(Received for publication, June 5, 1995; and in revised form, August 2, 1995)
From the
The chaperone SecB selectively binds polypeptides that are in a
non-native state; however, the details of the interaction between SecB
and its ligands are unknown. As a step in elucidation of the molecular
mechanism of binding, we have mapped the region of a physiologic ligand
(galactose-binding protein) that is in contact with SecB. The binding
frame comprises 160 aminoacyl residues and is located in the
central portion of the primary sequence. Comparison to the binding
frame within maltose-binding protein, which is similarly long and
positioned around the center of that polypeptide, reveals no similarity
in sequence or in folding motif. The results are consistent with the
proposal that the selectivity in binding exhibited by SecB is based on
the simultaneous occupancy of multiple binding sites, each of which
demonstrates low specificity, by flexible stretches of polypeptide that
are only accessible in non-native proteins.
SecB is a molecular chaperone in Escherichia coli dedicated to facilitation of the export of newly synthesized
proteins from the cytoplasm to their final destination in the
periplasmic space or in the outer membrane (Kumamoto and Beckwith,
1985; Collier, 1993). For the polypeptides to be transferred through
the cytoplasmic membrane, they must be in a competent state that can be
described as neither folded into a stable structure nor aggregated.
Binding by SecB to polypeptides either during or shortly after
completion of their synthesis maintains them in this competent state
(Randall and Hardy, 1986; Kumamoto and Gannon, 1988; Kumamoto, 1989;
Liu et al., 1989; Weiss and Bassford, 1990). As is true for
all molecular chaperones studied, SecB binds its ligands with high
selectivity for the non-native state, but with low specificity (Randall
and Hardy, 1995). The binding of several ligands to SecB has been
characterized, and it is known that, even though the binding affinity
is high, the ligand is in rapid equilibrium between the free and bound
state (Khisty and Randall, 1995; Randall and Hardy, 1995). It is of
great interest to understand the structural basis of the interactions
that provide the energy for the observed high affinity in the absence
of any consensus in sequence among the ligands. To this end, we
previously mapped the regions that are directly bound to SecB within
one physiologic ligand, maltose-binding protein. A binding frame was
identified that consisted of multiple contiguous sites positioned
around the center of the primary sequence and covering 170
residues (Topping and Randall, 1994). Here, we have determined the
binding frame within a second physiologic ligand, galactose-binding
protein. Although there is no obvious sequence similarity between the
two ligands, the binding frame for SecB within each polypeptide is
approximately the same length, and each is poised around the center of
the primary sequence.
The
concentrations of the proteins were determined by absorbance at 280 nm
using extinction coefficients for the SecB monomer and
galactose-binding protein of 11,900 and 37,700 M cm
, respectively. The
extinction coefficients were determined by measuring the absorbance at
280 nm of protein preparations that were also submitted for
determination of amino acid composition.
Galactose-binding protein is one of numerous proteins in E. coli that depend on SecB for efficient export to the periplasm (Powers and Randall, 1995). As was shown previously for the interaction of SecB with another of its natural ligands, periplasmic maltose-binding protein, SecB forms a complex with galactose-binding protein only if the protein is presented to SecB in a non-native state. Analysis by size-exclusion HPLC of a mixture of SecB and galactose-binding protein that was folded into its native state showed no detectable complex. The two proteins were well resolved from each other, with SecB and folded galactose-binding protein eluting at the positions characteristic of the free proteins, 14.5 min for SecB and 20 min for galactose-binding protein (Fig. 1, compare A, C, and D). However, if refolding of denatured galactose-binding protein was initiated by dilution of the denaturant, guanidinium chloride, in the presence of a 2-fold molar excess of tetrameric SecB, a complex of SecB and galactose-binding protein was formed. All of the galactose-binding protein coeluted with SecB at 13.5 min just ahead of free SecB (Fig. 1B) (the proteins present in each fraction were identified by SDS-gel electrophoresis (data not shown)). The approach we took to determine which portions of galactose-binding protein were directly bound to SecB was to subject complexes to proteolytic digestion and to determine which fragments of galactose-binding protein were protected from degradation and also remained bound to SecB. The same final results were obtained whether the complexes were formed as described above by diluting the denaturant to initiate folding of galactose-binding protein in the presence of SecB or by diluting the denaturant to allow galactose-binding protein to