(Received for publication, October 21, 1996, and in revised form, March 20, 1997)
From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 and the § Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011
Two high resolution crystal structures of
cytosolic aspartate aminotransferase from pig heart provide additional
insights into the stereochemical mechanism for ligand-induced
conformational changes in this enzyme. Structures of the homodimeric
native structure and its complex with the substrate analog
2-methylaspartate have been refined, respectively, with 1.74-Å x-ray
diffraction data to an R value of 0.170, and with 1.6-Å
data to an R value of 0.173. In the presence of
2-methylaspartate, one of the subunits (subunit 1) shows a
ligand-induced conformational change that involves a large movement of
the small domain (residues 12-49 and 327-412) to produce a
"closed" conformation. No such transition is observed in the other
subunit (subunit 2), because crystal lattice contacts lock it in an
"open" conformation like that adopted by subunit 1 in the absence
of substrate. By comparing the open and closed forms of cAspAT, we
propose a stereochemical mechanism for the open-to-closed transition
that involves the electrostatic neutralization of two active site
arginine residues by the negative charges of the incoming substrate, a
large change in the backbone (,
) conformational angles of two key
glycine residues, and the entropy-driven burial of a stretch of
hydrophobic residues on the N-terminal helix. The calculated free
energy for the burial of this "hydrophobic plug" appears to be
sufficient to serve as the driving force for domain closure.
It frequently has been observed that an enzyme will undergo a large conformational change to bring catalytic groups into functionally active orientations in response to substrate binding at the active site. This type of ligand-induced conformational change was proposed by Koshland in his "induced fit" model (1) and reflects the flexible nature of large proteins (2). X-ray crystallographic methods have been used to directly reveal ligand-induced structural changes in several enzymes (3-4); e.g. aspartate aminotransferase (see below), hexokinase (5), alcohol dehydrogenase (6), and citrate synthase (7).
Aspartate aminotransferase (AspAT)1 is one of the key enzymes in amino acid metabolism. The enzyme is responsible for the following reversible transamination reaction.
![]() |
![]() |
Pig heart cAspAT is a dimeric enzyme of identical 412-residue subunits
(molecular mass of 92,700 Da). Each subunit consists of a large and a
small domain, and the active site is located at the interface between
the two domains. At the active site, one molecule of the coenzyme
pyridoxal 5-phosphate is covalently linked to the
-amino group of
lysine 258 in the large domain through an aldimine linkage. Two
different conformations for the small domain were characterized
previously from a 2.7-Å resolution structure of native cAspAT and a
3.2-Å difference Fourier map of a cAspAT-substrate analog complex
(21). In the absence of substrates, the two identical subunits are
spatially related by 2-fold symmetry, adopting the so-called "open
conformation." The binding of substrate in the active site of one of
the subunits (referred to as subunit 1) induces the small domain of
that subunit to shift toward the active site, forming the "closed
conformation." In contrast to solution studies, which show that both
subunits are reactive and independent (22, 23), only subunit 1 in
crystalline cAspAT shows a conformational change on binding substrate
because crystal lattice contacts lock the other subunit (subunit 2) in the open conformation (Fig. 1).
Here we present the 1.74-Å structure of cAspAT and the 1.6-Å structure of cAspAT complexed with MeAsp. Analysis of these two structures reveals important elements of the stereochemical mechanism for domain movement in AspAT.
Highly purified cytosolic aspartate aminotransferase from pig heart was prepared as described by Yang and Metzler (24). Prior to crystallization, the native enzyme was dialyzed and concentrated against 40 mM sodium acetate at pH 5.4 by using a collodion bag apparatus (25,000 molecular weight cut-off).
Native cAspAT crystals were grown by vapor diffusion at 4 °C in a solution of 8% PEG 6000 buffered with 40 mM sodium acetate at pH 5.4 (25) and then increased in size with a seeding technique that has been described previously (26). The plate-shaped crystals have orthorhombic space group symmetry (space group P212121 with unit cell dimensions a = 125.0 Å, b = 130.8 Å, c = 55.8 Å), and each asymmetric unit consists of one dimer. This set of crystallization conditions will be referred to as the "standard crystallization solution" to distinguish it from other soaking solutions that change the ionic environment and the structure of the enzyme. Specifically, we have determined that under the standard crystallization conditions two bound acetate anions from the buffer induce the structural transition of the small domain in subunit 1 from the open to the closed conformation (27, 28). However, a concentration of 40 mM acetate is not sufficient to fully saturate the crystalline enzyme, and this results in a disordered image of the small domain of subunit 1. Fortunately, the disorder can be eliminated if crystals of cAspAT are soaked in a solution of 8% PEG 6000 and 40 mM sodium formate at pH 5.4. Analysis of high resolution electron density images shows that under these conditions formate anions do not bind at the active site of subunit 1, and they do not induce the movement of the small domain.2
Crystals of the cAspAT-MeAsp complex were prepared by equilibrating crystals of native cAspAT with a solution composed of 16% PEG 6000, 40 mM sodium acetate (pH 5.4), and 300 mM DL-MeAsp (pH 5.4).
