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J. Biol. Chem., Vol. 277, Issue 18, 15874-15880, May 3, 2002
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From the Departments of
Received for publication, December 21, 2001
Acyl carrier protein (ACP) performs the essential
function of shuttling the intermediates between the enzymes that
constitute the type II fatty acid synthase system. Mycobacterium
tuberculosis is unique in producing extremely long mycolic acids,
and tubercular ACP, AcpM, is also unique in possessing a longer
carboxyl terminus than other ACPs. We determined the solution structure
of AcpM using protein NMR spectroscopy to define the similarities and differences between AcpM and the typical structures. The amino-terminal region of the structure is well defined and consists of four helices arranged in a right-handed bundle held together by interhelical hydrophobic interactions similar to the structures of other bacterial ACPs. The unique carboxyl-terminal extension from helix IV has a
"melted down" feature, and the end of the molecule is a
random coil. A comparison of the apo- and holo-forms of AcpM revealed that the 4'-phosphopantetheine group oscillates between two states; in
one it is bound to a hydrophobic groove on the surface of AcpM, and in another it is solvent-exposed. The similarity between AcpM and
other ACPs reveals the conserved structural motif that is recognized by
all type II enzymes. However, the function of the coil domain extending
from helix IV to the carboxyl terminus remains enigmatic, but its
structural characteristics suggest that it may interact with the very
long chain intermediates in mycolic acid biosynthesis or control
specific protein-protein interactions.
There are two types of fatty acid synthase systems. The type I or
"hard-wired" system is found in metazoans and is carried out by a
multifunctional polypeptide with multiple active sites (1). In
contrast, the type II system found in bacteria and plants consists of a
set of discrete monofunctional proteins, each encoded by a separate
gene (2, 3). ACP1 is central
to both of these pathways because it functions to ferry the pathway
intermediates between active site centers or enzymes. ACPs are also
critical to the function of other metabolic pathways such as polyketide
synthases (4). The type II fatty acid synthase ACPs are abundant,
small, acidic proteins that carry the acyl intermediates attached as
thioesters to the terminus of the 4'-PP prosthetic group (2, 3). This
prosthetic group is added post-translationally to apoACP by
holo-(acyl carrier protein) synthase (AcpS), which transfers the 4'-PP
moiety of CoA to Ser-36 of apoACP. There are now over a hundred highly
similar ACP primary sequences in the molecular data bases and the NMR solution structures of Escherichia coli (Gram-negative) (5, 6) and Bacillus subtilis (Gram-positive) (7) ACPs are known, and there is a crystal structure of ACP bound to AcpS (8). There
is also a NMR structure for the apoACP involved in the actinorhodin polyketide synthase from Streptomyces coelicolor (9), an ACP similar to the type II fatty acid synthase ACPs. These proteins are
very similar and are composed of a four- In Mycobacterium tuberculosis, AcpM is one of the three
proteins that have an ACP signature sequence (12). However, in
Mycobacterium leprae, which has the smallest genome among
the mycobacteria (13), AcpM is the only ACP-like protein. The function
of AcpM in M. tuberculosis type II fatty acid synthase
system is supported by the location of the acpM (Rv2244) in
an operon with other genes that encode pathway enzymes (12), the
identification of mycolic acid precursors bound to AcpM (14), and the
cross-linking of AcpM to the elongation condensing enzyme, KasA (15).
AcpM is highly expressed in E. coli and is isolated as a
mixture of apoAcpM, AcpM, and AcpM acylated by long-chain fatty acids,
primarily palmitate (data not shown) (16, 17). Although AcpM functions
with the type II enzymes in E. coli (16), it is unique
compared with all other bacterial type II ACPs. The protein is similar
to the other ACPs in the amino terminus but contains an extended
carboxyl terminus of about 35 residues for which the structure and
function are unknown. In this report, we define the solution structure of AcpM by protein NMR spectroscopy. The goals are to determine whether
this novel ACP has a core structure similar to the typical bacterial
ACPs, to define the structure of the unique carboxyl-terminal extension
on AcpM, and to gain insight into the dynamics of the 4'-PP prosthetic
group, which the published ACP NMR structures have not addressed.