undergo an initial collapse followed within 10 s by the addition of SecB. In each case, the SecB tetramer was present in a 2-fold molar excess so that all of the galactose-binding protein would be in complex. The conditions of proteolysis were chosen such that SecB remained intact and all of the galactose-binding protein was fragmented. The pattern of peptides generated by digestion of galactose-binding protein in complex with SecB was compared with the pattern generated by proteolysis of denatured galactose-binding protein in the absence of SecB. The proteolyzed samples were subjected to reversed-phase HPLC (Fig. 2), and the peptides in each fraction were displayed by electrophoresis (Fig. 3) using a system designed to resolve small peptides (Schägger and von Jagow, 1987; Topping and Randall, 1994). It is clear that, while the peptide patterns are closely related, digestion of the complex results in the appearance of many peptides that are not present when galactose-binding protein is digested alone (Fig. 3). It should be recalled that, under the conditions of proteolysis used, SecB is not degraded, and furthermore, it is removed from the sample by precipitation before analysis of the peptides. Therefore, all of the peptides present should be derived from galactose-binding protein as was shown to be the case by direct determination of the aminoacyl sequence of the peptides as described below. The peptides that were unique to the samples derived from the digested complex represent regions of galactose-binding protein that were in direct contact with SecB and thus protected from proteolysis. To demonstrate that these peptides remained bound to SecB, the proteolyzed mixture was subjected to size-exclusion HPLC. After proteolysis, the complex eluted at 14.5 min, the position of free SecB; however, analysis by gel electrophoresis showed that those fractions containing SecB (eluting from 13.5 to 16 min) also contained fragments of galactose-binding protein. These fractions were pooled, and the peptides therein are referred to as bound, whereas peptides in the pool of fractions eluting between 16.5 and 30 min are referred to as free.
Figure 1: Binding of SecB to unfolded galactose-binding protein. HPLC of protein mixtures was carried out as described under ``Experimental Procedures.'' A, mixture of SecB and folded galactose-binding protein; B, mixture of SecB and unfolded galactose-binding protein; C, SecB only; D, unfolded galactose-binding protein only. The amount of protein applied to the column was 0.12 mg for galactose-binding protein and 0.48 mg for the SecB monomer.
Figure 2: Comparison of peptide patterns generated by proteolysis of galactose-binding protein either free or in complex with SecB. Peptides generated by proteolysis of unfolded galactose-binding protein (GBP; upper trace) or of a complex between SecB and galactose-binding protein (SecB-GBP; lower trace) as described under ``Experimental Procedures'' were analyzed by reversed-phase HPLC. Only the portion of the chromatograms that contained peptides is shown. The amount of intact galactose-binding protein initially present in each sample applied was 0.38 mg.
Figure 3: Comparison of peptides derived from proteolysis of galactose-binding protein either free (GBP) or in complex with SecB (SecB-GBP) displayed by gel electrophoresis. The peptides contained in the fractions from the reversed-phase HPLC shown in Fig. 2were resolved by gel electrophoresis. The standards for molecular mass were carbonic anhydrase (28.7 kDa), myoglobin (17.0 kDa), and fragments of myoglobin generated by cyanogen bromide cleavage (14.4, 8.2, 6.2, and 2.5 kDa; from Sigma).
Analyses of the bound and free sets of peptides by reversed-phase HPLC show that the set of bound peptides does account for the peptides that are unique to the complex (compare Fig. 2B and 4 and Fig. 3and Fig. 5). The pattern of the peptides recovered free from SecB is similar to that obtained when galactose-binding protein is digested alone. The sequence of the first 5 or 6 aminoacyl residues was determined for all material recovered in sufficient quantity to allow analysis from two separate experiments. This information, together with molecular weights estimated from the position of migration on peptide gels, allowed us to position the peptides within the sequence of galactose-binding protein ( Fig. 6and Table 1). The peptides recovered in the bound fraction cover approximately half of the primary sequence and are poised around the center of the sequence. The set of peptides recovered as free contains representatives of all regions of the sequence. Comparison of the relative recovery of material from along the sequence shows that the region of low recovery among the population of free peptides from aminoacyl residue 120 to residue 200 corresponds to the region represented by the peptides recovered as bound (Fig. 7).
Figure 5: Bound and free peptides displayed by gel electrophoresis. The peptides contained in the fractions from the HPLC shown in Fig. 4were subjected to gel electrophoresis. The standards for molecular mass were as described for Fig. 3.
