 h. The
solution was then dialyzed overnight against MilliQ water and
subsequently concentrated to 30 mg/ml using Amicon microconcentrators.
The final protein concentration was determined spectrophotometrically
using the extinction coefficient of A280 = 80,000 M
1 cm
1 (13).
radiation generated by a
Rigaku RTP500 RC rotating anode generator using a Marresearch imaging
plate. Reflection files were indexed using the program DENZO (15) and
subsequently scaled by the program SCALEPACK (16). A summary of data
collection statistics is shown in Table I. The structure of MAK33 Fab
was solved by the molecular replacement method using the anti-influenza
virus hemagglutinin Ha1
/IgG2A Fab fragment 17/9 (17) as a search
model in the program AMoRe (18). The sequence identity between the
search model and MAK33 Fab was 98 and 53% identical for the constant and variable domains of the light chain, respectively, and 87 and 89%
identical for the constant and variable domains of the heavy chain,
respectively. A rotational search using the entire Fab fragment was
carried out, resulting in a pair of clear solutions with correlation
coefficients significantly higher than those of the next highest
solutions. After an initial rigid body refinement based on this model,
a 2Fo
Fc map was calculated yielding strong electron density, and the molecular packing was checked
showing no conflicts between molecules related by translational symmetry. The elbow regions connecting the variable and constant domains of both the light and heavy chains could be easily discerned in
the electron density map, yielding an intact molecule for structure refinement.
-strands were mutated to alanine, and the
loops connecting the strands, including the six CDRs, were completely
deleted to minimize model bias. Structure refinement was carried out
using the program X-PLOR (19) utilizing noncrystallographic symmetry
restraints in the initial cycles. Side chains were rebuilt with the
program O (20). Iterative cycles of manual rebuilding employing
Fo
Fc and
2Fo
Fc maps in O were
alternated with automated positional refinement and grouped B-factor
refinement in X-PLOR in which the noncrystallographic symmetry
restraints were gradually removed. In the case of the CDR H3, the
longest loop in the heavy chain, the electron density was insufficient
to allow refinement of this segment within either molecule in the
asymmetric unit. Furthermore, residues His-133 through His-137,
forming the loop-connecting strands A and B of the heavy chain constant
domain, were also not included in refinement and model building due to
weak electron density. The final model geometry was analyzed with
PROCHECK (21). Overall refinement statistics and final model parameters
are given in Table I. Only 2 residues (0.6% of total) had phi-psi
angle combinations that fall just outside of allowed regions of the
Ramachandran plot. These are both located in loop regions on the
protein exterior. The final coordinates have been submitted to the
Protein Data Bank and have been assigned PDB ID 1FH5 and Research
Collaboratory for Structural Bioinformatics ID RCSB011583.
-32P]ATP
and ~4 µg of BiP in a total volume of 20 µl. Following different times of incubation at 37 °C, 3-µl aliquots were removed, and the
amounts of ATP and ADP were determined by thin-layer chromatography and
liquid scintillation.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel structure consisting of two
-sheets
connected by an internal disulfide bridge. The orientation of the two
interchain domain pairs with respect to each other is described by the
elbow angle between the pseudo-dyads VL-VH and
CL-CH1. In MAK33 the two molecules within the
asymmetric unit have very similar domain orientations, assuming typical
elbow angles of 161.9° and 161.3° for molecules 1 and 2, respectively.
Data collection and refinement statistics
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Fig. 1.
Overview and GRASP model of MAK33 Fab.
A, MOLSCRIPT (42) model of MAK33 Fab. The CDR regions
of both the light (in yellow) and the heavy (in
green) chains found within the variable domain of each chain
are highlighted. B, electrostatic surface representation of
MAK33 Fab with the basic and acidic surfaces highlighted in
red and blue, respectively. The view is looking
down upon the CDR loop region and is a 90° rotation of A.
Each of the six CDR loops is labeled.
/IgG family members and
belong to the same classes as those of the closely related antibody,
3D6, whose structure has been determined (25). The only possible
exception is the H3 loop whose conformation appears disordered in our
structure. Overall the surface comprised of the MAK33 CDR loops
contains mostly neutral or uncharged residues (Fig. 1B).
However, two strongly charged zones of only a few residues each are
localized in the heavy chain H1 and H2 loops. The only charged surface
potential found in the light chain is part of the CDR L2, whose surface
is partially directed away from the main antigen binding area of the
other CDRs (Fig. 1B).
