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INTRODUCTION |
The acetyl-CoA carboxylase
(ACC)1 (EC 6.4.1.2) of
Escherichia coli is a multisubunit enzyme belonging to the
biotin carboxylase family, a class of enzymes that use biotin to
transfer an activated carboxyl group from the carboxylation site to a
second site where carboxyl transfer occurs. The biotin moiety is
covalently attached to a specific lysine residue in the biotin carrier
domain of the carboxylase (1). In E. coli ACC the
biotin carrier function resides on a 156-residue protein, the
biotin carboxyl carrier protein (BCCP), of which the C-terminal half
comprises the biotin carrier domain (1, 2). The biotin group is
attached post-translationally to a lysine 34 residues from the C
terminus by the enzyme biotin ligase, which in E. coli is
the multifunctional BirA protein that is also the transcriptional
repressor of the biotin biosynthetic operon (3).
The first form of E. coli BCCP isolated was a 9.1-kDa biotin
carrier protein active in the carboxylation reaction (4) that was
subsequently shown to be a stable proteolytic fragment of the intact
subunit. A very similar protein was produced by digestion of BCCP with
subtilisin Carlsberg (5, 6). Subsequent in vivo experiments
showed that both the subtilisin fragment (BCCP-80), and the 9.1-kDa
protein (BCCP-82), comprising the C-terminal 80 and 82 residues,
respectively, contain all the sequences information necessary for
proper folding of the biotinoyl domain and hence for biotinylation (7,
8). BCCP-82 was used to obtain the three-dimensional structure of the
biotinylated (holo) form of the biotinoyl domain of E. coli
BCCP by x-ray crystallography (5), whereas the C-terminal 87 residues
of BCCP (BCCP-87) was used for multidimensional nuclear magnetic
resonance (NMR) (9-11) analyses. The two methods gave very similar
structures for the holo domain. (The differing N termini of the
proteins studied are of no consequence, since both N termini are highly
mobile and thus were not observed. Only the last 77 residues of BCCP are ordered). The protein forms a flattened 
-barrel structure with
the biotinoyl-lysine exposed on a tight -turn composed of the
conserved Ala-Met-Lys-Met biotinylation motif. The BCCP biotin domain
adopts a fold strikingly similar to those of several domains modified
by lipoic acid attachment (12). The structure of the unbiotinylated
(apo) form of the BCCP-87 biotinoyl domain determined by NMR (9, 10) is
very similar to that of the holoprotein. Both forms of the protein have
the same basic fold, although there are reported differences in the
more defined structures.
The biotinylated lysine residue of the BCCP biotin domain is at the tip
of a protruding hairpin turn (5, 9, 11), and thus modification of this
residue would not be expected to affect the overall structure or
dynamics of the protein. However, we previously reported that the holo
(biotinylated) form of the domain is 10-20-fold more resistant than
the apo form to attack by a variety of proteases and sulfhydryl
reagents (13). Thus, it appears that these reagents, almost certainly
as a consequence of the irreversible nature of their actions, provide
very sensitive indicators of changes in the dynamics of the biotinoyl
domain. In this paper we have investigated the mechanism of the
surprising stabilization bestowed by biotinylation and show that
stabilization is due to biotin attachment per se and that it
is the interactions of the attached biotin with a structural element
(called the "thumb") that protrudes from the otherwise symmetrical
biotinoyl domain of BCCP that is responsible for most of the
stabilization observed.
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EXPERIMENTAL PROCEDURES |
Bacterial Stains and Plasmids--
E. coli strain
CY1123 (
bioC::cml birA1) (14) and
its tolC::Tn10 derivative, strain
JS100, were transformed with the various plasmids and grown in
RB medium (10 g/liter tryptone, 1 g/liter yeast extract, and 5 g/liter sodum chloride). Strain JS100 was constructed by transduction
with phage P1vir grown on strain CAG12184 (15). Strain 2108 was
described previously (16). Antibiotics were added (in mg/liter) at 10, 25, 50, 100, and 100 for tetracycline, chloramphenicol, kanamycin,
trimethoprim, and sodium ampicillin, respectively.