Protein Expression and Purification--
The acpM
gene was amplified from M. tuberculosis Removal of Acyl Chains from Acylated AcpM--
The purified
protein sample was subjected to mass spectral analysis and determined
to be a mixture of apoAcpM, AcpM, acyl-AcpM (acylated with a variety of
saturated long-chain fatty acids consisting of 14, 16, and 18 carbon
acyl chains), and AcpM dimers. To get a sample containing only apoAcpM
and AcpM for NMR spectroscopy, protein purified by gel filtration
chromatography was treated with 100 mM DTT at 37 °C
overnight to reduce the dimer to monomer AcpM, and to remove acyl
chains, converting acyl-AcpM to AcpM. A precipitate formed during the
reaction because of the formation of acyl-DTT, which is insoluble. The
apo- and holoforms were separated from the precipitate by
centrifugation and dialyzed against 20 mM potassium
phosphate buffer, pH 6.5, at 4 °C overnight.
Conversion of ApoAcpM to HoloAcpM--
E. coli
His-tagged, purified AcpS protein was used to convert apoAcpM to
holoAcpM. The reaction contained 2 mM AcpM (a mixture of
apo- and holoforms), 10 mM CoA, 10 mM DTT, 10 mM MgCl2, and 3.6 µg of E. coli
AcpS in 20 mM potassium phosphate buffer, pH 6.5. After
incubation at 37 °C overnight, the AcpM was repurified.
NMR Samples--
Two types of AcpM samples were generated for
the structural studies. One was 15N-labeled, and another
one was dual labeled with 15N/13C. The
15N/13C-labeled sample was converted to 100%
holoAcpM as described above, and the 15N-labeled sample was
a mixture of apo- (about 40%) and holoforms (about 60%). Because
15N/13C-labeled protein was synthesized using
an 15N/13C enriched medium and unlabeled CoA
was used to convert apoAcpM to AcpM, only about 40% of the bound 4'-PP
groups in the sample were 15N/13C-labeled, and
the rest of them were unlabeled. Protein samples were dialyzed against
40 mM potassium phosphate buffer, pH 6.5, containing 0.1 mM DTT and 0.1% NaN3 at 4 °C. The dialyzed
samples were concentrated to about 2 mM for NMR
spectroscopy studies.
NMR Spectroscopy--
All NMR data were acquired with Varian
Inova 600-MHz spectrometers at 27 °C. Data were processed and
displayed by the program packages NMRpipe and NMRDraw (18) on an SGI
Octane work station. The programs XEASY (19) and CSI (20) were used for
data analysis and semiautomatic assignments. Backbone resonances were
assigned on the basis of three-dimensional HNCA, HNCACB,
CBCA(CO)NH, HNCO, and HNCOCA, and the automated program Assign2 (21)
was used. Side-chain resonances were assigned on the basis of
three-dimensional 15N-edited TOCSY, HCCH-COSY, and
HCCH-TOCSY spectra.
Structural Calculations--
The NOE connections were assigned
on the basis of three-dimensional 15N-edited NOESY and
13C-edited NOESY with the help of four-dimensional
15N/13C NOESY and four-dimensional
13C/13C HMQC-NOESY-HSQC. An automated program,
NOAH (22), was used for the NOE assignment and all the assignments were
manually checked. A total of 2043 meaningful distance constraints were
derived from NMR data. Integrated NOE peaks were calibrated and
converted to distance constraints with the program CALIBA (23). In the
final structural determination, the program DYANA (24) was used, and torsion angle dynamics (TAD) combined with a simulated annealing algorithm was employed in the calculation.
Sequence Uniqueness and Secondary Structure of AcpM--
A
sequence alignment of the ACPs from M. tuberculosis and
M. leprae with typical bacterial ACPs is shown in Fig.
1. The proteins have a high degree of
similarity, especially around the serine residue where the prosthetic
group is attached and extending along helix II. The most notable
difference between AcpM and other ACPs that function in the type II
fatty acid synthase systems is the carboxyl-terminal extension. A
search of current sequence data bases failed to detect any significant
similarity between the unique AcpM carboxyl-terminal extension and
other sequences.
