From the Department of Biological Sciences, Columbia University, New York, New York 10027
Received for publication, December 2, 2002
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ABSTRACT |
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BtuF is the periplasmic binding protein (PBP) for
the vitamin B12 transporter BtuCD, a member of the ATP-binding cassette (ABC) transporter superfamily of transmembrane pumps. We have determined crystal structures of Escherichia coli BtuF in
the apo state at 3.0 Å resolution and with vitamin B12 bound at 2.0 Å resolution. The structure of BtuF is similar to that of the FhuD and
TroA PBPs and is composed of two The ATP-binding cassette
(ABC)1 transporter
superfamily consists of mechanochemically coupled polypeptide
complexes in which ATP hydrolysis is coincident with transport of
solutes against cellular concentration gradients (1). Members of this
family share common structural features that are conserved across the phylogenetic spectrum. The prototypical ABC transporter consists of
four subunits, two of which are Structures have previously been reported for several PBPs (4), with the
maltose-binding protein (MBP) being the most widely studied. MBP is
composed of two globular domains joined by a hinge region. One maltose
molecule binds at the interface of the globular domains. During
binding, these domains undergo a substrate-dependent conformational change, rotating ~35° about the hinge region (5). Other studies suggest that this conformational change transmits a
signal to the TM domains of the maltose transporter promoting ATP-dependent uptake of maltose across the cytoplasmic
membrane (6). Most other PBPs have a similar molecular architecture to
MBP and presumably function using a similar ligand-induced "clamping" mechanism. PBPs of this kind are subdivided into types (called I and II) based upon the topology of their globular domains (7,
8). However, the structure of the TroA Zn2+ PBP displayed a
distinct fold wherein a rigid Vitamin B12 is a large organic cofactor with 93 non-hydrogen atoms that
is employed in a diverse array of biochemical reactions ranging from
methyl transfers to ribonucleotide reduction (11). B12 is an essential
cofactor in all kingdoms of life, and while some bacteria and archaea
have evolved the propensity for its synthesis, most prokaryotes and all
eukaryotes contain transport systems to import B12 (12). The products
of B12 biosynthesis are called coenzyme B12 and have either a
5'-deoxyadenosyl group or a methyl group as the axial ligand of the
cobalt atom on the catalytic face of the cofactor. The industrial
process used to prepare B12 for human consumption results in the
replacement of this axial ligand by a cyano group, leading to a species
called cyanocobalamin or vitamin B12 (13). As the biosynthesis of B12 requires ~30 enzymatic steps (14), the benefits of importing externally synthesized molecules are clear. In Escherichia
coli, the transmembrane transport of B12 is carried out by the Btu
(B twelve uptake) system composed of BtuB, an outer membrane
TonB-dependent transporter (15), and BtuCDF, an ABC
transporter located in the inner membrane. BtuC and BtuD compose
respectively the TM domain and the ABC (16), while BtuF is the cognate
PBP (17). The crystal structure of the BtuCD integral membrane protein
complex has recently been reported (18). We herein present the crystal structures of both vitamin B12-bound and free (apo) forms of BtuF.
Protein Expression and Purification--
Pre-BtuF including its
N-terminal signal peptide was cloned into pET24 (Novagen), and
periplasmic expression of the mature protein was induced for 4 h
at 37 °C in E. coli BL21(DE3) cells. A cleared lysate in
300 mM NaCl, 10 mM imidazole, 10% glycerol, 10 mM dithiothreitol, 50 mM potassium
phosphate, pH 7.0, was loaded on a nickel-nitrilotriacetic acid SF
(Qiagen) column and eluted with a linear gradient to 250 mM
imidazole. Following gel filtration on a Superdex 200 column (Amersham
Biosciences) in 100 mM NaCl, 10 mM
dithiothreitol, and 10 mM Tris-Cl, pH 7.5, the protein was concentrated to 10 mg/ml for crystallization. Selenomethionine-labeled protein was induced overnight at 37 °C in B834(DE3) cells grown in
M9 minimal media with 50 mg/ml DL-selenomethionine and the Kao and Michaluk vitamin supplement (Sigma).