The construction of pCY327, which encodes the 4K-4R BCCP-87 species,
was described previously (16). Plasmid pCY328, which encodes the wild
type form of BCCP-87, was made by exactly the same manipulations. The
thumbless BCCP-87 protein were produced by expression from plasmids
pQE-Th-C and pQE-Th-P, which encode shortened versions of the
Th-(C) and Th-(P) BCCP species studied previously (17). The
pQE-Th plasmids were constructed by digestion of pCY377 or pCY449
(17) with NcoI and BbuI followed by ligation to
pQE60 (Qiagen) digested with the same enzymes.
Protein Methods--
Purification of the various BCCP domains
was performed as described previously (16, 18). The production of the
different domains was induced by adding
isopropyl--D-thiogalactoside or arabinose to final
concentrations of 1 mM or 0.5%, respectively. N-Formyl-BCCP-87 was obtained either from strain 2108 as
described previously (16) or by adding actinonin (Sigma) to the medium as follows. Strain JS100 carrying pCY327 was grown to an
A600 of 0.5-0.6, and the production of
N-formyl-BCCP was induced by simultaneously adding arabinose (0.5%
final concentration) and actinonin (1 µg/ml final concentration).
Following 4 h of induction, the cells were harvested, disrupted by
sonication, and the biotin domain protein was purified. The protein
species were identified and evaluated for purity by polyacrylamide gel
electrophoresis and electrospray ionization mass spectrometry (13,
18).
Protein Biotinylation--
For in vitro enzymatic
biotinylation the apo domain (50 µM) was incubated at
37 °C with 1.25 nM E. coli biotin ligase (the kind gift of Dr. D. Beckett, University of Maryland) in the presence of
3 mM ATP, 5.5 mM MgCl2, 60 µM biotin, 100 mM KCl, and 5 µM dithiothreitol in 20 mM sodium phosphate (pH 7.0) as
described previously (19). Chemical biotinylation was done essentially as previously described (20). A sample (0.1 ml) containing 0.25 µmol
of biotinoyl-N-hydroxysuccinimide (Sigma) was added to a solution containing N-formyl apo-BCCP4K-4R (8 mg in 5 ml of phosphate-buffered saline (pH 7.0)). The solution was kept at
room temperature for 4 h and then dialyzed overnight at 4 °C
against phosphate-buffered saline with one buffer change. The
biotinylation reactions were monitored by polyacrylamide gel
electrophoresis and electrospray ionization mass spectrometry.
Protease Digestions--
The BCCP-87 species (0.35-0.5 mg/ml)
were treated with trypsin (sequencing grade from Roche Molecular
Biochemicals) at a BCCP-87 to trypsin ratio of 40:1 at 25 °C in 100 mM Tris-HCl (pH 7.9) containing 50 mM NaCl and
10 mM CaCl2 as described previously (13, 19).
The reactions were stopped by snap-freezing at 
80 °C. The
digestion products were separated either on sodium dodecyl sulfate-Tricine polyacrylamide gels (19) followed by Coomassie Blue
staining and densitometry (Molecular Dynamics) or by reverse phase high
pressure liquid chromatography on a C4 column (Brownlee Aquapore,
7-µm particle size, 2.1 × 100 mm) by elution with a linear
gradient of 0-50% acetonitrile (v/v) in 0.1% trifluoroacetic acid
(v/v) over 50 min (19).
| |
RESULTS |
Biotin Attachment by Chemical Acylation Is Sufficient to Stabilize
the BCCP Biotinoyl Domain--
Our first hypothesis (13) concerning
the stabilization of the BCCP biotinyl domain was that the increased
stability may not due to biotinylation per se, but rather
could be a consequence of interactions with the BirA biotin ligase, the
enzyme catalyzing the modification reaction. The rationale for this
hypothesis was that the reaction mechanism of E. coli biotin
ligase is known to involve major structural rearrangements of the
enzyme (21-23), and it seemed possible that these conformational
changes could be relayed to the biotinyl domain substrate. Moreover,
alterations in domain tertiary structure upon biotinylation could aid
in dissociation of the ligase-holo biotinoyl domain complex. These
putative ligase-domain interactions would result in changing the
conformation of the domain such that it became more stably folded than
the apo form. Subsequent determinations of the structure of the
apoprotein that allowed comparison with the holo form showed
that conformational changes would necessarily be small (9-11).
