From the Department of Biological and Environmental
Science, University of Jyväskylä, FIN-40351,
Jyväskylä, Finland, the § Department of
Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot,
Israel, and the ¶ Department of Biological Chemistry, The
Institute of Life Sciences, The Wolfson Centre for Applied Structural
Biology, The Hebrew University of Jerusalem, Givat Ram, 91904 Jerusaleum, Israel
Received for publication, August 30, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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Chicken avidin, a homotetramer that binds four
molecules of biotin was converted to a monomeric form by successive
mutations of interface residues to alanine. The major contribution to
monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4
interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin
binding, thermostability, and protease resistance. To our knowledge,
these unexpected results represent the first example of a small
monovalent ligand that induces oligomerization of a monomeric protein.
This study may suggest a biological role for low molecular weight
ligands in inducing oligomerization and in maintaining the stability of
multimeric protein assemblies.
Many proteins are composed of more than one subunit. The
principles and rationale for the spatial arrangement of subunits (or
monomers) into dimers, trimers, and tetramers are still not well
understood. The role of multimeric associations in binding and protein
activity is still a mystery, particularly for proteins in which it was
shown that each subunit contains the necessary information for binding
or activity. Consequently, interaction between subunits is apparently
unnecessary for binding of a ligand. An example of this phenomenon is
the aldolase homotetramer, in which each subunit exhibits identical
catalytic activity (1).
Avidin is a homotetramer (molecular weight ~63,000), each monomer of
which binds a single biotin molecule with the highest known affinity in
nature between a protein and a ligand (2, 3). The three-dimensional
structure of avidin has been solved, and the interactions among the
various residues at the intersubunit interfaces have been determined
(4-6).
The avidin tetramer is essentially a dimer of dimers consisting of
three types of monomer-monomer interactions (Fig.
1), as discussed in detail by
Livnah et al. (4). The 1-4 intermonomer interface exhibits
an excessive monomer-monomer interface (1780 Å2 per
monomer) and forms a structurally cohesive dimer. The two monomers are
so tightly integrated that this particular dimer can essentially be
regarded structurally as a single entity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structural basis for mutant selection.
A, ribbon diagram of the avidin tetramer. The
monomers are color-coded as follows: 1, light blue; 2, red; 3, dark blue; and 4, magenta. The
biotin molecule is represented in a ball and stick model. The figure
was created using MOLSCRIPT (20). B, interface between
monomers 1 and 3 (the 1-3 interface), consisting of hydrophobic
interactions between three identical residues from each monomer. The
indicated surface mesh represents the contact area of monomer.
C, structural disposition of monomer 1 versus
monomer 4 (the 1-4 interface), emphasizing the extensive interface
between them. Sidechains of residues Asn-54 and Asn-69 are color-coded
blue and red, according to the respective parent
monomer (1 and 4, respectively). Note the two pairs of interacting
residues on opposite sides of the interface. D, schematic
representation of the hydrogen bond network, in the vicinity of
residues Asn-54 (Monomer 1) and Asn-69 (Monomer
4). Note the conserved water molecule that bridges the hydrogen
bond array between the two monomers. The same interaction is duplicated
between residues Asn-54 (Monomer 4) and Asn-69
(Monomer 1) on the other side of the dimer interface.
In contrast, the 1-2 and 1-3 interfaces each comprise only a selected number of critical interactions (buried surface areas of 557 Å2 and 147 Å2 per monomer, respectively). Thus, the dimer-dimer interaction is dictated by the combined 1-2 and 1-3 interfaces. The 1-3 interface consists of a contribution of only three amino acids from each monomer (4), namely Met-96, Val-115, and Ile-117 (Fig. 1B). This clearly hydrophobic interaction has a contact surface of 147 Å2, which is a mere 20% of the dimer-dimer interaction.
The 1-4 interface is characterized by extensive polar and hydrophobic interactions. Some of the polar interactions consist of an intricate network of hydrogen-bonding interactions, sometimes involving conserved water molecules. The ten hydrogen-bonding interactions observed for the side chains of two of these residues, Asn-54 and Asn-69 (each from the opposite monomer), are particularly extensive (Fig. 1, C and D). Because of the 2-fold symmetry axis, perpendicular to the plane of the 1-4 interface, the site is duplicated on opposite poles of the overall interacting interface (Fig. 1C). The two residues are positioned within the same site but on opposite monomers (Fig. 1D).