Crystallization--
Crystals were grown using standard 1:1
hanging-drop vapor diffusion reactions. For the B12 crystals, 5 mM cyanocobalamin (Sigma) was added to the protein stock,
and the well solution contained 2.0 M NaCl, 3% ethanol,
100 mM sodium acetate, pH 4.6. Crystals grew to a maximum
size of ~2003 µm over 10 days and were cryoprotected by
incubation for 1 h in the well solution plus 5 mM B12
and 30% glycerol before freezing in liquid propane. Apo crystals grew
to a maximum size of ~4003 µm over 1 week using a well
solution containing 30% polyethylene glycol 4K, 200 mM
(NH4)Cl, and 100 mM sodium acetate, pH 4.6. They were cryoprotected by incubation for 30 min in 40% polyethylene glycol 4K, 100 mM NH4Cl, 10%
glycerol, and 50 mM sodium acetate, pH 4.6.
X-ray Data Collection and Structure Determination--
Data were
collected at NSLS (National Synchrontron Light Source) beamline X12C at
Brookhaven National Lab using the Brandeis-B4 detector.
Multi-wavelength anomalous diffraction datasets were collected in
365° sweeps at 0.9785, 0.9787, and 0.9500 Å using 1° oscillations.
Data processing and reduction were performed with DENZO and
SCALEPACK (19). The four selenium atom sites in the B12 crystals
were determined by SOLVE (20), and density modification with automated
model building was carried out by RESOLVE (21). Density modification
with non-crystallographic symmetry (NCS) averaging in DM (22) allowed
completion of this structure, which was use to solve the apo structure
by molecular replacement with COMO (23).
Model Building, Refinement, and Molecular Graphics--
Models
were built using O (24) and refined using CNS (25, 26) without
imposing NCS restraints at any stage. Rfree sets containing
5% of the reflections were selected at random independently for each
crystal form. Refinement comprised iterations of overall anisotropic
B-factor refinement, bulk-solvent correction, rigid-body refinement,
positional minimization, simulated annealing, and individual isotropic
B-factor refinement. Residues in the BtuF models are numbered according
to their position in the mature protein, i.e. after
proteolytic cleavage of the 22-residue N-terminal signal peptide in the
preprotein. Coordinates and structure factors for the B12-bound and apo
structures have been deposited in the PDB under accession codes 1N4A
and 1N4D, respectively. Molecular interactions were analyzed using the
program CONTACT from CCP4 (27) using a cut-off of 3.8 Å for van der
Waals contacts and 3.3 Å for H-bonds. Ribbon diagrams were created
with MOLSCRIPT (28) or BOBSCRIPT (29) and rendered with Raster3D (30). Surface-rendered images were created with GRASP (31).
Overall Protein Structure--
BtuF is composed of two globular
domains linked by a rigid interdomain
Domain I is an
Domain II is also an
Previous studies suggests a common mode of cobalamin binding by this
domain. In both MetE and MMCM, the bound B12 is in the "base-off" conformation (Fig. 2B) where the DMB
moiety has been displaced as one of the axial ligands of the central
cobalt atom by a histidine residue that is conserved in the enzymes but
absent in BtuF. This histidine modulates the reactivity of the other axial ligand of the cobalt atom on the opposite "catalytic surface" of the B12 molecule (11). However, B12 is clearly bound to BtuF in the
"base-on" conformation in which N2 of DMB remains coordinated to
the central cobalt, with the cyanide moiety of cyanocobalamin as the
other axial ligand on the opposite face (Figs. 2B and
3A). Presumably, this conformation was selected to preserve
the co-factor in an unreactive conformation until delivered to the
appropriate intracellular enzymes as dissociation of the DMB is a key
step in the activation of coenzyme B12 (11). Probably as a result of
this conformational difference in B12, domain II of BtuF binds the
opposite surface of the ligand as MetE and MMCM. Domain II in BtuF
contacts the catalytic surface of B12 (with bound cyanide), while the
equivalent domain in the structural homologues contacts the opposite
surface of B12 where the DMB has been displaced by the conserved
histidine residue. In all cases, the homologous domains interact with a
flat surface of the B12 ring (Fig. 2B). The reversal of
binding geometry in BtuF may be caused by obstruction of the face that
typically interacts with this domain by the DMB moiety in the base-on conformation.