However, there are several reported differences in the structures of
the holo and apo domains that could have been imparted by interaction
with BirA during the biotinylation reaction. For example, Yao and
co-workers (10) reported that the hairpin turn that contains the
biotinylated lysine is twisted in the holo form, but is not twisted in
the apo form.
The hypothesis of a conformational change imparted by interaction with
BirA was difficult to test, because biotin ligase treatment was the
only method available to specifically biotinylate the domain, and
we know of no method to remove the biotin moiety from the intact
domain. We now report a means to test this hypothesis by preparation of
a biotin domain modified such that the lysine residue targeted for
biotinylation was the sole amino group of the protein. This allowed
biotinylation by use of a chemical acylation reaction that was specific
to amino groups under the experimental conditions chosen.
The "sole-amino" form of the biotin domain was prepared in two
stages. In the first stage all of the lysine residues except that
targeted for biotinylation were converted to arginine residues by
oligonucleotide cassette mutagenesis of the BCCP-87 coding sequence
(17). Arginine was chosen to retain the sensitivity of the protein to
trypsin digestion, since this is a valuable conformational probe of
BCCP-87 structure (13, 19). The substitutions of arginine for lysine
also retained the charge and extended the aliphatic chain of the
original residues. As expected from the surface location of the lysine
residues, the modified protein (called 4K to 4R) seemed well folded,
since it was readily purified by our standard protocol and was a good
substrate for biotin ligase in vitro and in vivo.
More definitive was the observation that the 4K to 4R domain retained
full function in vivo when converted to a full-length BCCP
(17).
Although arginine residues cannot be chemically acylated at
physiological pH values (due to the very high pKa
values of guanidino groups) (24), the 4K to 4R domain retained a group in addition to that of the target lysine that could be acylated, the N
terminus of the protein. This amino group cannot be removed by
mutagenesis, and since N termini generally have lower
pKa values than lysine 
-amino groups (24),
selective acylation of the target lysine was precluded. Therefore, we
explored several in vivo modifications to block the BCCP N
terminus and found N-formylation to be the most effective
means. We took advantage of the pathway of initiation of protein
synthesis in bacteria (25) in which the N-terminal methionine is
incorporated as the N-formylated species by use of the
initiator tRNA carrying N-formylmethionine. In the normal
course of protein synthesis the N-formyl group is cleaved
from the nascent peptide by peptide deformylase. Following deformylation the methionine residue is removed by methionine aminopeptidase, but only if the penultimate residue has a small side
chain (25). Our prior work showed that methionine aminopeptidase is
inactive on formyl peptides, and thus methionine removal is dependent
on deformylase action (16). We also showed that an N-formylated biotinoyl domain accumulated in vivo
when peptide deformylase activity was blocked by mutation (16). However
since peptide deformylase is an essential enzyme, synthesis of the
N-formylated protein required several cumbersome
manipulations. Moreover, these manipulations generated large amounts of
a yellow compound (presumably a folate metabolite) that complicated
purification of the domain (16). Since that work was reported,
actinonin, a broad-spectrum antibiotic of previously unknown function,
has been shown to be a specific inhibitor of the E. coli
peptide deformylase targeted to the active site (26). Actinonin
addition has no effect on wild type E. coli strains, because
the efflux pump encoded by the acr genes rapidly exports the
inhibitor. However when the pump is inactivated by mutation of the
acr or tolC genes, actinonin becomes a
potent inhibitor of growth (26). Although accumulation of
N-formylated proteins had not been demonstrated in
actinonin-treated E. coli cells, the finding that growth
inhibition was overcome by overproduction of the deformylase indicated
that the primary target of actinonin was peptide deformylase (26).