The extensive structural information currently available on avidin renders the avidin-biotin complex an excellent model to study protein-protein interactions and how oligomerization may affect the structural and functional properties of the protein. It would be challenging to determine the minimal number of changes necessary to form functional protein dimers or monomers. In this context, we have recently described dimeric, biotin-binding forms of both chicken avidin and bacterial streptavidin. In both proteins, an unconventional single-residue mutation was performed, by which a binding-site tryptophan, which also forms a critical part of the 1-2 interface, was converted to a lysine (7). The biotin binding was reversible, and the dimeric structure was retained in both proteins before and after binding to biotin.
In the present study, we describe the construction of a set of avidin
mutants in which selected 1-3 and 1-4 interface residues were
modified to alanine. Successive mutation of these residues caused a
progressive weakening of the quaternary structure. In the absence of
biotin, two of the mutants proved to be stable soluble monomers.
Surprisingly, the presence of biotin regenerated stable avidin
tetramers in these two mutants. This is the first instance in which a
small ligand that binds to a single subunit causes subunit assembly
into a stable tetramer. The results suggest that even though a monomer
can bind a ligand, stability factors can favor formation of the native
oligomer. This study may be important for designing novel binding
proteins and for demonstrating the role(s) of the quaternary structure
in maintaining stability of multisubunit proteins.
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MATERIALS AND METHODS |
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Construction of Recombinant Bacmids and Baculoviruses-- Chicken egg white Avidin cDNA was mutated by the megaprimer method (8) using pGEMAV (9) as a template. When combined mutants were created, the former constructs were used as a template. After the a second PCR amplification, the products were digested with BglII and HindIII, extracted from agarose, and cloned into the BamHI/HindIII-treated pFASTBAC1 plasmid to construct recombinant vectors. The vectors were transformed into JM109 cells, and the nucleotide sequences were confirmed by dideoxynucleotide sequencing using an automated DNA sequencing instrument. The preparations of recombinant viruses were completed according to the manufacturer's instructions of the Bac-To-BacTM baculovirus expression system (Life Technologies, Inc., Gaithersburg, MD). The primary virus stocks were amplified for large scale production of mutants, and the titers of virus stocks were determined by a plaque assay procedure (10).
Production and Purification of Mutant Avidins-- Mutant avidins were produced in baculovirus-infected insect cells described previously (7). Purification on 2-iminobiotin-agarose column was performed from cell extracts grown in biotin-free medium as described by Airenne et al. (11).
Biotin Binding Assays-- The determination of binding constants for avidin and its mutants to iminobiotin were performed using an IASyS optical biosensor as reported earlier (12). Reversibility of biotin binding was determined by competitive binding to biotinylated biosensor surfaces and by competitive biotin-binding enzyme-linked immunosorbent assay as detailed earlier (7).
Structure Analyses--
The molecular mass of a mutant was
calculated from the known amino acid composition using the GCG package
program Peptidesort (Genetic Computer Group, Madison, WI).
SDS-PAGE,1 immunoblot
analyses, and assays for protease sensitivity were performed according
to Laitinen et al. (7). For stability analysis, protein
samples were combined with sample buffer and incubated at selected
temperatures for 20 min before being subjected to SDS-PAGE (13).
Quaternary states of untreated or biotin-treated avidin and mutant
samples were defined by FPLC as described recently (7).
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RESULTS |
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In a recent study (7), we mutated a critical tryptophan residue to a lysine in both avidin and streptavidin. This tryptophan (Trp-110 in avidin and Trp-120 in streptavidin) plays a dual role in both proteins. On one hand, it serves a biological role as one of the major biotin-binding residues. On the other hand, it plays a major structural role in characterizing one of the three intermonomer interactions (the 1-2 interface). The consequence of the tryptophan-lysine exchange was 2-fold; (a) the affinity constant of the biotin binding was reduced, and the binding could be reversed, and (b) the mutated protein formed a stable dimer in solution. Because dimer formation requires the combined counteraction of two interfaces, the inference was such that a drastic mutation also affected the weaker 1-3 interface leaving the extensive 1-4 interface intact. In the present work, we investigated the residues in avidin, which contribute to these latter two interfaces, with the intention of disrupting the tetramer into smaller components.
Construction, Production, and Purification of Mutant Avidins-- The three hydrophobic amino acids that line the 1-3 interface and selected polar residues that play a major role in the 1-4 interface were changed to alanine, thereby reducing the hydrophobic nature of the first set and eliminating hydrogen-bonding potential of the second. The target residues were selected on the basis of the 3D structure of avidin (4), taking into account their suspected contribution to the respective intermonomer interface. The resultant mutants are listed in Table I.
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All mutants were purified from cell lysates. Isolation of the mutants on iminobiotin-agarose resulted in pure products of high yields (data not shown), comparable with those reported earlier (11, 12). In general, the 2-iminobiotin purification scheme used was very efficient in terms of protein purity, because no contaminating proteins could be observed in Coomassie-stained SDS-PAGE gels.