Geometry of Vitamin B12 Binding--
The highly conserved (Fig.
3B) B12 binding site in BtuF
is strongly acidic (Fig. 3C) despite the fact that the
protein molecule as a whole is slightly basic (pI
A total of six residues in domain I make 50 direct contacts to B12
(Figs. 3A and 4B). The backbone oxygen of Ala-10
in helix
A total of five residues in domain II make 39 direct contacts to B12
(Figs. 3A and 4B). Four of these residues
interact with the propionamide side chain of Ring D (the side chains of
Trp-174 after strand
The relatively small number of direct atomic contacts between BtuF and
vitamin B12 (89 total) stands in contrast to situations observed in
vitamin B12-utilizing enzymes. Both methionine synthase (34) and MMCM
(35) make over 150 direct contacts to the ligand. BtuF has presumably
adapted to make fewer contacts because it must release the ligand to
the transporter in response to a relatively small conformational change
(see below), while the B12-utilizing enzymes will ideally permanently
immobilize the cofactor in a specific geometry. In addition, as most of
the contacts are to the periphery of the B12 molecule, e.g.
the propionamide groups, it is likely that bacteria have evolved the
ability to transport cobalamin variants with different axial ligands
including 5'-deoxyadenosine.
Conformational Consequences of B12 Binding and
Release--
Crystal structures were obtained of BtuF both in
the presence and absence of the vitamin B12 ligand (Table
I and Fig. 4). Both the
B12-bound and apo crystals contain two
BtuF molecules related by NCS in the asymmetric units of their
respective lattices. However, the refinements were performed without
imposing any NCS restraints so that the conformations of the two
different molecules in each lattice were determined independently.
Although the protein molecules in the B12-bound crystal have somewhat
similar crystal packing environments and therefore cannot be considered
to be completely independent, the molecules in the apo crystal have distinct packing environments (Fig. 4B and additional data
not shown) and therefore represent two independent views of the
conformation of apoBtuF that present a consistent picture of the
structural consequences of B12 release.
In Fig. 4A, the B12-bound and apo molecules are aligned by
least-squares superposition of their N-terminal domains. As expected, a
large conformational change of the type observed in MBP does not occur
as it is sterically prohibited by the rigidity of the interdomain helix
Comparing the protein structures within the individual domains, the
equilibrium conformation of domain I is tightly conserved in both of
the apoBtuF molecules compared with the B12-bound molecules (Fig.
4A). The backbone B-factors in this domain are also very similar in the apo structures and B12-bound structures, except for a
local elevation at two sites in the apo structures corresponding to
protein loops that directly contact B12 when it is bound (Fig. 4B).
In contrast, substantially larger differences are consistently observed
in both the equilibrium conformation and backbone B-factors in domain
II in the two NCS-related molecules in the apo structure (Fig. 4). The
backbone B-factors are elevated throughout the entirety of domain II in
the apo molecules (Fig. 4B), indicating a global increase in
the mobility of domain II upon ligand release. These high B-factors
lead to the presence of weak electron density at the corresponding
sites in the protein structure. The density is so weak in domain II of
the apoB molecule that it was not possible to interpret the
conformation of residues 195-211. Although the corresponding region
could be interpreted in the apoA molecule, it has the highest backbone
B-factors in this molecule (Fig. 4B). The least mobile
regions in domain II of the apo molecules (
The crystal packing contact plot above the B-factor plot in Fig.