Figure 6: Representation of the positions of the peptides within the linear sequence of galactose-binding protein. Mature galactose-binding protein comprises 309 aminoacyl residues, represented by the horizontal line at the center of the figure. All peptides that were sequenced are represented by boxes placed at their position within the sequence of galactose-binding protein. The length of each peptide was estimated from its position of migration on peptide gels. The heights of the boxes represent the recovery of peptides estimated from the yield of amino acids obtained upon determination of the sequence. The closed boxes represent those peptides that remained bound to SecB, and the open boxes represent those peptides that were recovered free from SecB (see Table 1for details).
Figure 7: Summation of bound and free peptides. The data from Fig. 6were used to calculate the recovery of amino acid residues at each position along the sequence by summing the yield of every peptide that contained that aminoacyl residue. aa, amino acid; GBP, galactose-binding protein.
Figure 4: Pattern of peptides resolved into bound and free fractions after proteolysis of the complex between galactose-binding protein and SecB. The complex was formed and proteolyzed, and the peptides were separated into those that remained bound to SecB and those recovered free from SecB as described under ``Experimental Procedures.'' The amount of intact galactose-binding protein initially present in each sample applied to the size-exclusion column was 0.78 mg. The chromatograms of the analyses by reversed-phase HPLC are shown. HPLC analysis of the unfractionated peptide mixture from which the free and bound fractions were isolated is shown in Fig. 2(lower trace).
The shortest of the peptides recovered as bound after proteolysis of
the complex that had been formed between SecB and full-length
galactose-binding protein had a M of
6000 (Table 1). To determine whether SecB could bind tightly to
fragments of this length that were presented directly to SecB, free
galactose-binding protein was proteolyzed first, and then SecB was
added to the heterogeneous mixture of peptides. Conditions of
proteolysis were chosen (proteinase K at 0.005 mg/ml, 5 min on ice) so
that a wide range of peptide lengths would be present. SecB was added
in a 2-fold molar excess calculated based on the amount of intact
galactose-binding protein initially present to ensure maximal binding
of the fragments, and the mixture was subjected to size-exclusion HPLC.
Each pool of peptides (total, bound, and free) was analyzed by
reversed-phase chromatography (Fig. 8), and the peptides in each
fraction were resolved by gel electrophoresis (Fig. 9). It is
clear that fragments having M
values of <6200
were recovered quantitatively in the free pool of peptides, and only
those fragments with a minimal length of between 55 and 80 residues
were bound with sufficiently high affinity to be isolated in complex
with SecB. There were peptides among those isolated as free that are
longer than the length defined as minimal for binding. It may be that
these fragments were long enough to acquire structure or that they
rapidly aggregated and thereby were not able to interact with SecB.
Figure 8: Proteolysis of free galactose-binding protein followed by the addition of SecB. Uncomplexed, denatured galactose-binding protein was subjected to proteolysis, and the peptides present were resolved by reversed-phase HPLC (upper trace). The amount of intact galactose-binding protein initially present in the sample applied was 0.38 mg. SecB was added to the peptide mixture, and the peptides that bound to SecB were separated from those that remained free (see ``Experimental Procedures'' for details). The amount of intact galactose-binding protein initially present in the sample applied to the size-exclusion column was 0.78 mg. The peptides present in the bound fraction (middle trace) and in the free fraction (lower trace) were analyzed by reversed-phase HPLC.
Figure 9: Comparison of peptides that bound to SecB with those that remained free displayed by gel electrophoresis. Peptides contained in the fractions from the HPLC shown in Fig. 8were resolved by gel electrophoresis. The standards for molecular mass were as described for Fig. 3.
