From the Center for Advanced Research In
Biotechnology, University of Maryland Biotechnology Institute,
Rockville, Maryland 20850, § The National Institute of
Standards and Technology, Gaithersburg, Maryland 20899,
Advanced Photon Source, Argonne National Laboratory, Argonne,
Illinois 60439, ** Department of Biological, Chemical,
and Physical Science, Illinois Institute of Technology, Chicago,
Illinois 60616, and
Department of Chemistry
and Biochemistry, University of Maryland Baltimore County, Baltimore,
Maryland 21250
Received for publication, December 23, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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D-Tyr-tRNATyr
deacylase is an editing enzyme that removes D-tyrosine and
other D-amino acids from charged tRNAs, thereby preventing incorrect incorporation of D-amino acids into proteins. A
model for the catalytic mechanism of this enzyme is proposed based on the crystal structure of the enzyme from Haemophilus
influenzae determined at a 1.64-Å resolution. Structural
comparison of this dimeric enzyme with the very similar structure of
the enzyme from Escherichia coli together with sequence
analyses indicate that the active site is located in the dimer
interface within a depression that includes an invariant threonine
residue, Thr-80. The active site contains an oxyanion hole formed by
the main chain nitrogen atoms of Thr-80 and Phe-79 and the side chain
amide group of the invariant Gln-78. The Michaelis complex between the
enzyme and D-Tyr-tRNA was modeled assuming a nucleophilic
attack on the carbonyl carbon of D-Tyr by the Thr-80
O Aminoacyl-tRNA synthetases transfer L-amino acids to
tRNA, and the aminoacyl-tRNAs are delivered to the ribosome where
protein synthesis takes place. These enzymes may also utilize
D-amino acids that if incorporated into a polypeptide chain
would impair correct folding. To maintain high fidelity of protein
synthesis, any incorrectly charged tRNA must be removed.
D-Tyr-tRNATyr deacylase hydrolyzes the ester
link between D-Tyr and tRNA (1, 2). Found in many bacteria
as well as in Saccharomyces cerevisiae, Caenorhabditis
elegans, Arabidopsis thaliana, mouse, and human, the
enzyme also acts on D-Asp, D-Trp,
D-Ser, D-Leu, D-Gln,
D-Phe, and D-Gly tRNAs (1-4). Whereas the
enzyme exhibits broad specificity toward D-amino acids, it
is inactive toward L-aminoacylated tRNAs (1) and
N-blocked D-aminoacylated tRNAs (2).
Recently, the crystal structure of
D-Tyr-tRNATyr deacylase from Escherichia
coli has been determined (5), revealing a dimeric structure and a
novel fold. Ferri-Fioni et al. (5) propose that the
active site is formed in a depression at the dimer interface that
contains a cluster of invariant residues including residues 77-81
(SQFTL) of one monomer and Arg-7 and residues 134-140
(NXG(V/F)T) of the second monomer. They also proposed
that conserved positively charged residues at positions 48, 53, 87, and
90 interact with negatively charged phosphate groups on the tRNA.
However, none of the publications so far present a proposal for the
catalytic mechanism.
The D-Tyr-tRNATyr deacylase structure from
Haemophilus influenzae (HI0670 according to the The
Institute for Genomic Research (TIGR) numbering scheme (6))
reported here is similar to that from E. coli. A comparison
of the two structures together with modeling of the
enzyme/D-Tyr-tRNA complex provides the structural basis for
a proposed catalytic mechanism and explains all of the enzyme
specificity data that have been reported.