4B shows that there are a significant number of
intermolecular contacts in the high B-factor regions in all four
crystallographically observed BtuF molecules. This observation,
combined with the different packing environments of the two
independently observed apo molecules, makes it very unlikely that the
increase in the mobility of domain II in the apo structure is a crystal
packing artifact.
The differences in the equilibrium conformation in domain II in the apo
structures occur near the surface expected to interact with the BtuCD
transporter (lower right in Fig. 4A) and are thus likely to inhibit the gratuitous binding of apoBtuF to the transporter. Moreover, the consistent increase in the mobility of domain II observed
in both of the independently observed NCS-related apo molecules suggest
that the conformational entropy of domain II increases after the
release of B12 into the transmembrane gate, and this effect could help
promote dissociation and recycling of apoBtuF (36).
Structural Aspects of BtuF-mediated Transmembrane
Transport--
The BtuCD structure (18) contains a B12-sized cavity in
the transmembrane region near the periplasmic surface of the
transporter at the interface between a pair of BtuC subunits related by
2-fold symmetry. However, computational analyses show that this cavity is sealed from the periplasm (yellow surface in
Fig. 4 of Ref. 18) indicating that a large conformational change must
take place in the BtuC domains upon BtuF-mediated delivery of B12. This
delivery process is likely to involve stable binding of BtuF to the
transmembrane BtuC subunit based on the results observed with other
PBP-dependent bacterial ABC transporters (37). Given the
2-fold symmetry of the BtuC subunits flanking the putative B12-binding
site in the transporter, BtuF might be expected to exhibit 2-fold
pseudo-symmetry in the regions flanking its B12 binding site. However,
detailed examination of the surface features shows no detectable
symmetry in hydrophobicity (not shown), electrostatic potential (Fig.
3C), or sequence conservation (Fig. 3B) in the two lobes of BtuF. While the back surface of the molecule on the opposite face from its B12-binding site shows the lowest level of
sequence conservation (not shown), relatively strong conservation is
observed on the surfaces surrounding the B12-binding site (Fig. 3B). However, even stronger conservation is observed at the
periphery of the BtuF molecule on the surface of domain I furthest away from the B12 ligand (on the far left as shown in Fig.
3B), suggesting that this region that is relatively remote
from the B12-binding site might interact directly with BtuC. Future
studies will be required to determine how the asymmetrical vitamin B12
and BtuF molecules bind to the symmetrical BtuCD complex during the
active transport reaction.
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domains linked by a rigid
-helix. B12 is bound in the "base-on" or vitamin conformation in
a wide acidic cleft located between these domains. The C-terminal domain shares structural homology to a B12-binding domain found in a
variety of enzymes. The same surface of this domain interacts with
opposite surfaces of B12 when comparing ligand-bound structures of BtuF
and the homologous enzymes, a change that is probably caused by the
obstruction of the face that typically interacts with this domain by
the base-on conformation of vitamin B12 bound to BtuF. There is no
apparent pseudo-symmetry in the surface properties of the BtuF domains
flanking its B12 binding site even though the presumed transport site
in the previously reported crystal structure of BtuCD is located in an
intersubunit interface with 2-fold symmetry. Unwinding of an
-helix
in the C-terminal domain of BtuF appears to be part of conformational
change involving a general increase in the mobility of this domain in
the apo structure compared with the B12-bound structure. As this
helix is located on the surface likely to interact with BtuC, unwinding
of the helix upon binding to BtuC could play a role in triggering
release of B12 into the transport cavity. Furthermore, the high
mobility of this domain in free BtuF could provide an entropic driving force for the subsequent release of BtuF required to complete the
transport cycle.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helical transmembrane (TM) domains
presumed to determine substrate specificity and trajectory. The other
two subunits (the ABCs) are peripherally associated with the
cytoplasmic region of the TM domains and mechanically couple ATP
hydrolysis to solute translocation (2). In addition, most bacterial
importers employ a periplasmic
substrate-binding protein (PBP) that delivers
the ligand to the extracellular gate of the TM domains. ABC
transporters have been linked to a number of human diseases, the most
notable of which are cystic fibrosis and tumor multidrug resistance
(3).