The structure of AcpM from M. tuberculosis was studied by
multidimensional NMR spectroscopy. The resonance assignments were obtained from an array of heteronuclear experiments. The backbone assignments were achieved through conventional strategy by using CBCANH
and HN(CO)CBCA experiments (25-27). The side-chain assignments were
mainly derived from 13C-HCCH-TOCSY and
13C-HCCH-COSY data. The NOE constraints were obtained from
both 15N-edited NOESY and
15N/13C-edited NOESY spectra. A total of 2043 unique NOE distance restraints were identified; a summary of these
constraints is illustrated in Fig. 2.
On the basis of resonance assignments using the program CSI (20) and
the chemical shift values (mainly the C Solution Structure of AcpM--
The structure of the AcpM was
determined based on 2043 NOE distance constraints and 48 hydrogen bond
restraints derived from NMR measurements. In the final calculation, a
total 320 structures were calculated using the program DYANA (24); 20 structures with the lowest target functions were selected and
superimposed (Fig. 3). The structural
statistics are given in Table I. All experimental NMR constraints were well stratified, and there were no
NOE constraint violations of more than 0.35 Å. Although no dihedral
torsion angle constraint was applied in the structural calculation,
most residues have
The structure of AcpM consists of a folded amino-terminal region and a
highly flexible and structurally undefined carboxyl terminus. The four
The four-helix bundle is the central architectural feature of the AcpM,
and this helical bundle is maintained by hydrophobic interactions
between the helices. The helix I contains seven hydrophobic residues.
Ile-12, Ile-15, and Val-19 form hydrophobic contacts with Met-44 and
Ile-47 (contacts with both Ile-15 and Val-19) from helix II, and
residues Ile-8 and Ile-12 are in close contact with Val-74 and Val-73
of helix IV. Helix III, which is the shortest helix, has contacts with
Ala-48 in helix II and Tyr-76 in helix IV through residue Leu-64.
The three loops that connect the four helices in the ACP domain are all
highly ordered. Loop 1, which connects helices I and II, is the
longest, and despite the large number of residues, it is not flexible
and has a defined structure. Several hydrophobic contacts between the
residues within the loop and residues in the helical core keep the loop
in close contact with the helical bundle. Notably, the side chains of
residues Ile-27 and Pro-29 within the loop have contacts with Ile-16
and Ile-9 in helix I. The loop is further stabilized by an intra-loop
salt bridge, Glu-26 and Lys-31. Loop 2 and Loop 3 connect helix III to
the main helical bundle, and residues in both loops have contacts to
the main helical core. For example, Ile-59 in Loop 2 and Leu-67 in Loop
3 form hydrophobic contacts with Ile-77 and Tyr-76 in helix IV,
respectively. Furthermore, Lys-58 forms a salt bridge with Glu-81 in
the carboxyl terminus of helix IV. These interactions function to
reduce the flexibility of the helix III, the shortest helix in the structure.
Comparison of the AcpM with Other ACP Structures--
NMR
structures of the E. coli ACP (29, 30), actinorhodin apoACP
(9) and Bacillus subtilis ACP (7), as well as the crystal
structure of the B. subtilis ACP in the ACP·AcpS
complex (8) have been reported. The secondary structure elements and overall folding of these proteins are very similar. Comparison of the
AcpM solution structure with these ACP structures indicates that the
structured amino-terminal domain of AcpM possesses the common ACP fold.
The superposition of AcpM with each of the known ACP structures over
backbone atoms of the helical core is illustrated in Fig.