Protein Production--
The gene encoding HI0670 was amplified
from chromosomal DNA of H. influenzae Rd KW20 and cloned
into the pET17b (Novagen) for expression of the native polypeptide in
E. coli strain BL21(DE3). Cells were grown at
37 °C in LB medium containing 100 µg/ml ampicillin. At mid-log
phase (A600 = 0.8), expression was induced by
the addition of 1 mM
isopropyl-1-thio-
After clarification of the cell lysate by high speed centrifugation,
the protein was purified with the use of a BioCAD 700E chromatography
work station (PerSeptive Biosystems). The purification protocol
consisted of two steps. The first step employed an anion exchange
column (Poros HQ 50) at pH 8.4, and the second step employed a cation
exchange column (Poros HS 20) at pH 8.0. The integrity of the purified
protein was assessed by polyacrylamide gel electrophoresis in the
presence of SDS. The pooled fractions of HI0670 were concentrated (~3
mg/ml) and dialyzed at 4 °C against solution containing 50 mM Tris-HCl, pH 7.5, 0.1 mM dithiothreitol, and
0.1 mM EDTA. Protein crystals suitable for x-ray
diffraction work were formed during dialysis. The molecular weight of
HI0670 was verified by MALDI mass spectrometry (Voyager, PerSeptive
Biosystems) (Mr = 15,861.7 observed and 15,862.3 calculated). Thermal stability was examined with the use of circular
dichroism at 208 nm, revealing a stable protein with a
Tm = 64 °C. The protein exists in solution predominantly in dimeric form as determined by sedimentation
equilibrium measurement.
It is worth noting that our initial attempts to express the HI0670 as a
thrombin-cleavable His-tagged protein resulted in an insoluble product.
With hindsight, the folding of the polypeptide chain was impaired
because the N-terminal amino group is buried and forms essential
contacts with three carboxylate groups.
Structure Determination--
The HI0670 crystals belong to space
group P41212, and the asymmetric unit contains
1 molecule and 55% solvent. For diffraction data collection, crystals
were flash-cooled at 100 K from a solution containing the dialysis
buffer and 20% glycerol. Data were collected using both a home x-ray
facility and the IMCA-CAT 17-ID beamline at the Advanced Photon Source
(Argonne National Laboratory, Argonne, IL). The home CuK
A single crystal was soaked with buffer containing 4 mM
ethyl mercury phosphate for 2 days, and 2.0-Å resolution diffraction data were collected on the home x-ray source. The data were
non-isomorphous with the native data; thus, the phases were determined
using solely the mercury anomalous signal. The statistics of data
collection are shown in Table I.
Computations leading to phase determination were carried out with the
CCP4 suite of computer programs (8). The mercury-derivative anomalous
difference Patterson map revealed a single peak (>8
Following the structure determination, a native data set was collected
to a resolution of 1.64Å on the IMCA-CAT beamline (Table I) and used
for structure refinement. Refinement of the HI0670 structure was
carried out using the CNS program (13) with the data between 20.0 and
1.64 Å for which F Modeling--
A model of the
enzyme/D-Tyr-tRNATyr complex was built on a
Silicon Graphics Octane work station using the computer program
TURBO-FRODO (14). Because no structure for tRNATyr is
available, the structure of yeast tRNAPhe (Protein Data
Bank code 1ehz) (15) was used. H. influenzae tRNATyr contains 85 nucleotides, whereas
tRNAPhe of both yeast and H. influenzae contain
76 nucleotides (www.tigr.org). Thus, structural differences between the
two are expected. Yet, all of the known tRNA structures have a similar
L-shaped structure with an unpaired 3'-terminal CCA
sequence at the acceptor stem. It is the 3'-CCA region that is expected
to undergo conformational changes to fit in the active site of the
deacylase. The D-Tyr-tRNA was built by linking
D-tyrosine to the O3' atom of adenosine 76 (A76). After
manual adjustments to the 3'-CCA-D-Tyr conformation as
described under "Results and Discussion," energy minimization of
the modeled tRNA was carried out using CNS.
HI0670 Structure--
The final model of HI0670 includes all 144 amino acid residues and 207 water molecules. The quality of the
electron density map is shown in Fig. 1,
focused on the region of the proposed active site. For the
mercury-soaked crystal, the data show that the mercury atom is bound to
Cys-133. The N-terminal amino group (Met-1) is buried, interacting with
the carboxyl groups of Glu-34, Asp-37, and the C terminus.
The molecules pack into closely associated dimers around the 2-fold
crystallographic symmetry axis (Fig. 2).
The fold is the same as that of the E. coli
D-Tyr-tRNA deacylase (Protein Data Bank code 1jke) (5).