Data CollectionPrior to data collection, the cAspAT crystals were cut to a length of about 1 mm and mounted in quartz capillary tubes. The initial x-ray diffraction data sets for native cAspAT were collected to a resolution of 2.4 Å with an Enraf-Nonius CAD4 diffractometer. Later, a much higher resolution data set was collected on a single crystal of cAspAT using the multiwire area detectors at the University of California at San Diego Resource for Protein Crystallography. A total of 112,786 unique reflections were collected to a resolution of 1.6 Å. At this point, the effect of 40 mM acetate on cAspAT structure was not recognized, and this data set was collected using cAspAT crystals grown under the standard crystallization conditions. Subsequently, high resolution diffraction data were collected on cAspAT crystals soaked in 40 mM sodium formate, eliminating the acetate-induced movement of the small domain in subunit 1 (27, 28). A Rigaku AFC6R diffractometer fitted with a San Diego Multiwire Systems area detector was used to collect this data set. The data set from two cAspAT crystals soaked in 40 mM sodium formate (91,745 reflections out of 444,811 measurements) contains 95.9% of the possible reflections to a resolution of 1.74 Å. The Rsymm value for this data set is 5.4%. All diffraction data were scaled and merged according to the procedure of Howard et al. (29).
The initial x-ray diffraction data sets for the complex with MeAsp were collected to a resolution of 2.5 Å with an Enraf-Nonius CAD4 diffractometer. Later, a 1.6-Å resolution data set was collected on the Rigaku AFC6R diffractometer fitted with a San Diego Multiwire Systems area detector. A total of 114,162 unique reflections (476,759 measurements) were collected on the cAspAT-MeAsp crystals. This data set has an Rsymm of 6.0% and is 94% complete out to a resolution of 1.60 Å.
Refinement of cAspATTable I contains a summary of the final refinement statistics for the native cAspAT structure and the cAspAT-MeAsp complex as reported in the restrained least squares refinement program PROLSQ (30, 31).
|
The 2.7-Å resolution atomic model of Hyde et al. (32) was
used as the starting point for subsequent refinement of the native enzyme. This unrefined model had an R value of 34.2% at
3.5-Å resolution and relatively poor stereochemistry. A global
temperature factor of 30 Å2 initially was applied to all
atoms. In subsequent refinement cycles, individual atomic temperature
factors were refined, and a 2-fold noncrystallographic symmetry
restraint was employed. The resolution was gradually increased during
the refinement process so that all of the data between 20.0- and 2.4-Å
resolution with magnitudes greater than 4 were included. After the
entire model was rebuilt twice using 2Fo
Fc and Fo
Fc "omit" maps (i.e. maps with the
atoms of the section being viewed deleted), the crystallographic
R value decreased to 22.5% at 2.4-Å resolution. At this
stage, the restraint on 2-fold noncrystallographic symmetry was
removed.
The 1.6-Å resolution data next were included in the refinement, and
after several rounds of refinement and manual rebuilding the model
included 408 water molecules and two acetate ions, and the R
value decreased to 17.6% for the 2 data between 8.0- and 1.6-Å
resolution. At this resolution the two acetate ions were observed very
clearly in the active site of subunit 1 (but not in subunit 2) at the
positions of the carboxyl groups of a bound substrate molecule. It is
now clear that acetate can mimic the binding of substrate and induce
the movement of the small domain in subunit 1. Detailed analysis has
shown that under our standard crystallization conditions
(i.e. in 40 mM acetate), both the open and
closed conformations are present at approximately equal occupancy in
subunit 12 (27, 28). Therefore, subsequent refinement was
carried out using the 1.74-Å diffraction data set collected on cAspAT
crystals soaked in 40 mM formate. The last stages of
refinement included removing the two acetate ions in subunit 1 and
rebuilding two alternative conformations of the side chain of
Arg292 in subunit 2 (see below). Water molecules were
retained in the model only if their temperature factors were less than
50 Å2.