4. The structural homology is obvious and
extends to all four helices. Variations in the structures are apparent
in the loop regions, although it is not clear whether any of these
differences could be attributed to the conditions of data collection
and structural refinement. Despite the differences in sequences among
the ACPs (Fig. 1), a detailed examination revealed that the
interhelical hydrophobic interactions, which stabilize the helical
bundle of ACPs, are very similar. For example, in our AcpM structure,
Ala-48 in helix II forms hydrophobic contacts with Val-73 in helix IV. In the three other bacterial ACP structures, the residue corresponding to the AcpM Ala-48 in helix II is valine (Val-43 in E. coli
ACP sequence), whereas the residue in helix IV corresponding to Val-73 is alanine (Ala-68 in E. coli ACP). Therefore, the nature of
the alanine-valine contact is the same in all the ACPs.
The DSL motif (Asp-40, Ser-41, and Leu-42 in the AcpM sequence) is
conserved in all of the ACP sequences, and the 4'-PP prosthetic group
is covalently linked via a phosphodiester bond to the serine residue.
Closer examinations of all four ACP structures revealed that a high
degree of structure homology exists in the region adjacent to the DSL
motif. In all four structures, the DSL sequence is present at the amino
terminus of helix II (Fig. 4). This strong structural homology of the
DSL region in AcpM to other bacterial ACPs provides a rationale for why
AcpS and the enzymes of E. coli type II fatty acid synthesis
use ACPs from other species as well as the species-specific ACP. The
similarities are strongest along helix II, a domain of the protein
referred to as the recognition helix, which is responsible for the
interaction of ACPs with the enzymes of type II fatty acid synthesis
(10).
Structure of the Molten Carboxyl-terminal Domain in
AcpM--
Increased mobility in the carboxyl-terminal segment is clear
from both the relaxation (Fig. 5) and
solvent exchange/accessibility data (data not shown), suggesting that
the carboxyl-terminal domain of AcpM exists as a "molten domain."
The steady-state heteronuclear 15N[1H] NOE
value versus the residue number is shown in Fig. 5. Because the lengths of the N-H bonds are fixed, the
15N[1H] NOE values report information about
the dynamics of N-H bonds and are used to determine whether a
particular amide is in a folded or unfolded region of a protein. The
value of the heteronuclear 15N[1H] NOE for
folded residues is ~0.7-1; the NOE value for a flexible loop is
~0.3-0.5; and the NOE value for the unstructured residues is between
A special term, "natively unfolded," is applied to protein domains
that have little or no ordered structure under physiological conditions
(32-34); many intrinsically unstructured proteins have been identified
and studied (34). Unstructured proteins are inherently flexible, and
their conformations are readily shaped through interactions with other
molecules (32, 35). The intrinsic plasticity of "natively folded"
proteins offers several important advantages in regulatory systems in
which the unstructured protein is induced to interact with a number of
different partners (32). Indeed, the requirement for a folding
transition upon binding of a disordered protein domain to a target
contributes significantly to the specificity of the interaction
(32).
The molten down, unfolded structure of the carboxyl-terminal extension
of AcpM has the hallmark features of a natively folded domain,
suggesting that it plays multiple roles in the function of the AcpM.
The mycobacterial type II fatty acid synthase produces very long chain
mycolic acids that give rise to the unique cell envelope structure of
this group of organisms (36). Thus, one possible function of the unique
AcpM carboxyl-terminal domain is to interact with the variety of
long-chain fatty acid intermediates carried by the protein (15, 37).
More than half the residues in the unstructured AcpM domain are
classified as hydrophobic, and the domain may function to sequester
from solvent the steadily elongating acyl chain bound to the prosthetic
group. In this scenario, the unstructured carboxyl-terminal domain in
AcpM would fold to a different extent to accommodate the spectrum of
long-chain and very long chain acyl intermediates in mycolic acid
biosynthesis. Experimental validation of this idea awaits the
development of technologies that allow the synthesis of very long-chain
acyl-AcpM.