To use actinonin as a peptide deformylase inhibitor we inactivated the
acr efflux pump by introduction of a
tolC::Tn10 mutation into the host
strains used for protein expression. By judicious addition of actinonin
to cultures of the tolC strain induced for expression of the
4K to 4R BCCP-87 domain we obtained large amounts of the
N-formylated apo species of the protein that was readily purified to homogeneity by ion exchange chromatography and had a mass
of 9474.1, a value in excellent agreement with the calculated mass of
9473.9 (Fig. 1A). (This to our
knowledge is the first demonstration of the accumulation of an
N-formylated protein due to actinonin treatment.) It should
be noted that we were unable to completely inhibit deformylase action
even at very high actinonin concentrations and thus had to separate the
N-formyl apo-BCCP-87 species from the other BCCP-87 species.
A possible explanation for the partial inhibition observed is that
peptide deformylase can use a variety of metal ions that impart greatly
differing actinonin sensitivities (26). It seems possible that in
vivo a mixture of different metal ion forms of peptide deformylase is present. As expected the N-formyl apo-BCCP-87 had a
trypsin sensitivity quite similar to that of the unformylated protein. Indeed by reversed phase high pressure chromatography all of the BCCP
species examined in this work were found to release marker tryptic
peptides in the same pattern and relative amounts as previously demonstrated for the wild type and several mutant proteins (13, 16, 19)
(data not shown). We then used
biotionyl-N-hydroxysuccinimide to biotinylate the sole amino
group of the N-formyl apo-BCCP-87 protein, that of the
lysine 122 side chain. Nondenaturing gel electrophoresis showed that
the chemical modification procedure shifted the N-formyl
apo-BCCP species to an electrophoretic mobility identical to that of
the protein biotinylated in vivo, indicating that a positive
charge had been neutralized and mass spectral analysis (Fig.
1B) gave a mass of 9699.8 for the chemically biotinylated protein, a value in excellent agreement with the calculated mass of
9700.2. These results showed that each biotinylated N-formyl BCCP molecule contained only a single amide-linked biotin moiety, thereby demonstrating the specificity of the acylation reaction. It
should be noted that BCCP-87 contains a single cysteine residue that
upon reaction with biotionyl-N-hydroxysuccinimide would give a biotin thioester. However, cysteine biotinylation was not observed presumably due to the inaccessibility of the cysteine residue (which
resides in the hydrophobic core of the domain) plus the conditions used
for the acylation reaction (which were conducive to thioester
hydrolysis).
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Fig. 1.
Electrospray mass spectral analysis of the
N-formylated and chemically biotinylated BCCP-87
species. A, the purified N-formyl apo form
of BCCP-87 4K-4R was prepared for mass spectral analysis as described
under "Experimental Procedures." The peak with mass 9474.1 corresponds to the mass of the apo-BCCP87-4K-4R plus a formyl
modification of the N terminus of the protein. The minor species of
greater mass represent adducts of ammonium and sodium ions bound to the
many acidic groups of BCCP87-4K-4R. B, the product of
chemical biotinylation of the protein of A by treatment with
biotinoyl-N-hydroxysuccinimide.
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We then compared the trypsin sensitivity of the chemically biotinylated
protein to that of biotinylated protein obtained by biotinylation
in vivo (Fig. 2, A
and B). Several sites in BCCP-87 have very similar
sensitivities to trypsin cleavage in both the apo and holo species (13,
16, 19) such that the digestion appears as an all-or-none reaction on
denaturating gel electrophoresis (13), and thus the only bands present
on these gels are those of the full-length proteins. Therefore,
quantitation of the rate of digestion by loss of band intensity was
straightforward. The trypsin sensitivities of the two proteins were
very similar, but more importantly upon chemical biotinylation the
N-formyl 4K to 4R protein became as stable as the holo form
obtained by biotinylation in vivo. In both cases the holo
forms were only slightly digested upon 21 h of trypsin treatment,
whereas the apoproteins were largely or completely digested.