Binding that involves recognition of non-native structure is
the hallmark of the class of proteins termed chaperones. We are
investigating complexes between SecB and its ligands in an attempt to
understand the molecular basis of such interaction. The defining
characteristic of the binding is that it occurs with low specificity,
but when the ligand is a long polypeptide, the affinity is high
(dissociation constants are in the range of 1-100 nM (Randall, 1992)). A model based on studies with synthetic peptide
ligands attributes the high affinity to multiple binding sites on the
SecB tetramer for separate stretches of the polypeptide ligand
(Randall, 1992). It was proposed that the initial interaction with a
potential ligand is the binding of an extended flexible stretch of
polypeptide. Non-native proteins would have many flexible regions and
thus could occupy several separate sites on the SecB tetramer. A study
of peptide binding showed that there is likely to be at least one site
on each monomer (Randall, 1992). Furthermore, multiple occupancy of
these sites by flexible regions of the ligand was shown to induce a
conformational change in SecB that was proposed to result in additional
interaction with hydrophobic regions of the ligand. The binding energy
at each of the individual sites could be low, but the sum of the
binding energies, resulting from simultaneous occupancy, would be high
and would account for the low probability of dissociation. The binding
frame for SecB within the ligand galactose-binding protein, as
determined here, and the binding frame within maltose-binding protein,
determined previously (Topping and Randall, 1994), are consistent with
this model. Each of the binding frames is sufficiently long to fill the
four putative sites for extended polypeptide chains and the postulated
hydrophobic site. The minimal length of peptides that bind with
dissociation constants in the micromolar range is 12 residues
(Randall, 1992). This is likely to be the length required to fill one
site, but the binding energy of interaction at one site is too low to
allow isolation of complexes. The observation that fragments of
galactose-binding protein must be at least 60-80 residues in
length to be bound tightly enough to be isolated in complex with SecB
by column chromatography is consistent with the idea that the high
affinity for long polypeptide ligands results from simultaneous binding
of separate stretches of the ligand at multiple sites on SecB. In the
case of maltose-binding protein, the smallest peptides found bound
after column chromatography were
25 residues long.
The binding
frame for SecB within galactose-binding protein and that within
maltose-binding protein are not only similar in length (150 residues
for galactose-binding protein and 170 residues for maltose-binding
protein), but both are similarly positioned in the center of the
primary sequence of each polypeptide. Comparisons of the two
polypeptides with respect to aminoacyl sequence and to structure
provide no clue as to why the binding frame covers the central portion
of each polypeptide. Sequence analysis using the COMPARE program with
the SIMPLIFY algorithm showed no significant sequence similarity within
the binding frames. Furthermore, a comparison of the distribution of
charges along the polypeptides did not reveal any common pattern
(Randall and Hardy, 1995). The two proteins do have similar tertiary
structures. Each contains two domains that comprise central cores of
-sheets flanked by
-helices. Thus, one might think that SecB
recognizes a specific array of secondary structure in an intermediate
along the folding pathway. However, the proteins display different
connectivities between the elements of secondary structure. The
stretches of amino acids covered by the binding frames, although
located similarly along the linear sequences, are disposed differently
among the elements of secondary structure (Fig. 10). Thus, it is
not clear why SecB binds to the middle portion of the polypeptides. As
suggested previously (Topping and Randall, 1994), the binding frame
might lie at the center of the polypeptide ligand simply because the
probability of multiple interactions resulting in tight binding would
be higher if potential binding sites existed on each side of the first
site of contact, a situation that would not occur if the initial
contact were at either end.
Figure 10:
Comparison of the topology of
maltose-binding protein and galactose-binding protein. The topological
diagrams were generated using the TOPS (Flores et al., 1994)
and DSSP (Kabsch and Sanders, 1983) programs and the x-ray
crystallographic coordinates for maltose-binding protein (Spurlino et al., 1991) and galactose-binding protein (Mowbray et
al., 1990). -Helices are depicted as circles, and
-sheets as triangles. The amino and carboxyl termini are
indicated by N and C, respectively. The binding frame
for SecB within each polypeptide is indicated by the filled
symbols.
The large size of the binding frame,
covering 50% of each of the physiologic ligands studied to date,
may be crucial to the function of SecB as a chaperone. SecB facilitates
export of polypeptides through the cytoplasmic membrane into the
periplasm by binding the polypeptides before they can either fold into
their thermodynamically stable state or aggregate. Since both folding
and aggregation are rapid, the rate at which SecB binds its ligands
must be high. As discussed previously (Randall and Hardy, 1995), SecB
could have a rate constant of association that is 10
M
s
or even higher since
the target for collisions with SecB that lead to binding covers as much
as 50% of the surface of the ligand. The ability to bind non-native
ligands rapidly and selectively with high affinity is common among
chaperones. It is likely that chaperones other than SecB also make use
of multiple binding sites, each having a low specificity, and have
large overall binding frames on the ligands to achieve rapid and high
affinity binding in the absence of consensus in sequence among the
polypeptides bound.