Each monomer contains a five-stranded mixed twisted
The H. influenzae and E. coli deacylases share a
67% sequence identity, and their polypeptide chains align well with
one another. Excluding one residue that is deleted in H. influenzae HI0670 compared with the E. coli enzyme
(residue 117), the superposition of the C
The positions of some water molecules are also conserved in the
H. influenzae and E. coli enzyme structures. The
superposition of HI0670 and the E. coli AD dimers shows
seven pairs of water molecules within 0.2 Å, 15 pairs within 0.2-0.5
Å, and 25 pairs within 0.5-1.0 Å. For the BC dimer, there are 4, 18, and 23 paired water molecules within the corresponding distances. The
best matched water molecules are either buried or involved in bridging
hydrogen bonds between protein main chain atoms.
Active Site--
As reported earlier (5), multiple sequence
alignment (Fig. 3) and structure analysis
show a cluster of invariant residues located in a surface depression at
a dimer interface region. The dimer contains two such sites ~20 Å apart on the opposite sides of the dimer interface, and these are
probably the active sites of the enzyme. In the following discussion,
residues located on the symmetry-related molecule are labeled by an
asterisk.
Much of the postulated active site depression is lined with
hydrophobic residues including four phenylalanines, Phe-79, Phe-93, Phe-124, and Phe-140* (Fig.
4A). Invariant polar residues
in the active site are Arg-7*, Ser-77, Gln-78, and The-80.
Ferri-Fioni et al. (5) also noted four conserved positively
charged residues, two at the edge of the putative active site
(Arg/Lys-53* and Lys-90) and two at a more remote
location (Arg/Lys-48* and Lys-87, although position
87 is sometimes occupied by a serine or an alanine), which may play a
role in tRNA recognition. Although they identify the location of the
active site, the authors did not suggest which are the catalytic
residues nor did they propose a catalytic mechanism. Structural
considerations, modeling, and inferences from the mechanisms of other
hydrolytic enzymes enabled us to develop a proposal for the catalytic
mechanism as described below.
Proposed Michaelis Complex and Catalytic
Mechanism--
Enzyme-catalyzed hydrolysis often involves a general
base mechanism with an enzyme group mounting a nucleophilic attack on a
carbonyl carbon atom of the substrate and a group that serves as a
proton park, enabling shuttling of protons from and to the nucleophilic
group during catalysis. This is a two-step reaction with an acyl enzyme
intermediate and two tetrahedral transition states, one en route to the
acyl enzyme intermediate and the second along the deacylation step when
the hydrolytic water molecule attacks. We propose that
D-Tyr-tRNATyr deacylase utilizes such a general
base mechanism. Two invariant residues in the active site need to be
considered as potential nucleophilic groups, Ser-77 and Thr-80. Ser-77
is rather buried, whereas Thr-80 is accessible to solvent. Moreover, a
striking oxyanion hole is located adjacent to Thr-80 (but not adjacent to Ser-77) formed by the side chain amide group of the invariant Gln-78
and the main chain nitrogen atoms of Phe79 and Thr80 (Fig. 4A). The presence of an oxyanion hole is reminiscent of the
catalytic machinery of other hydrolytic enzymes such as the serine and
thiol proteases and the serine
With these considerations in mind, the Michaelis complex between HI0670
and the aminoacyl-tRNA was modeled following the hypothesis that Thr-80
is the nucleophilic group. Note that in the E. coli enzyme
structure, the oxyanion hole of molecule A is shielded from solvent by
Phe-79 side chain adopting a conformation different from that seen in
molecule D or in the structure of HI0670. We propose that the active
conformation is the one exposing the oxyanion hole to solvent.
To enable nucleophilic attack, the
The key interactions in the active site are shown in Fig.
4B. The base-stacking interactions of the C74, C75, and A76
seen in the crystal structure of free tRNA are eliminated. Instead, a
cluster of edge-to-face interactions is formed between the bases and
the active site phenylalanine residues. The aromatic ring of the
D-Tyr is also part of this cluster, stacking parallel to A76 and perpendicular to the benzene ring of Phe-79.