/IgG1 antibody, is a broad and relatively flat area
with no obvious grooves or cavities but instead one extended region,
comprising residues from CDRs H1 and H2, that appears to interact with
the lysozyme active site (26). The CDR loop H3 is very short in this
antibody and contributes only 1 residue to the interaction. The binding
surface of monoclonal antibody F9.13.7, as seen in complex with a cross
reacting antigen, Guinea fowl lysozyme, includes a shallow surface
groove one side of which is mainly composed of the CDR H3 loop. In this
antibody CDR H3 is relatively long and positioned such that its length and composition directly influence the binding interaction (27). This
longer CDR loop displays conformational changes upon antigen binding.
In both native and complexed structures, the binding of anti-peptide
Fab 17/9 to various segments from its antigen, influenza virus
hemagglutinin, also utilizes a mostly wide flat surface. Comparison of
these structures has shown that the CDR H3 loop exhibits a large
conformational change upon antigen binding resulting in two very
distinct structures for the bound and free H3 loop (28).
-strands of each individual domain (29). As shown in Fig. 2, the
intersubunit contact surfaces of variable and constant domains differ
despite the overall similarity in domain structure. In the MAK33
structure the variable domains interact along the edge of the sheet
made up of
-strands HGCD such that the contact residues
extend along
C and
H and include those of the adjacent loops. The
constant domains, however, wrap around each other and interact face to
face such that their respective
sheets comprising strands
DEBA are at an angle of roughly 60 degrees. Direct contact
extends along all these
-strands. These differential interactions
are likely to influence the folding pathway of the intact antibody and
might be related to differences in sequential ordering or rate-limiting
effects of certain steps as compared for single domains or multidomain
chains.
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Fig. 2.
Model of the intersubunit contact surfaces of
MAK33 Fab. Strands involved in interface formation are labeled for
both the light (on the left) on heavy (on the
right) chains. This view is made by pulling the two chains
apart and rotating the interface of each chain 45° toward the reader.
This figure was made in GRASP (43).
segment of the light chain and is
generally exposed to the solvent with a solvent-accessible surface of
18 Å2. The cis conformation appears to
be stabilized by an additional H-bond to O
1 of Thr-L103 on an
adjacent
strand (Fig. 3A).
The equivalent position in the heavy chain is occupied by a glycine residue that is also important in maintaining intradomain contacts. However, near the C terminus of the heavy chain there is a somewhat analogous cis proline residue at Pro-H191. This residue is
also exposed to the solvent, with an accessible surface area of 17 Å2, and would appear to be able to readily interact with
potential isomerases. The cis conformation of this residue
is most likely stabilized by stacking on the preceding residue Trp H190
(Fig. 3B).
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Fig. 3.
Stereo models of cis-proline
environments. Oxygen atoms are red, carbon atoms are
gray, and nitrogen atoms are blue. Subsets of
hydrogen bond distances are shown by dotted lines.
A, cis-ProL9; B,
cis-ProH191; C, cis-ProL142;
D, cis-ProH149 and cis ProH151.
-strands B and C of the constant domain and extends into
the elbow region. The cis isomer is stabilized by an H-bond from the carbonyl to the neighboring His-L199. In addition, further stability is afforded by the improved stacking arrangement on the
residue immediately preceding it, Tyr-L141 (Fig. 3C). This cis proline residue has an accessible surface of only 10 Å2, suggesting that interaction with a PPIase is
restricted once the light chain has assumed its tertiary fold. This
highly conserved cis proline is critical for maintaining the
intrastrand domain orientation.
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Fig. 4.
Location of BiP-binding sites within MAK33
Fab. Potential in vivo BiP-binding sites of the light
and heavy chains identified by the ability of the corresponding
heptapeptide to stimulate BiP-ATPase activity are highlighted on the
alpha carbon backbone trace of the Fab.