On the other hand, it is also plausible that the carboxyl-terminal
extension may be involved in protein-protein interactions. In the type
II fatty acid synthase, ACP interacts with a variety of functionally
different enzymes (10). The unstructured domain may allow AcpM to
interact specifically with pathway enzymes, perhaps by presenting a
slightly different structure for each acyl intermediate and thereby
directing the cargo to the appropriate pathway enzyme. The
carboxyl-terminal domain may also act as an inhibitor of the
interaction of acyl-AcpM with acyltransferases. It is interesting that
long-chain acyl-AcpM accumulate in E. coli, suggesting that
AcpM is able to productively interact with the enzymes of type II fatty
acid synthesis but is not a substrate for the glycerol-phosphate
acyltransferases involved in the formation of membrane phospholipids.
Thus, the carboxyl terminus may function to prevent the acyl chains
destined for mycolic acids from being diverted to membrane phospholipid
formation. Clearly, further studies of the AcpM structure(s) in the
presence of proteins that it interacts with will shed the light on the
role of the carboxyl-terminal domain mycolic acid synthesis.
Interactions between AcpM and the Prosthetic Group--
The 4'-PP
prosthetic group is covalently attached to the Ser-41 in AcpM. Based on
the observation that no NOE was detected between the 4'-PP and ACP
residues in B. subtilis ACP, it was suggested that the 4'-PP
is readily accessible at the protein surface. Because our
15N/13C-labeled AcpM sample had about 60% of
the 4'-PP molecules unlabeled, we were able to perform a number of
"filtered-edited" experiments (38) to study the interaction
between 4'-PP and protein. Consistent with the observations reported in
previous NMR ACP structures (7), no inter-NOEs between the
prosthetic group and protein itself were detected in our experiments.
Our analysis of the differences in the amide proton and nitrogen
chemical shift of residues in apoAcpM and AcpM revealed a clearer
picture of the prosthetic group dynamics. 15N-HSQC spectrum
of the mixture of apo- and holoforms of 15N labeled AcpM
showed that majority of the residues in the AcpM have same amide proton
and nitrogen chemical shift. However, about a dozen residues had
different amide proton and/or nitrogen chemical shifts in the apo-
compared with the holoform. The differences were small enough so that
we were easily able to assign every residue in the apo form
based on the resonance assignment in the holoprotein. No significant
chemical shift differences were noted between apoAcpM and AcpM,
suggesting that the structures of the two forms are essentially the
same. Nevertheless, the observation of the chemical shift differences
between the two forms shows that the bound 4'-PP group interacts with
the protein.
Using the equation provided by Kurt Wuthrich and co-workers (39) to
combine the chemical shift difference of proton and nitrogen shift
together, we summarized the chemical shift changes between apoAcpM and
AcpM (see Fig. 6). The diameter of the
sausage corresponds to the sum of 1H and 15N
shift perturbation due to the bound 4'-PP group. The chemical shift
perturbations were detected in residues that range from helix I to the
loop that connects helix III and helix IV. No perturbations were
observed for helix IV and the carboxyl-terminal coil. Large perturbations were observed for Phe-33, Asp-40, Ser-41, Leu-42, Thr-51,
Glu-52, Ala-65 and Gly 66. These residues, with the exception of Thr-51
and Glu-52, are structurally close to Ser-41, which is covalently
linked to the 4'-PP group. Thr-51 and Glu-52, located in the carboxyl
terminus of the helix II, were also perturbed, indicating transient
interaction with the prosthetic group. Small perturbations were also
found in the loop region that connects helix II with helix III (Fig.
6). Together, those residues form an extended hydrophobic region around
Ser-41. The chemical shift of a nucleus is sensitive to changes in the
local environment, including aromatic ring-current effects, peptide
bond anisotropy, electrostatic interactions, and hydrogen bonding. When
a ligand binds to a protein, the interactions between them cause
changes in the environment of the nuclei at the interfaces, resulting in chemical shift changes. The analysis of the AcpM chemical shift data
clearly points to the 4'-PP group spending part of the time bound to
the hydrophobic pocket. It is likely that the attached 4'-PP group
experiences a rapid exchange between two states, one that is bound to
the hydrophobic pocket and another that is solvent-exposed. These two
states are consistent with the dual function of ACPs, which need to
both load and unload cargo (acyl intermediates) into the active sites
of the type II enzymes while protecting the cargo from solvent
when shuttling the fatty acyl intermediates between the enzymes of type
II fatty acid synthesis.