An ion pair interaction is formed between the invariant
Arg7* and the A76 phosphodiester group. The O2'
hydroxyl of ribose 76 forms a hydrogen bond with the carbonyl oxygen of
Pro-137*. Most strikingly, the amino nitrogen of
D-Tyr is buried in an environment that is largely
hydrophobic (Phe-93, Phe-140*,Val-138*, and Met-62*) with the exception
of its interactions with the O2 of the C75 base and with
the Thr-80 O
The model of the complex reveals that the basis for discrimination in
favor of D-amino acids is space exclusion, i.e.
L-amino acids would clash badly with protein groups. On the
other hand, any D-amino acid may be accommodated because
the side chain projects toward solvent. The model explains another
experimental observation, that is the enzyme is inactive toward
N-acetyl blocked D-aminoacylated tRNAs (2)
because the amino group is buried in a crowded environment and
additional atoms would not fit in the space.
The model indicates that catalysis is substrate-assisted (Fig.
6). The hydroxyl group of Thr-80 mounts a
nucleophilic attack on the D-Tyr carbonyl carbon atom. The
amino nitrogen atom of D-Tyr is deprotonated and is located
in an appropriate position to accept a proton from Thr-80, thus serving
as a general base. A negatively charged tetrahedral transition state is
formed at the D-Tyr carbonyl carbon stabilized by the
oxyanion hole. The D-Tyr-tRNA ester bond breaks, yielding
an acyl enzyme. The tRNA molecule diffuses out of the active site,
whereas the D-Tyr remains bound to the enzyme. A water
molecule next attacks the acyl enzyme, replacing the departing tRNA
ribose 76 O3' atom, and the amino nitrogen atom of
D-Tyr serves again as the base that accepts a proton from
the activated water molecule. The ester bond between the enzyme and
D-Tyr is cleaved, and free enzyme is generated for the next
catalytic cycle. We note that substrate-assisted catalysis has been
reported previously (19), and deprotonated amino groups have been
observed in proteins when shielded from solvent (20).
The role of the invariant Ser-77 remains unclear. In both the H. influenzae and E. coli enzymes, Ser-77 is
hydrogen-bonded to a water molecule (Wat265 and Wat1115 in the Protein
Data Bank entries 1j7g and 1jke, respectively), which in turn is
hydrogen-bonded to a second water molecule (Wat223 and Wat1384 in 1j7g
and 1jke, respectively). Wat265 is also hydrogen-bonded to the main
chain nitrogen of Ala-125, a residue on a type II reverse turn located
on the edge of the active site depression. According to our model,
neither of the water molecules needs to be displaced when the substrate
binds and Wat223 is located ~5.5 Å from the modeled
D-Tyr carbonyl carbon atom. It is tempting to speculate that Wat223 may serve as the hydrolytic water molecule and that Ser-77
hydroxyl group together with the main chain amide of Ala-125 and Wat265
help maintain the polar environment despite being somewhat shielded
from the bulk solvent by the side chains of Phe-79 and Phe-124.
Detailed structural analyses and modeling provide the basis for
the proposed catalytic mechanism of an important enzyme that helps
maintain the fidelity of protein synthesis,
D-Tyr-tRNATyr deacylase. The modeled
enzyme-tRNA complex is consistent with all of the available enzyme
specificity data. The proposed catalytic pathway follows the general
base mechanism seen in other hydrolytic enzymes. In the deacylase,
Thr-80 hydroxyl serves as the nucleophilic group, there is an oxyanion
hole to stabilize the negatively charged tetrahedral transition state,
and the reaction is substrate-assisted by the amino group of
D-Tyr, providing a "proton park" for the Thr-80
hydroxyl group. The enzyme-tRNA complex model and the proposed catalytic mechanism could not be developed without an accurate atomic
structure of the enzyme. Moreover, the model suggests future experiments that should be carried out to elucidate the mechanism experimentally. Finally, the crystal structure of the enzyme-tRNA complex will reveal the exact interactions in the active site and
validate or refute the proposed mechanism.
atom and a role for the oxyanion hole in stabilizing
the negatively charged tetrahedral transition states. The model is
consistent with all of the available data on substrate specificity.