The native cAspAT
atomic model at 2.4 Å (see above) was the starting point for refining
the cAspAT-MeAsp structure. This model resulted in an R
value of 26.6%. Several rounds of refinement and model rebuilding
produced an atomic model with good stereochemistry and reduced the
R value to 21.3% at 2.5-Å resolution. Since high resolution (1.8 Å) diffraction data became available first for the
isomorphous enzyme-glutamate complex (28), refinement continued with
this data set before the final stages of the refinement process were
carried out with a 1.6-Å enzyme-MeAsp data set. During this last phase
of refinement, 147 cycles of PROLSQ were run, and the entire model was
inspected and rebuilt three times. The final model for the MeAsp
complex contains 325 water molecules (all with temperature factors less
than 50 Å2) and has an R value of 17.3% for
the diffraction data between 8.0 and 1.6 Å resolution with magnitudes
greater than 2 .
If the atoms of an atomic model are assumed to behave as
isotropic harmonic oscillators, then the crystallographic atom
temperature factor (B) is related to the average
displacement (µ) of an atom by the relationship B = 82µ2. While the isotropic harmonic
oscillator assumption is frequently inaccurate, crystallographic
B values are, nevertheless, an empirical measure of atomic
mobility (4, 33). In the case where groups of atoms move between two
low energy conformations, the temperature factors can also be a measure
of atomic occupancies.
In Fig. 2 the temperature factors of the backbone atoms
of two cAspAT atomic models are compared; one is the result of
refinement against diffraction data collected on crystals equilibrated
with 40 mM acetate at pH 5.4, and the other is the result
of refinement against data collected on crystals equilibrated with 40 mM formate at pH 5.4. In both atomic models the small
domain of each subunit was positioned in the open conformation. The
large differences in temperature factors between the two models are
isolated to the residues of the small domain of subunit 1, and they
reflect the acetate-induced disorder of the small domain of subunit 1. More detailed studies have shown that the open and closed conformations in subunit 1 are present at about equal occupancy in 40 mM
acetate, whereas this equilibrium shifts almost completely to the open conformation in 40 mM formate2 (27, 28). Full
occupancy of the closed conformation in subunit 1 can be achieved with
very high concentrations (~300 mM) of acetate or
dicarboxylate ligands. However, subunit 2 is always locked in the open
conformation due to lattice contacts that inhibit its motion.
Secondary Structure
The secondary structure assignments shown
in Fig. 3 are based on the stereochemical rules defined
by Kabsch and Sander (34) as implemented in the program DSSP. With the
exception of Ser296, the cAspAT and the cAspAT-MeAsp
complex backbone torsion angles (the and
angles of all of the
nonglycine amino acids) fall in the stereochemically allowed region of
the Ramachandran plot as produced by the program PROCHECK (35).
Ser296 is located in the middle of an eight-residue loop
that connects helix 11 and helix 12 (see Fig. 3), and its position is
well defined in the cAspAT electron density map (with individual atomic
temperature factors ranging from 7 to 12 Å2). From a
survey of well refined high resolution protein structures, Herzberg and
Moult (36) found that nonglycine residues that fall in the prohibited
regions of the Ramachandran plot are usually associated with some
aspect of protein function. This is clearly the case for
Ser296, since it is part of the coenzyme binding site.
Specifically, in both subunit 1 and subunit 2 the side chain hydroxyl
group of Ser296 interacts via a water molecule with the
phosphate group of the coenzyme on the opposite subunit. The same
strained conformation has been observed for Ser296 in
chicken mAspAT (16), chicken cAspAT (18), and E. coli AspAT
(20, 37). However, in the case of the mitochondrial isoenzyme, the
authors also listed the following residues as falling in a disallowed
region of the Ramachandran plot: Ser107,
Ser167, Lys183, Tyr263, and
Tyr295. In contrast, all of these residues fall within the
allowed (
,
) regions in pig cAspAT. The only other unusual
residues in the pig cAspAT structure are cis prolines 138 and 195, residues that are located in the loops under the active site.
As with Ser296, the cis conformations of
Pro138 and Pro195 also have been reported in
the high resolution structures of AspAT from different sources (16, 18,
20, 37).
The secondary structures of mAspAT (16), E. coli AspAT (19,
20, 37), and cAspAT differ only in the presence of two additional
two-residue -strands in the cytosolic isoenzyme (A8 and A9 in Fig.