Biotin Binding Assays--
To study the reversibility of biotin
binding, an assay based on optical biosensor technology was designed.
The results showed that only native avidin and Avm-4a were completely
irreversible (Fig. 2). The three mutants
exhibited levels of reversibility between 30 and 45%. In this group of
mutants, reversible biotin binding was highest for Avm-[3,4a]. To
measure the actual binding constants of native avidin and the mutants
for 2-iminobiotin, we used an IASyS optical biosensor. The measurements
indicated that interaction of mutants Avm-3 and Avm-4a with
2-iminobiotin surfaces was similar to that of native avidin (Table
II.). Calculation of the
Kd values from ka and
kd and direct determination of
Kd from the binding curves gave comparable values. Because of the monomer-tetramer transition following biotin binding, we
could not use the IASyS biosensor to evaluate the actual binding constants for mutants Avm-[3,4a] and Avm-[3,4b]; in qualitative terms, however, these mutants also showed strong binding to
2-iminobiotin (not shown).
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Structure Analysis--
The stability of the mutants was analyzed
by SDS-PAGE as described previously (7). When compared
with native avidin, all the mutants
showed decreased stability (Figs. 3 and
4). Avm-3 and Avm-4a exhibited partial
tetrameric structure in SDS-PAGE at ambient temperatures in the absence
of biotin. In contrast, Avm-[3,4a] and Avm-[3,4b] migrated as
monomers, even at room temperature in the absence of biotin. In the
presence of biotin all mutants formed tetramers that displayed
stability characteristics similar to those of native avidin.
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In protease assays, all mutants showed lowered resistance to proteinase
K degradation compared with native avidin (Fig.
5). In the absence of biotin, all mutants
were rapidly degraded. Upon addition of biotin, however, complete
resistance (similar to native avidin) was restored to all mutants
except Avm-[3,4a], which exhibited only 75% resistance to
degradation.
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The quaternary status of the mutants was analyzed by gel filtration
FPLC. The theoretical molecular mass for all mutant monomers without
the oligosaccharide side chain is between 14,127 and 14,300 daltons.
According to the FPLC data, the observed mass for Avm-[3,4a], in the
absence of biotin, was 13,990 daltons. After saturation with biotin,
the mass was calculated to be 58,900 daltons. The corresponding masses
for Avm-[3,4b] were 14,280 and 55,960 daltons. These values suggest
that in the absence of biotin, Avm-[3,4a] and Avm-[3,4b] were
monomers but that they formed tetramers upon biotin binding. In
contrast, Avm-3 and Avm-4a formed tetramers even in the absence of
biotin (Fig. 6).
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DISCUSSION |
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The aim of this study was to elucidate the minimal number of changes in the interface residues among the subunits of the avidin tetramer, which would lead to disruption of the avidin tetramer into monomers without destroying the strong binding specificity for biotin. Such a monomeric avidin would be a valuable tool for studying the contribution of different residues (and their bonding interactions) to the extreme stability of the tetrameric structure and biotin-binding capacity of the avidin molecule. In the broader sense, such a study may also shed light on protein-protein interactions in general.
The avidin tetramer was thus probed by converting selected interface residues to alanine via site-directed mutagenesis. For this purpose, all three hydrophobic residues in the 1-3 interface were mutagenized (Fig. 1B). In addition, two critical 1-4 interface residues, Asn-54 and Asn-69, were also modified. We considered that alteration of the latter two residues to alanine would preclude the formation of the extensive hydrogen-bonding network involving these residues, thus abolishing the conserved water molecule and perturbing the entire 1-4 interface (see Fig. 1D). The combination of these mutations may thus result in a substantial weakening of the tetrameric assembly of avidin. Indeed, in the absence of biotin, both Avm-[3,4] mutants were found to exist in the monomeric state in solution. When biotin was introduced, however, a stable avidin tetramer was assembled, presumably because of the additional interaction in the 1-2 interface between Trp-110 (from the adjacent monomer) and biotin in its binding pocket. This result demonstrates the critical importance of the 1-2 interaction for the stability of the avidin molecule. It is known that biotin enhances the stability of native avidin, and in the present study we have shown that biotin not only stabilizes the native protein but that it can also play a role in the assembly of tetrameric avidin from the monomer.