Based on this model, we propose a substrate-assisted
acylation/deacylation-catalytic mechanism in which the amino group of
the D-Tyr is deprotonated and serves as the general base.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
-D-galactopyranoside and growth
continued for 4 h. Harvested cells were suspended in 20 mM Tris-HCl, pH 8.4, and lysed with three passes at
8,000-10,000 p.s.i. in a French press.
x-ray source was a Siemens rotating anode equipped with MAR345
image plate. The IMCA-CAT beamline was equipped with a MAR CCD
detector. Data processing was performed using the HKL program suite
(7).
Data collection and phasing statistics
). The computer
program MLPHARE (9) was used to refine the position and occupancy of
the heavy atom and to calculate phases. The phases were further
improved by solvent-flattening using the computer program DM (10). The
resulting electron density map was excellent, showing the molecular
boundaries, unambiguous secondary structural elements, and clear
density for the side chains. Most of the 144-residue polypeptide chain
(93%) was traced automatically using the computer program Arp/Warp
(11). Building of the remaining residues and adjustments to the model
were carried out with the interactive computer graphics program O (12)
on a Silicon Graphics Octane work station.
2
(F) (Table
II). In the final stages of the
refinement, water molecules were added to the model based on the
Fo
Fc difference electron
density map (where Fo and Fc are
the observed and calculated structure factors, respectively) using peaks with a density of
3
as the acceptance criteria.
Refinement statistics
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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Fig. 1.
Stereoscopic representation of the
final electron density maps at a 1.64-Å resolution together with the
model. The active site region is shown. The coefficients
(2Fo Fc) and
calculated phases are used. The map is contoured at a 1
level.
-sheet and a
three-stranded anti-parallel
-sheet. The dimer packing results in
the three-stranded
-sheets of two monomers forming a continuous
six-stranded anti-parallel
-sheet, and the pair of five-stranded
-sheets forms a
-sandwich with a central axis roughly
perpendicular to the six-stranded
-sheet. Two parallel
-helices
cover each of the five-stranded
-sheets. A large loop (residues
83-94) extends from each monomer to interact with the neighboring
molecule. Although Ferri-Fioni et al. (5) list structural
similarities between portions of the molecule and segments of other
proteins (5), we note that these correspond to small common motifs and
are not extensive enough to imply evolutionary relationship. Thus,
based on Dali (16), the fold is unique to D-Tyr-tRNA
deacylase.
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Fig. 2.
Crystal structure of HI0670.
A, ribbon diagram of a dimer with each monomer shown in a
different color. B, stereoscopic representation of the C
trace. Every 20th residue is labeled for one monomer. The figure was
generated with the computer programs MOLSCRIPT (21) and RASTER3D (22,
23).
atoms of the monomers
results in root mean square
(r.m.s.)1 deviation of 0.6 Å, and for the respective dimers (using dimer AD of the E. coli enzyme structure), the r.m.s. deviation is 0.5 Å. When dimer
BC of the E. coli deacylase structure is compared, the
r.m.s. deviation values for monomers and dimers are 0.7 and 0.6 Å, respectively.
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Fig. 3.
Multiple sequence alignment of
D-Tyr-tRNATyr deacylase. At the time of
writing, the first iteration of PSI-BLAST (24) using the NCBI
non-redundant data base generates 57 homologous sequences. For brevity,
only 33 sequences are shown. The alignment was constructed using
ClustalW (25). The secondary structural elements of HI0670 are shown as
arrows ( -strands) and cylinders (
-helices). Invariant
residues are shaded dark gray, and conservatively replaced
residues are shaded light gray. The proposed catalytic
residue is Thr-80.
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Fig. 4.
Proposed active site of HI0670.
A, spectroscopic representation of the residues forming the
active site depression. Coloring scheme is as follows: oxygen atoms are
shown in black; nitrogen atoms are shown in gray;
and carbon atoms are shown in white. B,
spectroscopic representation of a modeled D-Tyr-tRNA
segment docked in the active site. The Thr-80 O atom is
oriented appropriately for nucleophilic attack on the carbonyl carbon
of D-Tyr, and the carbonyl oxygen of D-Tyr is
located in an oxyanion hole formed by the main chain nitrogen atoms of
Phe-79 and Thr-80 and the side chain amide of Gln-78. These
interactions, the two interactions of the amino group of
D-Tyr (with Thr-80 O
atom and the
O2 of C75), and interactions between phosphate groups and
positively charged protein residues are shown in broken
lines. Coloring is as in noted in A, and in addition,
phosphodiester phosphorus atoms are shown in gray. Wat223 is
the water molecule closest to the carbonyl carbon of D-Tyr,
which may perform the hydrolytic function. L-Tyr cannot be
docked in this manner because its side chain would replace the C
proton and clash with several residues, in particular with the main
chain of Thr-80.