3) and in a one- or two-residue difference in the length of a few
-helices and
-strands. Almost identical elements of secondary
structure, including A8 and A9, were identified in chicken cAspAT (18).
Thus, with 47 and 41% amino acid sequence identity between cAspAT and
mAspAT and between cAspAT and E. coli AspAT, respectively
(38), the enzymes from different sources display essentially the same
secondary structure.
The sieve-fitting procedures described by Gerstein and Chothia (39) and Lesk (40) were used to characterize structural differences between cAspAT and cAspAT-MeAsp complex. Specifically, the backbone atomic coordinates of subunit 1 of the native enzyme and of the cAspAT-MeAsp complex were superimposed with the least-squares method of Kabsch (41) as implemented in the program BMFIT (42). This superposition included all 412 residues and had an overall r.m.s. deviation of 1.24 Å. Of these, 160 backbone atoms (40 residues) of the two structures had deviations of 2.0 Å or more and were not included in the next superposition cycle. Several more iterations of superposition were carried out with progressively smaller threshold values until no backbone atoms showed a deviation of more than 0.4 Å, leaving a total of 1088 backbone atoms in each of the two structures.
This sieve-fitting protocol identified the following residues as structurally isomorphous in the native enzyme and the cAspAT-MeAsp complex: Val5-Gln11, Leu50-Thr139, His143-His193, Thr198-Ala224, Glu234-Asp312, and Glu318-Met326. Thus, the small domain is defined as the N-terminal residues Ala12-Val49 and C-terminal residues Ala327-Gln412, and the large domain as residues Leu50-Met326. The N-terminal peptide, residues Ala1-Gln11, was not assigned to either domain because it extends away from each subunit to interact with the adjacent subunit (see Fig. 1).
Gerstein and Chothia (39) defined the "static core" of lactate dehydrogenase as those "residues that are both part of secondary structure and that remained after sieve-fitting." Using this definition, the static core of the large domain consists of 632 backbone atoms that include the residues of all of the helices except H13 (i.e. residues 51-61, 77-88, 93-96, 107-122, 143-149, 170-179, 202-215, 234-245, 277-292, and 301-311) and all of the strands (residues 100-106, 133-137, 155-159, 161-162, 167-168, 185-189, 218-223, 250-255, and 267-273). The r.m.s. deviation between the backbone atoms of the large domain static core in the open and closed conformations is 0.14 Å, indicating that these residues are in fact motionless with respect to substrate binding within the error limits usually associated with high resolution structures.
After the large domain static cores of subunit 1 of the native enzyme
and the cAspAT-MeAsp complex are superimposed, positional differences
in the small domain of as much as 5 Å are observed (Fig.
4A). While the largest movements occur in
small domain residues, large domain residues Gly227,
Phe228, and Ala229 (which are located in the
domain-domain interface) also show positional changes of as much as 2.5 Å.
When the small domains of subunit 1 in cAspAT and the cAspAT-MeAsp
complex are superimposed, the r.m.s. residual of the backbone atoms is
0.86 Å. When compared with the corresponding residual of 0.29 Å for
the large domain, this indicates that the tertiary structure of the
small domain is clearly altered between the open and closed
conformations. To determine which parts of the small domain change, the
sieve-fitting analysis was carried out using the same criteria that
were applied to the large domain. This allowed a static core for the
small domain to be defined as most of the residues of four out of five
helices (H14, H15, H16, and the second half of H13) and three
-strands (B1, B2, and B4). Since superposition the backbone atoms of
these static core residues results in an r.m.s. deviation of only 0.23 Å, it follows that the residues outside the static core undergo
significant tertiary structural changes as a result of the
open-to-closed transition. In particular, residues in helix 1 (Leu16-Glu26) and the loop residues 36-49 at
the N terminus of the small domain show the largest positional
differences relative to those of the static core residues (see
Fig. 4B).
By forming an
array of noncovalent interactions with the coenzyme-MeAsp adduct,
active site residues Gly38, Lys258,
Arg386, and Arg292 stabilize the external
aldimine of the cAspAT-MeAsp complex (Fig. 5). In
particular, the -carboxyl group of the substrate interacts with both
Gly38 and Arg386. The interaction of
Gly38 is brought about by a large shift in the loop
containing Gly38 that positions the backbone nitrogen atom
of Gly38 within hydrogen bonding distance (about 3.0 Å) of
the
-carboxyl group. In the case of Arg386, its
-carbon shifts by about 1.2 Å toward the active site, and its side
chain shifts by as much as 2.5 Å so that an ionic interaction neutralizes
-carboxyl group. The environment around
Arg386 remains relatively unchanged in both open and closed
conformations due to the coordinated movement of Gly36 and
other small domain residues.