Binding of synthetic heptapeptides to BiP
B of the heavy and light chain
constant domains, respectively. These peptides, located within the core
of their respective domains and ~9 Å apart, both show moderate BiP
scores but very low ATPase stimulation. In contrast, examination of
another set of analogous sites (HM166 and LM176) reveals a tendency
toward high BiP score and increased ATPase stimulation at interface
positions nearer to the intrachain elbow regions. However, the two
overlapping peptides located on
D of the heavy chain constant
domain, HM164 and HM166, have very different BiP scores and ATPase
stimulation values. HM166, which is closer to the elbow region of the
heavy chain and the interchain contact surface, has significantly
higher values for both, suggesting that binding affinity in this region
is high and that this region might be directly involved with the
potential conformational change that is associated with substrate
binding by BiP. Moving away from this immediate elbow region by even a
few residues (as seen with HM164) would then be consistent with
significantly reduced binding affinity as evidenced by the negative BiP
score and further supported by the lack of ATPase stimulation. On the
other hand, the light chain peptide LM176 (
E) is also located close
to its respective elbow region at the interface and is roughly 7 Å from the heavy chain
D strand. This peptide shows values close to those of HM166, and its ATPase stimulation is the highest among the
light chain peptides tested.
H strand of the variable domain and are positioned progressively
farther away from the elbow and the core of the domain and closer to
the protein surface. LM93 actually comprises part of a CDR loop. Due to
the orientation of variable and constant domains, this progression in
primary sequence is also leading into the area of the light intrachain
domain interface. Because these sequences are at the end of the
variable domain, the chaperone binding might be required to stabilize
the growing light chain so that its final domain can fold properly.
This would be analogous to binding at HM14, as mentioned above, and
perhaps more importantly, would also help to orient the growing chain
such that the two domains are positioned properly with respect
to each other. In contrast, LM81 and LM84 are also located
within the elbow region but on the outer side of the light
chain V-C junction. These peptides are far from the dimer
interface and do not appear to bind BiP.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-strand
formation. However, structural comparison of potential BiP-binding
sites within MAK33 also emphasizes that, in addition to the presence of
an individual secondary structure feature, it is the protein
environment with respect to intra- and interdomain orientations that is
critical for recognition. The overall three-dimensional structure of
such a recognition site is determined by key characteristics conserved
among proteins of a given class despite differences in the details of
individual examples. In addition, the characteristic manner of pairing
in antibodies of variable versus constant domains is
significantly different in general, and therefore, the interaction with
BiP may be influenced by the different quaternary organization as well.
Because interchain contacts are in fact essential for the pairwise
domain orientations, binding of BiP to "hot spots" within such
contact regions might be expected to compete with the proper antibody
subunit interactions needed to complete the native molecule. The
inability of the heavy chains to fold, as observed in vitro,
may be compensated for by chaperone binding in vivo.
-strand between chaperone and target (39, 40). The
crystal structure of the peptide binding domain of the BiP homologue,
DnaK, suggests that in both proteins this domain is largely
-structured (41). Although this
domain has a topology that is
different from that in a typical immunoglobulin fold, the presence of
sheet motifs in both partners suggests that in this case the heavy
chain binding mode of BiP may utilize a
-strand donor or
complementarity as seen with PapD and FimC. Thus this more specific
function of assisting antibody assembly might utilize the structural
requirements of the pairing of two
domains.
sheet and dimer formation as well as extrinsic signals, i.e.
for interaction with BiP. The three-dimensional structure of MAK 33 Fab
provides an important starting point for further analyzing these
signals by a systematic structure-based mutagenesis approach.
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ACKNOWLEDGEMENTS |
---|
We thank Alfred Engel for resequencing the MAK33 cDNA, Susanne Modrow for the synthesis of peptides, Hauke Lilie for helpful discussions and unpublished data, Mary-Jane Gething for insightful discussions of the BiP score, Helmut Lenz for sharing his knowledge on immunoglobulins throughout this work, and Juia-huai Wang for helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to J. B.) and DFCI-Barr Foundation (to C. A. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1FH5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Dana-Farber Cancer
Institute, Harvard Medical School, SM1036, 44 Binney St., Boston, MA
02115. Tel.: 617-632-3984; Fax: 617-632-4393; E-mail:
caf@red.dfci. harvard.edu.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M005221200
2 Nomenclature for sequences: L, light chain; H, heavy chain; e.g. Pro-L9.
3 In earlier biochemical studies this proline is referred to as pro H159.
4 H. Lilie and J. Buchner, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: CK-MM, muscle-type human creatine kinase; BiP, immunoglobulin heavy chain binding protein; CDR, complementarity-determining region; CH3, the third constant domain of the heavy chain; ER, endoplasmic reticulum; Fab, antigen binding fragment of an antibody comprising the two variable domains and two constant domains of the heavy (CH1) and light (CL) chains; Fc, dimeric antibody fragment comprising the constant domains CH2 and CH3; LM, light chain of MAK33; HM, heavy chain of MAK33; PPIase, peptidyl prolyl isomerase.
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