-helix connects the two ligand-binding
domains, suggesting that this subclass of PBP does not undergo a large
scale conformational change of the same nature as type I and II PBPs;
comparison of free (9) and ligand-bound (10) TroA structures supports
this hypothesis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix (
6) (Fig.
1). The N-terminal domain I (residues 1-106 of the mature protein) and C-terminal domain II (residues 130-242) possess a similar topology (Fig. 1B) and display
19% sequence identity and a root mean square deviation (r.m.s.d.) of
4.4 Å for the superposition of 106 C
atoms (32). As in other types
of PBPs, the vitamin B12 transport ligand is bound between the two
domains and makes contacts to both.
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Fig. 1.
Structure of E. coli BtuF bound to Vitamin B12. A, ribbon
diagram of the BtuF·B12 structure color-coded by domain organization,
with domain I colored cyan, domain II tan, and
the interdomain helix ( 6) green. Vitamin B12 is
represented as a space-filling model with carbon atoms colored
black, nitrogens blue, oxygens red,
and phosphorus magenta. B, topology diagram of
BtuF colored as in A with arrows representing
-strands and cylinders representing
-helices. C,
sequence alignment of BtuFs (from E. coli, Salmonella
typhimurium, Vibrio cholerae, Yersinia
pestis, and Ralstonia solanacearum) and
structural homologues FhuD, TroA, the B12-binding domain of methionine
synthase (1BMT), and malonyl-CoA mutase (1REQ). Residues that contact
B12 directly are colored pink, while residues that make
water-mediated contacts are colored lime. Pink
asterisks indicate residues that H-bond directly to B12. The
classification of well ordered
-turns (40) is indicated above the
structural schematic.
/
sandwich composed of a twisted
five-stranded parallel
-sheet with an
-helix connecting each
strand (Fig. 1, A and B). Helix
5 of domain I
packs against the interdomain helix
6 to form a partial helical
bundle. This domain makes contacts to both the
dimethylbenzimidazole (DMB) and
propionamide groups on B12 (Fig. 1, B and C)
using residues in helix
1, strand
4 and the loops following
strands
2 and
4 (see below). A portion of the topology is
conserved between domain I of BtuF and the N-terminal lobes of both
type I and II PBPs (Fig. 1B). Specifically, these domains
all contain a super-secondary structural element at their N termini
composed of three parallel
-strands linked by two
-helices.
Thereafter, both types I and II PBP topologies are divergent from BtuF
(7). However, the entirety of domain I of BtuF is structurally similar
to the N-terminal domain of FhuD (33), the iron-siderophore PBP
(residues 35-127), and these regions share 20% sequence identity and
an r.m.s.d. of 2.5 Å for superposition of 101 C
atoms. This
superposition (Fig. 2A)
reveals an expanded ligand-binding cavity of BtuF relative to FhuD,
presumably to accommodate the increased ligand volume of B12
versus gallichrome. Furthermore, BtuF shares the same fold
as the TroA Zn2+-binding protein (10) both in domain I and
also in domain II where their topologies diverge from that of FhuD
(Fig. 1B). However, TroA has an additional
-hairpin
inserted into domain I, and its mode of ligand recognition and possibly
release is distinct from that of BtuF (see below).
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Fig. 2.
Structural homologues of BtuF.
A, stereo pair of the structural alignment of the N-terminal
domains of BtuF and E. coli FhuD bound to gallichrome (PBD
accession number 1EFD). BtuF is colored by domain as above, and
vitamin B12 is shown in ball-and-stick representation and colored
magenta. FhuD is colored gold with the bound
gallichrome shown in bright green. B, stereo pair
of the structural alignment of domain II of BtuF with the B12-binding
domain of E. coli methionine synthase (MetE, PDB accession
number 1BMT). The corrin ring and DMB of B12 are colored
brown and red, respectively. MetE is colored
blue with the corrin ring and DMB of its bound B12 colored
aqua and cyan, respectively.