The substrate-Arg292 interaction involves a shift in the
position of the side chain of Arg292 on the neighboring
subunit. In the absence of substrate, two different conformations of
the Arg292 side chain are observed, but only one
conformation exists in the active site of the cAspAT-MeAsp complex,
where a strong (3.0 Å) electrostatic interaction forms between the
-carboxyl group of MeAsp and the Arg292 guanidinium
cation. The strength of this interaction greatly reduces the mobility
of the side chain of Arg292 (its atomic temperature factors
range from 8 to 15 Å2). Unlike Arg386, the
substrate-induced movement of Arg292 does not involve a
change in the position of its backbone atoms. The movement of the
Arg292 guanidinium group by as much as 7.9 Å is
accomplished by changes in two side chain dihedral angles
(
2 = 130°, and
3 = 165°).
Transaldimination from the internal to the external aldimine liberates
the side chain of Lys258. The free -amino group of
Lys258 has been proposed as the base that accepts a proton
from the
-carbon atom of the substrate and as the acid that donates
the proton to the formyl carbon to form the ketimine intermediate (43).
Consistent with this proposal is the observation that the
-amino
group of Lys258 in the MeAsp complex is only 2.9 Å from
the C-4
atom of the external aldimine. Because of the importance of
this interaction, bias in positioning the side chain of
Lys258 was reduced by omitting it in the penultimate stage
of least squares refinement. Then the side chain of Lys258
was carefully fit into a 2Fo
Fc electron density map before its atomic
coordinates and temperature factors were refined in the final series of
least squares refinement cycles.
Although the ligand-induced movement of the small domain is large, many of the interactions between the large and small domains in the MeAsp complex are the same as those of native cAspAT. In particular, the number of hydrogen bonds (14 and 13 for the open and closed forms, respectively, including the direct and water-mediated hydrogen bonds) and the number of van der Waals contacts (16 and 14 for the open and closed forms, respectively) do not change much in the two structures. In addition, little change is seen in the number of water molecules associated with the domain interface (10 in the open conformation versus 11 in the closed conformation), and the buried surface area of the interface remains constant in the two structures. This high degree of structural isomorphism in the domain-domain interface suggests that there is only a small energy barrier between the open and closed conformations, which is consistent with the estimated minimum value of 2-3 kcal/mol estimated by Pfister et al. (44).
Most of the domain-domain interface interactions are formed by residues that are elements of loop structure. Two of these loop regions come from the small domain (residues 48-49 and 356-360), and three come from the large domain (residues 192-199, 225-229, and 258-263). In particular, small domain residues 356-360 and large domain residues 192-199 form an extensive set of interactions in both the open and closed conformations, creating the major framework of the interface. It is also interesting to note that unlike other residues of the large domain, the peptide composed of residues Phe228, Ala229, and Ser230 (residues that are not part of the active site) has two distinct conformations that depend on the ligation state (see Fig. 4A). The structural basis for this ligand-induced movement is unclear at present.
To a first
approximation, domain closure in cAspAT can be described by a rigid
body rotation of the small domain relative to the large domain. This
rigid body movement of the small domain core, expressed as a screw
rotation about a unique axis, was calculated after the large domain
static core residues of subunit 1 in the native enzyme and the MeAsp
complex were superimposed (Fig. 6). The components of
the screw rotation amount to a rotation of 10.3° and a translation of
0.4 Å. The screw rotation axis is almost parallel to the molecular
dyad (it actually makes an angle of 2° with the dyad), and it passes
through the domain-domain interface near Met326 in helix 13 and residues from two turns (residues 357-360 and 193-196). Since the
very long helix 13 bridges between the large and small domains, the
location of the screw axis next to helix 13 allows domain closure to
take place without a large (energetically costly) distortion of the
helix. Instead, the open-to-closed movement of the small domain takes
requires only a small bend in helix 13 at Met326 (Fig.
6).
The ligand-induced movement of the small domain is not a pure rigid body motion, however. Superposition of the open and closed forms of the small domain shows that the movement of the loop from Gly36 to Val49 is not completely linked to the motion of the core residues of the small domain (Fig. 4B). This deviation from pure rigid body motion has its origin in large changes of the conformational angles of two residues, Gly36 and Gly38.