We thank Tuan Tran for technical assistance
and Dr. Weixing Zhang for NMR technical support.
*
This work was supported by National Institutes of Health
Grants GM34496, AI49320, and GM61739, Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates of the final 20 structures
(code 1KLP) have been deposited in the Protein Data
Bank, Research Collaboratory for Structural Bioinformatics, Rutgers
University, New Brunswick, NJ (http://www.rcsb.org/)
and the BioMagResBank.
§
These authors contributed equally to this work.
**
To whom correspondence should be addressed: Dept. of Structural
Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Tel.: 901-495-3168; Fax: 901-495-3032; E-mail: jie.zheng@stjude.org.
Published, JBC Papers in Press, February 1, 2002, DOI 10.1074/jbc.M112300200
The abbreviations used are:
ACP, acyl carrier
protein;
AcpM, ACP from M. tuberculosis;
4'-PP, 4'-phosphopantetheine;
AcpS, holo-(ACP) synthase;
NOE, nuclear
Overhauser effect;
NOESY, nuclear Overhauser effect spectroscopy;
HSQC, heteronuclear single quantum coherence;
MOPS, 4-morpholinepropanesulfonic acid;
DTT, dithiothreitol.
The Solution Structure of Acyl Carrier Protein from
Mycobacterium tuberculosis*
§,§,
, and**
Structural Biology and
¶ Infectious Diseases, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105 and the Department of Molecular
Sciences, University of Tennessee, Memphis, Tennessee 38163
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix bundle with the
prosthetic group attached to a conserved Ser-36 located in helix II.
This helix is proposed to be the site for interaction of ACP with
target proteins based on the analysis of mutant proteins (10), and
indeed helix II is the portion of ACP that associates with AcpS in the
crystal structure and becomes distorted to position Ser-36 to accept
the incoming prosthetic group (8). An extended loop connects helices I
and II; helix III is short and was not detected as a structural
element in the earliest NMR structure determinations (5, 9, 11). The
high degree of similarity among these structures is consistent with the
ACPs from one species being used efficiently by the fatty acid synthase
enzymes from another species.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
gt11
genomic library using primer pair 5'-CATATGCCTGTCACTCAGGAAG-3'
and 5'-GGATCCAAGGCTGACTCACTT-3' to introduce a NdeI
site at the initiator methionine and a BamHI site downstream
the stop codon. The PCR product was ligated into pCR2.1 (Invitrogen).
The clone with the correct DNA sequence was digested with
NdeI and BamHI and ligated into the pET11a
vector. The uniform 15N or
15N/13C-labeled AcpM was expressed in E. coli strain BL21 CodonPlus (DE3)-RP growing in MOPS minimal medium
containing 0.1% 15NH4Cl or 0.1%
15NH4Cl and 0.2%
D-[U-13C]glucose. Cell pellet was resuspended
in buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1 mM DTT. Cells were lysed by two
passages through a French pressure cell at 20,000 p.s.i. The AcpM
protein in the cell-free extract was purified using gradient elution
from a DEAE-cellulose column followed by gel filtration on a HiLoad
26/60 Superdex 75 column.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Sequence alignment of ACPs. The
alignment was produced by ClustalW and modified manually according to
the structures. Residues that are conserved in all ACPs are
highlighted. The consensus secondary structure of the ACPs
is shown above the sequence.
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Fig. 2.