Interpretation of these results was straightforward; interaction of the
biotin domain with the BirA biotin ligase was not required to obtain a
protein having the reduced trypsin sensitivity of the holo domain.
Chemical biotinylation was sufficient to stabilize the domain, and thus
the presence of biotin attached to Lys-122 was solely
responsible for the increased stability of the holo form relative to
the apo form. Note that prior comparisons showed no differences between
BCCP-87 biotinylated in vivo or biotinylated by biotin
ligase in vitro (13). The BCCP residues first cleaved by
trypsin are Arg-84, Arg-93, Lys-100, and Lys-136, whereas cleavage of
Lys-122 was observed only at higher trypsin:apo-BCCP-87 ratios (13).
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Fig. 2.
Protease sensitivities of BCCP-87
species. In each panel the black bars represent the
holo form, and the gray bars represent the apo form of each
protein examined. The amount of intact protein was measured by
denaturating gel analysis (see "Experimental Procedures"). The BCCP
species examined were all BCCP-87 derivatives (thumbless proteins have
less than 87 residues). The holoproteins were obtained either by
in vivo biotinylation or by treatment of the apoprotein with
the BirA biotin ligase except in B where the apoprotein was
modified by treatment with biotinoyl-N-hydroxysuccinimide.
A, BCCP 4K-4R; B, N-formyl-BCCP 4K-4R;
C, BCCP-Th(C); and D, BCCP-Th(P). There was
no detectable BCCP-87 remaining at the 21-h time point of
B.
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Stabilization by Biotinoylation Is Largely Due to Interactions
between the Biotin Moiety and the Protruding Thumb--
As outlined in
the introduction there is direct evidence for interactions between the
biotin uriedo ring and the protruding thumb structure formed
by residues 91-97. Since these interactions are the major differences
between the apo and holo forms, it seemed plausible that they could
account for the greater overall stability of the holo form. However,
since strong hydrogen bonds could not be detected between the
interacting partners, it was unclear whether the cumulative effects of
the detected weak interactions were sufficient to impart the observed
stabilization. We have approached this question by examining the
comparative stabilities of the apo and holo forms of two proteins
missing the thumb structure. If the stabilization of the biotin domain
is due to biotin-thumb interactions, deletion of the thumb should mimic
the absence of biotinylation. That is, biotinylation should do little
or nothing to stabilize the thumbless BCCPs, and therefore, the apo and
holo forms of the "thumbless" proteins would show similar
sensitivities to trypsin digestion.
One of these thumbless BCCP-87 species was constructed from the wild
type gene during the ligase specificity studies of Reche and Perham
(27), whereas the second was constructed in the 4K to 4R background in
this laboratory (17). In the Reche/Perham thumbless construct (called
BCCP-Th (P)) the thumb was cleanly excised, whereas in our construct
(called BCCP-Th (C)) the thumb was replaced by a single alanine residue
inserted to avoid a putative unfavored Ramachandran angle predicted by
molecular modeling. We found the two proteins to behave similarly
except that BCCP-Th (P) was the more stable protein toward trypsin
digestion (see below) and a better substrate for in vitro
biotinylation by BirA, although the two proteins showed comparable
biotinylation in vivo (17).
The two thumbless proteins were produced in both the apo and holo
forms, purified to homogeneity, and their relative trypsin sensitivities tested (Fig. 2, C and D). We found
that the apo forms of both thumb deletion proteins were slightly more
sensitive to trypsin digestion than the 4K-4R (Fig. 2A) or
wild type (13) apoproteins, which probably reflects low levels of
disturbed residue packing resulting from the deletions. However, in
both cases the holo form of the protein was only slightly more stable
to trypsin digestion than the apo form. That is, the degree of
stabilization toward proteolysis resulting upon biotinylation was
much less than that seen for BCCP species that retained the thumb
structure. BCCP-87 became much less stable when either partner in the
interaction was missing (Fig. 3).