The Importance of Gly36 and Gly38 in Small Domain MovementThere are many examples of ligand-induced domain
closure (e.g. adenylate kinase (45) where the open-to-closed
transition includes significant changes in the backbone ,
angles
of "joint residues" between the mobile domain and the remainder of
the protein (46)). To detect joint residues in cAspAT, the magnitudes
of the changes in backbone torsion angles between the open and closed conformations were calculated as a "distance" in
,
space
(
,
distance = (
2 +
2)1/2) and plotted versus residue
number (Fig. 7). By far the largest changes in
,
angles were observed for two glycine residues, Gly36
(
= 8.4°,
= 94.3°) and Gly38 (
= 44.7°,
= 60.3°). The
,
angles of Gly36
change from the allowed region for nonglycine residues in the Ramachandran plot (
=
95.3°,
=
11.7°) to the forbidden
region for nonglycine residues (
=
86.9°,
=
106°) in
response to ligand binding, while the corresponding large
,
changes for Gly38 take place within the allowed region for
nonglycine residues (
=
125.5°,
= 28.1° to
=
80.8°,
= 88.4°). All other residues, including small domain
residues at the domain-domain interface (i.e. residues
49-50 and 326-327), showed much smaller or no significant changes in
backbone torsion angles. Similar backbone conformational changes in
Gly36 and Gly38 have been observed in E. coli AspAT (37) and mAspAT (47).
These observations and the fact that residues 36 and 38 have been
conserved as glycine in all AspATs sequenced (38) indicate that the
Gly36-Val37-Gly38 peptide is a very
interesting target for mutagenesis experiments. To date, studies have
been carried out with the site-directed mutants G36A, V37A, G38A, and
G38S (48, 49). Relative to wild-type cAspAT, the G38A and G38S
mutations, respectively, decrease kcat from 230 s1 to 11.6 s
1 and 1.44 s
1 and
increase Km for aspartate from 1.85 mM
to 7.9 mM and 50 mM (48). Thus, the catalytic
efficiency of the enzyme (kcat/Km) decreases by a
factor of 0.012 for G38A, and by 0.00023 for G38S, confirming the
predicted requirement for glycine at this site. X-ray diffraction
studies of G38A and G38S (48) showed that these mutations do not
perturb the structure of the unliganded enzyme in the open
conformation, implying that the altered kinetic parameters reflect
impairment of the substrate-induced closure of the small domain and/or
the interactions that stabilize the enzyme-substrate complexes. In
fact, x-ray studies of G38A and G38S crystals soaked in 300 mM MeAsp (28) have shown that the open-to-closed
equilibrium of the small domain is shifted greatly toward the open
conformation relative to the MeAsp complex of the wild-type enzyme
under the same conditions. Furthermore, modeling the G38A-MeAsp and
G38S-MeAsp complexes with the small domain in the closed conformation
indicates that the
-carbon of Ala38 or Ser38
would be only 3.0 Å from the MeAsp 2-methyl group. Thus, the prevention of direct steric conflict with substrate-pyridoxal 5
-phosphate intermediate complexes is very likely to be the reason residue 38 has been conserved as glycine.
In the case of G36A, kcat is decreased 11-fold,
but the Km for aspartate is increased only by a
factor of 1.4. Although crystallographic data are not yet available for
G36A, analysis of the wild-type enzyme (see above) predicts that an
alanine mutation should inhibit transition to the closed conformation
because residue 36 normally assumes ,
angles in the closed
conformation that only are appropriate for glycine. Therefore,
inhibiting the open-to-closed transition in this way has a large impact
on the enzyme's catalytic efficiency.
In contrast to the Gly36 and Gly38 mutations,
the V37A mutation has no measurable effect on substrate turnover as
measured by kcat (48), suggesting that the
stereochemical mechanism for domain closure has not been compromised.
The crystallographic data are consistent with this interpretation of an
unaltered kcat value in that the structure of
the unliganded enzyme in the open conformation is not perturbed (48),
and MeAsp induces closure of the small domain in V37A as it does in the
wild-type enzyme (28). However, the V37A mutation does increase the
Km for aspartate by a factor of 6.81, and the
Kd for MeAsp is increased 4.1-fold (48). This may
reflect an increase in local dielectric constant (Val37 is
3.8 Å from the MeAsp -carboxyl group) that weakens polar and ionic
interactions between the
-carboxyl group of the incoming substrate
molecule and Gly38 and Arg386.