This study also sheds light on the importance of the major 1-2
interface and Trp-110 for tetramer formation. Indeed, we have shown
recently that (in both avidin and streptavidin) the unconventional mutation of this tryptophan to a lysine residue resulted in a stable
dimeric protein in solution, both in the presence and absence of biotin
(7). In this context, previous studies with streptavidin have
demonstrated that even conservative mutation of this tryptophan (Trp-120) to phenylalanine or alanine reduced the biotin binding and
weakened intersubunit association (14). The results of the present
study strengthen our previous claims that to preserve high affinity
biotin binding, the binding-site residues should be retained,
particularly the aromatic ones. In contrast, a certain amount of
latitude is permissible in modifying the other residues, including the
loop and -strand residues (12, 15). The present study also
demonstrates that it is possible to manipulate the interfacial residues
quite freely to alanine, thereby reducing both hydrophobic interactions
(e.g. in the 1-3 interface) and hydrogen bonding
(in the 1-4 interface).
The efficient isolation of all mutants on 2-iminobiotin-agarose may
indicate that the monomeric mutants formed tetramers upon interaction
with the immobilized ligand. Under acidic conditions the mutated
proteins were converted to the monomers, which could be reassociated to
the tetramer upon interaction with free or immobilized ligands. This
indicates that constant interaction with the ligand is a prerequisite
for tetramerization of the interface mutants. These results also imply
that biotin is not a necessary element for proper folding of the
monomeric avidin. Together with our previous results concerning the
dimeric avidin and streptavidin mutants, this also indicates that the
avidin -barrel per se is a stable tertiary structure and
capable of folding correctly without the involvement of neighboring monomers.
According to Ellison et al. (16), the mechanism that
protects the native avidin-biotin complex against proteinase K
treatment appears to be the closure and consequent rigidity of the loop between -strands 3 and 4. Compared with native avidin, however, all
the mutants used in this study were rapidly digested in the absence of
biotin by this enzyme but were all stable in the presence of biotin.
This result was not surprising for mutants Avm-[3,4a] and
Avm-[3,4b], which were monomers in the absence of biotin. These data
are also in good agreement with our recent findings regarding the
dimeric avidin mutant (W110K) that remains dimeric in both the absence
and presence of biotin (7). This dimeric mutant was rapidly degraded
both in the presence and absence of biotin. It thus seems possible that
the tetrameric form of avidin is important for rendering the molecule
stable to proteolytic digestion. On the other hand, tetramerization
alone may not suffice, as evident from the observed protease
sensitivity of the tetrameric Avm-3 and Avm-4a in the absence of
biotin. Consequently, biotin binding may be the major factor in
protecting the tetramer against proteolytic digestion.
Weakening of the quaternary structure of the tetramer seems to correlate well with susceptibility to protease digestion, as well as reduced resistance against denaturation in SDS-PAGE. Upon regeneration of the Avm-[3,4a] and Avm-[3,4b] tetramers by adding biotin, the extreme stability characteristics (similar to those exhibited by the native protein) were restored.
The results of this study may have broader implications regarding why avidin and streptavidin appear as tetramers in the native state and how proteins form oligomers in general. We can thus conclude that unusually high ligand-binding affinity and the exceptional stability of avidin and streptavidin are two sides of the same coin. We do not yet know which of these properties is more important, but it seems that the two have coevolved, and it is difficult to separate them.
Many intracellular processes are mediated through inducible
protein-protein interactions (17). These can also be achieved by using
bivalent ligands (18). In most cases, monomeric ligands can cause
dissociation of such protein-protein interactions (19). The
biotin-induced monomer-tetramer transition is the first example of a
monomeric ligand that causes association of protein components. This
phenomenon may eventually be amenable to biotechnological application.
The capacity to produce a monovalent form of avidin, which can then
undergo oligomerization upon addition of biotin, iminobiotin, or other
avidin-binding ligands, may open new avenues for the production of
different intracellular fusion proteins that can be switched on by
oligomerization. The possibility of subsequent coupling of such a
fusion protein with a natural counterpart or substrate may enable new
perspectives in controlling protein-protein interactions and the
importance of oligomerization to cellular processes. The mutated
avidins may also be applicable for reversible assembly and dismantling
of working proteins in living cells.
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ACKNOWLEDGEMENTS |
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We thank Irene Helkala, Anne Ettala, Satu Pekkala, and Henri Nordlund for expert technical assistance.
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FOOTNOTES |
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* This study was supported by grants from the Academy of Finland (SA 29783) and the Israel Science Foundation (administered by the Israel Academy of Sciences and Humanities, Jerusalem, Israel).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.
To whom correspondence should be addressed: Dept. of
Biological and Environmental Science, University of
Jyväskylä, P. O. Box 35 (YAB), FIN-40351,
Jyväskylä, Finland. Tel.: 358-14-2602272; Fax:
358-14-2602221; E-mail: kulomaa@csc.fi.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M007930200
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography.
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