Summary of NOE distance constraints obtained
in the NMR experiments. Sequential NOEs are indicated by
thick horizontal bars; the thickness of each bar is in
proportion to the NOE intensity. Thin horizontal bars
indicate long distance NOEs. The vertical bars indicate
helical regions detected by the CSI.
and CO chemical shifts),
four helical regions were clearly identified. This result was
consistent with the distribution of NOEs; within these regions, helical
characteristic medium range, Hi-HNi+3
and Hi-H
i+3, as well as strong sequential
HNi-HNi+1 NOEs were clearly observed. In the
carboxyl-terminal extension, a few helical elements were detected by
CSI. However, other than intra-residence and sequential NOE distance
constraints, few NOEs were found, which indicates that there was no
defined structure in this section of the protein.
and 
dihedral angles in the most favorable or
favorable regions of the Ramachandran plot (Table I). The only
residue that is in the disallowed region is Phe-33, which is located in
the long loop between two helices. The solution structure was of high
precision. The average root mean square deviation from the average
structure for backbone heavy atoms (C', C, N) of the first 90 residues was 0.42 Å. Within the four well folded helical regions, the
average root mean square deviation for backbone heavy atoms was 0.31 Å.
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Fig. 3.
Solution structure of AcpM.
Stereo view of the backbone (N, C , C') of 20 superimposed AcpM
structures (residues 3-96). Helices I-IV are shown in red,
green, orange, and magenta,
respectively.
Statistics for AcpM structure
-helices in the structure of AcpM comprise a "right-turn"
helical bundle (Fig. 1). Its topology is "square," as classified by
Cohen and co-workers (28). In antiparallel helix packing, all four
helices (I-IV) are connected by "underhand" loops. Helix I
(residues Gln-5 to Val-19), II (Asp-40 to Lys-54), and IV (Val-70 to
Glu-81) are of approximately equal length with an up-down-down
topology. Helix III (Asp-61 to Leu-67) is relatively short compared
with the other helices. The length of the loops that connect these four
helices is also variable. The loop between helices I and II is the
longest and contains about 20 residues.
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Fig. 4.
Comparison with other ACP structures.
a, ribbon diagram of the average of the 20 best AcpM structures. The Roman numerals indicate the four
helices in AcpM, and the red arrows point to the location of
the serine residue where the prosthetic group is attached to the
proteins. b, superposition of the AcpM structure
(dark green) with the E. coli ACP structure
(gold) (5, 6). c, superposition of the
AcpM (dark green) structure with the actinorhodin
(polyketide synthase) apoACP structure (magenta) (8).
d, superposition of the AcpM structure (dark
green) with the x-ray ACP structure in the B. subtilis ACP·AcpS binary complex (yellow) (9).
1.0 and 0 (31). Although we did not detect any defined secondary
structural elements in the carboxyl-terminal domain, the structure of
this region remains relatively ordered until Glu-96. Past this point,
the values of 1H[15N] NOEs are less than
zero, indicating a completely melted down random coil. The
dynamic study showed the gradual loss of NOEs in this region, which is
characteristic for an unfolded structure. Nevertheless, the AcpM NOESY
data indicate several close contacts between the carboxyl-terminal
domain and helix I in the amino-terminal ACP fold, mainly in the first
few residues of the sequence. Specifically, we observed NOE contacts
between Thr-4 and Gln-5 in helix I with Gln-89.
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Fig. 5.
Plot of backbone amide heteronuclear
15N[1H] NOE values versus
residue number for the AcpM. The steady-state heteronuclear
15N[1H] NOE value is plotted
versus the residue number. Because the lengths of the N-H
bonds are fixed, the 15N[1H] NOE values
provide information about the dynamics of N-H bonds that can be used
to determine whether a particular amide is in a folded or unfolded
region of a protein. The value of the heteronuclear
15N[1H] NOE for folded residues is 0.7 to 1;
the NOE value for a flexible loop is between 0.3 and 0.5; and the NOE
value for the unstructured residues is between 1.0 and 0. The
carboxyl-terminal region of the protein becomes progressively more
disordered as it approaches the end of the protein.
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Fig. 6.
Interaction between 4'-PP prosthetic group
and AcpM. The figure shows a worm representation of AcpM compared
with apoAcpM. The thickness of the worm is proportional to the sum of
the 1H and 15N shift differences between the
holo- and apoforms of AcpM. The width and color (blue to
red) of the sausage shows the regions of highest chemical
shift differences between the two protein forms and the residues on the
protein that interact with the 4'-PP prosthetic group.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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