/
sandwich composed of a four-stranded
parallel
-sheet with connecting
-helices. The C-terminal helix (
11) packs against the interdomain helix (
6) as observed for helix
5 in domain I. Clearly, domain II has diverged from the iron-siderophore class of PBPs, as the FhuD structure contains a
five-stranded
-sheet with mixed topology (33). However, a DALI (32)
search of the PDB revealed that domain II has significant structural
homology to the B12-binding domains in methionine synthase (MetE, PDB accession number 1BMT) (34) and methylmalonyl-CoA mutase (MMCM, PDB accession number 1REQ) (35), two enzymes that
catalyze methyl transfer reactions. These two enzymes share only 9 and
13% sequence identity to domain II of BtuF, respectively. Nonetheless,
there is 3.4 Å r.m.s.d. for the superposition of 88 C
s in domain II
with residues 583-721 in MetE (Fig. 2B) and similar
structural homology with MMCM.
7.9). The
DMB-binding surface of B12 interacts exclusively with residues in
domain I in BtuF, while the catalytic surface interacts exclusively
with domain II (Fig. 3A). In contrast to the type I and II
PBPs and TroA, there is significant solvent exposure of the bound
ligand as also observed in the FhuD complex with gallichrome (33).
Nonetheless, ~820 Å2 of the solvent-accessible surface
area of the vitamin B12 is buried in the complex corresponding to 62%
of the total. Eleven residues in BtuF (shaded pink in Fig.
1C) make direct contacts to the bound vitamin B12 (Figs.
3A and 4B). In addition, six residues (shaded
green in Fig. 1C) mediate water-mediated hydrogen
bonds (H-bonds) to the ligand that are conserved between the two
NCS-related BtuF monomers in the crystal structure (see the green
spheres in Fig. 3A).
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Fig. 3.
Structure of the Vitamin B12 binding
site of BtuF. A, stereo pair of the B12-binding site of
BtuF colored as in Fig. 1A, with conserved waters
represented as green spheres. The side chains of relevant
residues are depicted in ball-and-stick representation colored by
domain with associated nitrogen and oxygen atoms blue and
red, respectively. Direct and water-mediated H-bonds are
represented by red and green dotted
lines, respectively. B, surface representation of the
likely BtuCD-interacting face of BtuF color-ramped according to
sequence conservation, with white indicating no conservation
and burgundy indicating 100% conservation in the five known
BtuFs. C, surface representation of the same face of BtuF
color-ramped according to electrostatic potential, with red
indicating negative potential, blue indicating positive
potential, and fully saturated colors indicating potential ± 5 kT (assuming an ionic strength of 100 mM).
1 forms an H-bond to the propionamide nitrogen of Ring A in
the corrin, while the side chains of Ser-8 and Pro-9 in the same helix make van der Waals contacts to other atoms in the same propionamide group. The side chain of the phylogenetically invariant residue Tyr-28
in strand
2 makes an H-bond to the propionamide oxygen of Ring B,
while the side chain of the phylogenetically invariant residue Trp-63
in strand
4 makes van der Waals contacts to the DMB group. The
carbonyl oxygen of the highly conserved residue Gly-65 following strand
4 contacts the sugar group of the nucleotide.
7, Ser-219 in strand
9, and Asp-220 and
Arg-224 in helix
10). Finally, the side chain of Glu-223 makes van
der Waals contacts to the cyanide group of B12.
E. Coli BtuF refinement statistics
I
/
I
was 3.6 in
the limiting resolution shell, which was 70% complete for all measured
reflections and 49% complete for reflections with I
2
I. For the apo structure, the value of
I
/
I
was 3.8 in the limiting resolution shell,
which was 71% complete for all measured reflections and 43% complete
for reflections with I
2
I. Coordinates and structure
factors for the B12-bound and apo structures have been deposited in the
PDB under accession codes 1N4A and 1N4D, respectively. The refinement
statistics come from CNS, and the Ramachandran analysis was performed
with PROCHECK (39).