Therefore, interactions between the thumb and the attached biotin
moiety are responsible for most, but not all, of the stabilization
imparted by biotinylation.
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Fig. 3.
Diagrams of the effects of biotinylation and
removal of the thumb. The left figure is that of
holo-BCCP-87 with the biotin moiety in an interacting position; the
central figure is that of apo-BCCP-87 with the lysine side
chain in the same rotamer conformation as that of the left figure; and
the right figure is a generic model of the thumb deletion
forms of holo-BCCP-87 with the biotin moiety in the same position as
that of the left figure.
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| |
DISCUSSION |
Our previously reported data demonstrated that the holo-BCCP-87
domain is considerably more stable toward proteolysis and cysteine
modification than the holo form. These data could be explained by the
apo and holo forms having different structures or differing dynamics
(or both). Since that report two groups have examined the structures of
the apo and holo forms of BCCP-87 by NMR techniques (9, 10). Although
there are some disagreements between the two studies, both report that
the apo and holo biotinoyl domains have the same overall structure.
Therefore, our prior data must indicate that the apo form has a more
dynamic structure than the holo form. This is reasonable since the
chemical and proteolytic probes we used were irreversible and thus well
suited to detection of altered dynamics (28). Altered dynamics were also shown by one of the NMR studies that specifically compared the
final energies of the structural ensembles of the apo and holo forms
and found that final energy of the holo form ensemble to be 507
kJ/mol, whereas that of the apo-BCCP-87 ensemble was significantly
greater (241 kJ/mol) (9). Another NMR study showed that the order
parameters calculated from 15N-relaxation times of the
protein backbone were generally smaller for the apo form, thus
indicating a lower degree of stability, although no values for the
overall stabilities of the apo and holo forms were given (11).
Our chemical biotinylation results indicate that the differences
between the holo and apo form are due solely to the presence of the
covalently attached biotin. These data specifically exclude models in
which interaction with biotin ligase converts BCCP-87 from one
conformer to another of similar or lower overall energy. Therefore, the
observed stabilization of the biotin domain structure conferred by
biotinylation must be understood in terms of biotin-protein interactions. In the first structure of the BCCP biotin domain, the
crystal structure of Athappilly and Hendrickson (5), two hydrogen bonds
were reported to link atoms of the biotin uriedo ring and
Thr-94 of the protruding thumb structure. Specifically, the Thr-94 side
chain and main chain oxygen atoms formed hydrogen bonds with the
carbonyl and N1'-H atoms of the biotin uriedo ring, respectively. There were a priori reasons to doubt the
biological relevance of the reported bonds. The angle of the hydrogen
atom in the first of these reported bonds was only 111°, the bond was rather long (3.8 Å), and Thr-94 is not a conserved residue in other
BCCPs, some of which are known to functionally replace that of E. coli in vivo (17). The second reported hydrogen bond would utilize
the hydrogen of the biotin N1' atom, which is replaced by a carboxyl
group during the ACC reaction. Therefore, the biotin carboxylase
component of ACC could function only after the reported hydrogen bond
had been broken and thus free biotin (at saturating concentrations)
might be expected to act as a better biotin carboxylase substrate than
the holo biotin domain. The converse is observed, and the domain is
much the better substrate (1, 4, 29), which suggests that the biotin
N1' atom is freely accessible for carboxylation. A recent triple
resonance NMR study of BCCP-87 specifically addressed the issue of this
hydrogen bond by measuring the rates of deuterium exchange of the
uriedo N1' and N3' amide protons (9). The two protons
exchanged at similar rates (<5 min), and these rates were comparable
with those seen for free biotin, and thus the exchange rate was not
retarded by several orders of magnitude as expected for strong hydrogen
bonds (9). Moreover, in the solution structure the Thr-94 side chain
oxygen was more than 5 Å from the uriedo carbonyl group,
making a hydrogen bond unlikely (9). Taken together, these data
strongly suggest that the hydrogen bonds observed by Athappilly and
Hendrickson (5) are not relevant to the solution structure and may be
artifacts of the crystal state.