Different values for the magnitude of movement of the small domain have been reported for the various forms of AspAT that have been studied crystallographically. In chicken mAspAT, a ligand-induced rotation of 13.6° was observed (16), while in the case of E. coli AspAT the binding of MeAsp is accompanied by only a 5-6° rotation of the small domain (20, 37, 50). Smith et al. (50) and Okamoto et al. (37) concluded that the basis for this difference is that the unliganded small domain of E. coli AspAT is not opened as much as it is in mAspAT. On the other hand, Malashkevich et al. (18) compared the closed conformations of cAspAT and mAspAT from chicken and concluded that ligand binding induces the small domain of mAspAT to close more fully than it does in cAspAT, with the movement of N-terminal helix 1 showing the biggest difference between the two isoenzymes. McPhalen et al. (47) also noticed that helix 1 in mAspAT moves differently relative to the rest of the small domain residues, and they showed that helix 1 is rotated further by 10° relative to the core of the small domain. Our analysis of pig cAspAT indicates that helix 1 rotates only an additional 3.0° relative to the static core of the small domain. Differences in the movement of helix 1 between isoenzymes may to be due to a sequence difference at position 15 (i.e. Asp15 in mAspAT versus Val15 in cAspAT), a residue in the small domain located just before the helix. In the closed conformation of mAspAT, the side chain carboxyl group of Asp15 maintains an ionic interaction with the guanidinium group of the buried residue Arg292 in the active site. This interaction could stabilize helix 1 of mAspAT in a more closed position.
Coenzyme Movement in AspATDuring transaldimination the
coenzyme undergoes a large conformational change in cAspAT (Fig. 5 and
Table II). Specifically, the and
torsional
angles (as defined in Fig. 8) vary, respectively, from
values of +36° and +41° in the internal aldimine (the average of
three values as described in Table II) to
27° and +68° in the
external aldimine (subunit 1 of the cAspAT-MeAsp structure). The
internal-to-external aldimine transition, however, does not result in
large changes of the
and
angles (Table II). The
rotation
(i.e. the apparent rotation about the C-4
-C-4 bond) is
simply the result of the exchange of the Lys258 N-
-C-4
internal aldimine bond for the new external aldimine MeAsp N-C-4
bond. On the other hand, the change in
reflects a true rotation
about the C-5-C-5
bond. As first predicted by Karpeisky and Ivanov
(11), it is this rotation that shifts the position of the C-5
carbon
of the coenzyme about 2 Å from a noncovalent N ... C-4
distance
in the enzyme-substrate Michaelis complex to the covalent N-C-4
bond
in the external aldimine intermediate.
|
The conformations of the internal and external aldimines as described
above are significantly different from the corresponding coenzyme
conformations described in an earlier report that was based on lower
resolution x-ray studies of cAspAT and the cAspAT-MeAsp complex (21,
51, 52). In particular, an unrefined 2.7-Å electron density image of
the internal aldimine was fit to an atomic model with a angle of
36°, placing the O-5
oxygen "in front of" the pyridine ring
and C-4
(as viewed from the direction shown in Fig. 5), and a 3.5-Å
image of the external aldimine was fit with a
angle of 89°,
placing the O-5
oxygen "behind" C-4
(51). If the
internal-to-external aldimine transition takes place by a simple
rotation about the C-5-C-5
bond as predicted by Karpeisky and Ivanov
(11), then these two conformations require the C-4
-H group to pass
through an intermediate eclipsed position with O-5
. To avoid this
eclipsed conformation, a "phosphate rotor" mechanism was proposed
that involved 120° rotations about the O-5
-P bond (51). However,
since the much higher resolution images of cAspAT and the cAspAT-MeAsp
complex now show that the low resolution analysis of the internal
aldimine conformation was incorrect, there is no need to invoke the
more elaborate phosphate rotor mechanism for the internal-to-external
aldimine transition. The simpler mechanism of Karpeisky and Ivanov (11)
seems more likely in the case of the cytosolic isoenzyme.