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Fig. 4.
Conformational changes associated with
B12 binding. A, stereo pair of the two apo and two
B12-bound molecules aligned by a superposition of their N-terminal
domains. The B12-bound molecules are represented as gray
semitransparent worms. The apo molecules are color-ramped by main chain
B-factors with molecule A going from blue to red
and molecule B from cyan to yellow for B-factors
from 25-110 Å2. B, the lowest panel
shows the average main-chain B-factors, while the upper
panels show the number of atomic contacts made by each residue in
each molecule either to the bound B12 (pink circles) or to
the adjacent protein molecules in the crystal lattice (colored
consistently with the traces in the lowest panel).
6. However, both apo molecules show a rigid body rotation about
residue Pro-105 at the N terminus of the interdomain helix, leading to
a net displacement of both this helix and domain II compared with the
B12-bound structure (Fig. 4A). The magnitude of this
rotation is larger in the "B" molecule (apoB) than the "A"
molecule (apoA), but a similar trajectory of movement is observed in
both. In apoB, the interdomain helix appears to rotate ~10° upward
upon ligand release, causing helix
8 and strand
9 in domain II to
move slightly outward from the B12-binding site. The movement of these
secondary structural elements leads to a modest ~1 Å expansion of
the ligand-binding cavity (Fig. 4A) that is likely to
facilitate ligand exchange. The residue experiencing the largest
displacement is Pro-132 at the C terminus of the interdomain helix,
which moves ~6 Å in apoB. This global conformational change is
consistently observed in both apoBtuF molecules and contrasts with the
local change observed in the apo structure of TroA (7, 8), where the
release of Zn2+ results in an ~1 Å net movement of the
two domains toward one another without any obvious conformational
changes in peripheral regions.
6,
10, and
11) are
those in closest proximity to the interdomain helix, so the entire
domain would appear to pivot around this point of attachment. However,
there also seems to be an increase in the internal mobility of this
domain in the absence of B12 as differences in its equilibrium
conformation are observed in both apo molecules in the regions with
high backbone B-factors (Fig. 4A). The largest
conformational change occurs in the region of BtuF that has the highest
B-factors in the apoA molecule (and was uninterpretable in the apoB
molecule) and involves the unfolding of both termini of helix
9 in
the absence of ligand (Fig. 4B). The relatively remote
location of these conformational changes compared with the
ligand-binding site reinforces the inference that they are coupled to a
general increase in the flexibility/mobility of domain II.
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ACKNOWLEDGEMENTS |
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We thank Anand Saxena and Sal Sclafani of the National Synchrontron Light Source (NSLS) for assistance with data collection and Gerwald Jogl for helpful discussions.
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FOOTNOTES |
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* This work was supported by grants from the March of Dimes and Cystic Fibrosis Foundation (to J. F. H.) and by the National Institutes of Health Protein Structure Initiative Grant to the Northeast Structural Genomics Consortium.
The atomic coordinates and the structure factors (code 1N4A and 1N4D) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Supported by a predoctoral training grant in biophysics from
National Institutes of Health.
§ To whom correspondence should be addressed: Dept. of Biological Sciences, 702A Fairchild Center, MC2434,Columbia University, New York, NY 10027. Tel.: 212-854-2775; Voice mail: 212-854-5236; Fax; hunt{at}sid.bio.columbia.edu.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M212239200
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ABBREVIATIONS |
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The abbreviations used are: ABC, ATP-binding cassette; TM, transmembrane; PBP, periplasmic substrate-binding protein; MBP, maltose-binding protein; Btu, B twelve uptake; apo, apoenzyme; r.m.s.d., root mean square deviation; DMB, dimethylbenzimidazole; PDB, protein data bank; MMCM, methylmalonyl-CoA mutase; NCS, non-crystallographic symmetry.
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REFERENCES |
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