Although the NMR data argue against strong hydrogen bonds linking the
biotin moiety with the thumb, other NMR data indicate that the biotin
rings do interact with the thumb residues. The amide protons of thumb
residues Thr-94 and Ser-96 show slower solvent exchange rates in the
holo form than in the apo form of BCCP-87 (9). In agreement with these
data, several weak nuclear Overhauser interactions are found between
the protons of the biotin moiety and protons of most of the thumb
residues. The proton chemical shifts of the thumb residues also differ
in the apo and holo forms. Finally, the dynamics of the thumb residues
differ in the two forms in that the thumb of the apoprotein is more
mobile than the thumb of the holo form (9, 11). Therefore, although it is clear that the biotin rings and the thumb interact, the interactions cannot be restricted to a specific geometry since virtually all of the
thumb residues show interaction with biotin (9, 11). Biotin is too
small to interact with all of these residues simultaneously unless the
thumb structure collapses around the biotin moiety. However, the
structure of the thumb is the same in the apo and holo forms, so
collapse is excluded. Finally, residues distant from the thumb show
weak interactions with biotin ring protons showing that the biotin
rings can access regions distant from the thumb region (9) and
therefore cannot be tightly bound in place. It should be noted that
early circular dichroism data indicated that the biotin moiety of the
holo biotinoyl domain interacted with the body of the protein (30).
We are therefore left with a dynamic picture in which the biotin rings
have restricted mobility, but are not frozen in place. Our analyses of
the thumbless biotinoyl domain proteins indicate that most, but not
all, of the observed stabilization is due to biotin-thumb interactions
that both slow the dynamics of the thumb (11) and prejudice the
location of the biotin rings. The interactions seem to be heterogeneous
within the population, such that in one molecule the biotin
uriedo ring may be interacting with Tyr-92, whereas in a
neighboring BCCP-87 molecule the interaction may be with Thr-94,
Ser-92, or a nonthumb residue. What we lack are values for the
strengths of the various biotin-thumb interactions and whether or not
these interactions are sufficiently large to give the observed greater
stability of the holo form (9). NMR studies of a thumbless BCCP domain
would be very useful in this regard.
Since the thumb is required for the acetyl-CoA carboxylase reaction
(17), the biotin-thumb interactions may have a physiological role. If
the thumb requirement is due to the interactions of this protein
segment with the biotin moiety, then the reason may be an example of
the "hot potato" hypothesis of Perham (31) in which the
carboxylated form of the biotin moiety must interact with the thumb to
stabilize the unstable enzyme intermediate until it can be delivered to
the active site where it is consumed. However, carboxylbiotin has a
long half-life (>100 min at pH 8) (32), and other biotinylated
proteins lack the thumb (5, 17), and thus the hot potato hypothesis
seems unlikely to apply in this case. Another hypothesis seems more
attractive. In this hypothesis the biotin-thumb interactions serve to
align the biotin moiety for accesses to the ACC active sites. In this
scenario the diversity of biotin-thumb interactions might suggest that
different biotin-thumb alignments are required to access the biotin
carboxylase and carboxyltransferase active sites. These aligning
interactions would result in decreased mobility of both partners giving
the observed greater stability of the holo form of the BCCP biotioyl
domain. Since the thumb structure is thus far found only in the
biotinoyl proteins of bacterial and chloroplast ACCs (17), it seems
that in other biotinylated enzymes, the postulated alignment role must
be provided elsewhere in the enzyme structure.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Microbiology, University of Illinois, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.:
217-333-7919; Fax: 217-244-6697; E-mail:
j-cronan@life.uiuc.edu.
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