-lactamases (17, 18). Similarly to
Gln-78 of the deacylase, a glutamine or an asparagine amide group
participates in the formation of the oxyanion hole in papain and
subtilisin, respectively. The oxyanion hole plays a role along the
reaction pathway by stabilizing the negatively charged transition states.
1 dihedral angle of
Thr-80 was rotated by 120° so that the O
atom is
accessible to solvent. The tRNA molecule (consisting of the
76-nucleotide-long tRNAPhe) linked to D-Tyr via
an ester bond on the O3' of ribose 76 was manually positioned so that
the carbonyl group of the D-Tyr faced the Thr-80
O
atom and the oxygen atom was accommodated in
the oxyanion hole. The dihedral angles of the 3'-terminal segment,
C74-C75-A76-D-Tyr, were modified to avoid clashes and to
form favorable electrostatic and hydrophobic interactions. A tRNA
orientation that permits potential interactions between phosphate
groups and the conserved positively charged residues at positions
48*, 53*, 87, and 90 were selected (Fig.
5). Modeling was followed by energy
minimization, moving only the tRNA 71-76-D-Tyr segment and
imposing harmonic restraint on the distance between the Thr-80 O
atom and the D-Tyr carbonyl carbon atom
(modeled somewhat arbitrarily at 2.5 Å) to prevent repulsion because
of the short contact expected to precede nucleophilic attack.
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Fig. 5.
A model of the complex between HI0670 and
D-Tyr-tRNA. The molecular surfaces of the two monomers
are shown in blue and yellow colors, and the tRNA
molecule is shown as a green coil. The D-Tyr and
four positively charged residues that interact with tRNA phosphate
groups outside of the active site are shown as stick models.
The molecular surface of the protein dimer was calculated with the
computer program MCMS (26). The figure was generated with the computer
program RASTER3D (22, 23). Note that the overall structure of the tRNA
is that of tRNAPhe, which is expected to be somewhat
different from the structure of tRNATyr. Thus, this is a
rough model intended to demonstrate the feasibility of the proposed
complex and to develop the proposed catalytic mechanism.
atom. The position and orientation of
D-Tyr amino group suggest that it binds in a deprotonated
state by either dissipating a proton to solvent or donating it to the
O2 atom of C75. Therefore, D-Tyr amino group
may serve as a general base, accepting a proton from the catalytic
Thr-80 O
atom.
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Fig. 6.
A scheme of the proposed catalytic mechanism
of D-Tyr-tRNATyr deacylase deduced by docking a
model of D-Tyr-tRNA in the proposed enzyme active
site. Note that because the scheme is two-dimensional, the
orientations and interactions of the D-Tyr side chain and
of A76 are not depicted properly.
CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank John Moult and Eugene Melamud for the use and help with their bioinformatics web site (s2f.carb.nist.gov). We thank the staff of Industrial Macromolecular Crystallographic Association-Collaborative Access Team (IMCA-CAT) at the Advanced Photon Source for help during data collection. The IMCA-CAT facility is supported by the companies of the Industrial Macromolecular Crystallographic Association through a contract with Illinois Institute of Technology. The use of the Advanced Photon Source was supported by the U. S. Department of Energy, Basic Energy Sciences, Office of Science under contract W-31-109-Eng-38. The Keck foundation provided generous support for the purchase of x-ray equipment at Center for Advanced Research in Biotechnology.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant PO1 GM57890.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Dept. of Physical Science, Eastern Connecticut State University, Willimantic, CT 06226.
§§ To whom correspondence should be addressed. Tel.: 301-738-6245; Fax: 301-738-6255; E-mail: osnat@carb.nist.gov.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M213150200
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ABBREVIATIONS |
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The abbreviation used is: r.m.s., root mean square.
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REFERENCES |
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