This issue is still not completely resolved, however. High resolution
structures of the mAspAT-maleate complex and the mAspAT-MeAsp complex
indicate that in the mitochondrial isoenzyme varies from
16° to
+85° during the internal-to-external aldimine transition, thus
requiring the C-4
-H group to pass through an intermediate eclipsed
position with O-5
if the Karpeisky and Ivanov mechanism is assumed
(13, 20). In the case of E. coli enzyme, one high resolution
(1.8-Å) study indicates that the internal-to-external aldimine
transition involves a
rotation of
16° to +110° (37), whereas
another study at 2.5-Å resolution (under slightly difference crystallization conditions) documents a corresponding
rotation of
+46° to +73° (20). Assuming all of the current, highly refined AspAT structures are reasonably accurate, the diversity of coenzyme conformations implies that a range of conformations are energetically accessible in the native enzyme. If one assumes the Karpeisky and
Ivanov mechanism is valid in all forms of AspAT, then the movement
of the C-4
-H group through an intermediate eclipsed position with
O-5
may not be energetically prohibitive. Alternatively, the phosphate
rotor mechanism may be operative in some cases.
The changes in solvent-accessible surface area between
cAspAT and cAspAT-MeAsp are displayed in Fig. 9.
Although the ligand-induced conformational changes are large, with
movements of more than 5 Å in subunit 1, less than a 2% increase in
buried surface area occurs as a result of the open-to-closed transition
(i.e. a change from 15,300 Å2 in the open
conformation to 15,030 Å2 in the closed conformation).
Most residues show very small, insignificant differences in buried
surface area, with even the mobile surface residues displaying changes
of less than ± 20 Å2 for the most part. As expected
from the above discussion of domain-domain interactions, the
interdomain residues do not show any noticeable differences in buried
surface area, confirming that these residues maintain a closely packed
interface in both the open and closed conformations.
However, the open-to-closed transition does result in some large localized changes in buried surface area for a few residues of subunit 1 as well as for a pair of residues (Tyr70 and Arg292) in the large domain of subunit 2. With the exception of Arg166 in subunit 1, all of these large changes are the result of increases in buried surface area that are coupled to the binding of substrate and the movement of small domain residues over the active site. (Arg166, a surface residue of the large domain, is more exposed to solvent after the open-to-closed conformational change, and its side chain torsion angles change significantly.)
The largest increases in buried surface area occur for N-terminal residues Pro14-Phe18, Arg25-Glu26, and Gly38 of subunit 1 and for Tyr70 and Arg292 on subunit 2 (Fig. 9). Arg25 and Glu26 are surface residues that undergo side chain rearrangements, Gly38 on subunit 1 is buried in the active site as the result of direct interactions with the substrate, and the surfaces of subunit 2 residues Tyr70 and Arg292 are buried by a combination of interactions with both the substrate and the shifted residues of the small domain of subunit 1. Residues Pro14-Val15-Leu16-Val17-Phe18, on the other hand, form a "hydrophobic plug" that could provide the main driving force for domain closure (53, 54). Together these residues have 222 Å2 more accessible surface area in the open conformation than in the closed conformation. This clearly should have a destabilizing influence on the open conformation, since ordered solvent molecules will form around the apolar atoms. On the other hand, without the compensating negative charges of the substrate carboxylic groups, the positively charged arginines 386 and 292 require the open conformation in order to have access to water and counterions in the solvent. Thus, Arg386 and Arg292 are not buried in the open conformation, but this is at the expense of exposing the apolar side chains of the hydrophobic plug residues to solvent. The binding of substrate shifts this equilibrium by electrostatically neutralizing the positively charged side chains of Arg386 and Arg292, allowing them to be buried by the hydrophobic plug as it closes over the active site. Hydrophobic forces are thought to be the dominant forces in protein folding and in protein-ligand binding, and the free energy associated with the burial of hydrophobic atoms has been estimated to be approximately 24 cal/(mol Å2) (55, 56). In the case of domain closure in cAspAT, the calculated free energy for burying the 222 Å2 of the hydrophobic plug amounts to 5.3 kcal/mol. This is more than enough energy to drive the conformational change, which has been estimated to require 2-3 kcal/mol subunit (44). It is important to also note that the apolar character of the hydrophobic plug is well conserved (except for one residue) for the 11 known vertebrate AspAT sequences as well as for E. coli AspAT (38). The sequence of the hydrophobic plug is of the form H-X-H-H-H, where H represents a hydrophobic residue, and X corresponds to Val in cAspAT or Asp in mAspAT and E. coli AspAT. In 4 out of 5 cytosolic enzymes, the sequence Pro14-Val15-Leu16-Val17-Phe18 is found, with alanine replacing Leu16 in the chicken enzyme.
The atomic coordinates (codes 1ajr and 1ajs) and structure factors (codes 1ajrsf and r1ajssf) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.