From the University of Texas, Southwestern Medical
Center at Dallas, Texas 75390, the ¶ Génétique
Moléculaire et Cellulaire, CNRS-URA 1925, INRA-UMR216, INAP-G,
F-78850 Thiverval-Grignon, France, and the
** Max-Planck-Institut für Biochemie, Am Klopferspitz
18a, D-82152 Martinsried, Germany
Received for publication, December 11, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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It is generally assumed that in proteins
hydrophobic residues are not favorable at solvent-exposed sites, and
that amino acid substitutions on the surface have little effect on
protein thermostability. Contrary to these assumptions, we have
identified hyperthermostable variants of Bacillus
licheniformis Some general rules for increasing the stability of proteins have
been derived from a large number of comparative structural and
mutagenesis studies (1, 2). Among the most generally recognized
strategies for protein thermostabilization are: increasing the
hydrophobic packing in the interior, decreasing surface hydrophobicity, extending networks of salt-bridges and hydrogen bonds, engineering disulfide bonds or metal binding sites, shortening or strengthening solvent-exposed loops and termini, increasing the extent of secondary structure formation, and replacing residues responsible for
irreversible chemical alterations of the protein structure. Yet, these
general rules may not always be applied successfully and examples of
engineered mutations resulting in effects opposite to the ones expected
are legions. Such studies have also failed to reveal outstanding
features associated with the adaptation of proteins to a given
temperature range, i.e. psychrophilicity, mesophilicity,
thermophilicity, and hyperthermophilicity. While most natural proteins
seem to achieve their respective stability by accumulating a large
number of weakly stabilizing interactions that result in a large net effect, some have acquired specialized structural features that cannot
easily be transferred in a general way into other proteins (3, 4).
Elucidating the origin of thermal stability for a given protein and
finding ways to increase it remains a specific and challenging task.
Highly thermostable Bacillus licheniformis When subjected to high temperatures, BLA unfolds irreversibly and
precipitates (11, 12). The derivation of comprehensive and accurate
thermodynamic parameters for the folding/unfolding process of BLA is
therefore not straightforward. Nevertheless, a wealth of information
has been acquired in various laboratories providing insights into the
origin of BLA stability (11-16). Several hundred BLA variants have
been constructed and characterized and have led to the identification
of protein regions and residues that are important for thermostability
(13, 17-20). BLA is also one of the few proteins for which detailed
biochemical studies on the mechanism of irreversible thermal
inactivation have been carried out. It is suspected that the main cause
of irreversible BLA inactivation at high temperature is the deamidation
of Asn/Gln residues (11), although the particular residues involved
have not been identified.
BLA is a secreted metallo-enzyme that requires the presence of calcium
for structural integrity and enzymatic competence, but the metal ions
are not directly involved in catalysis. The wild-type BLA species that
could readily be crystallized was depleted of the intrinsic metal ions
and proteolytically nicked (14). The first detailed views of the
structure of BLA were available for this particular inactive form (14,
21). This model served as a basis for a broad mutagenesis study (19).
In the course of this work, we discovered that variants that included
the mutations Q264S and N265Y gave crystals of the native, uncleaved
enzyme in the presence of calcium and reported the crystal structure of
such a variant solved to a resolution of 1.9 Å (22). This structure
revealed the presence of a calcium-sodium-calcium metal triad instead
of the single conserved calcium ion that is usually found at the
interface between domains A and B. The metal triad is a unique feature
among metallo-proteins in general, and is only present in BLA and its
closest relatives from B. amyloliquefaciens (23) and
B. stearothermophilus (24). BLA provides a rare example of a
secretory metalloprotein for which crystal structures of both the
metal-containing form and the metal-free form are available. A
comparison between these two structures shows that metal loss causes a
number of drastic structural changes in the vicinity of the active
site, although more than 95% of the structure is unaffected.
Here we describe the crystal structure of a BLA variant containing five
mutations that increase the kinetic stability of BLA. The effects of
the single mutations are cumulative and result in a protein that
unfolds 32 times more slowly under conditions where the wild-type
enzyme is rapidly unfolded. Four of five mutations involve the
incorporation of seemingly unfavorable hydrophobic residues at
solvent-exposed sites. Structural analysis indicates how the mutations
exert their stabilizing effect. The structural integrity of
cooperatively formed substructures, especially the central ternary
Ca-Na-Ca metal binding site, proves particularly crucial for the
overall kinetic stability of BLA.
Unfolding and Inactivation Kinetics--
BLA variants containing
various combinations of the mutations, H133V, N190F, A209V, Q264S, and
N265Y were constructed as described (18). The thermally induced
irreversible unfolding of BLA was followed by circular dichroism (CD)
spectroscopy using a Jasco J-715 CD-spectrometer equipped with a
peltier thermostatting cuvette holder. Samples were incubated at
85 °C in 50 mM sodium acetate, 0.1 mM
CaCl2, pH 5.6 and the CD signal recorded at 222 nm for up
to 800 min. The concentration of the samples was routinely around 0.1 mg/ml with a pathlength of 0.1 cm. To investigate the influence of
intermolecular interactions on the unfolding behavior, the
concentration of wild-type BLA was varied over about three orders of
magnitude (0.85 mg/ml with 0.1-cm pathlength, 0.1 mg with 0.1-cm
pathlength, and 0.02 mg/ml with 0.2-cm pathlength).
Pseudomelting curves were recorded by heating BLA with a rate of
1°/min from 20 to 95 °C and following the CD signal at 222 nm.
Because of the extreme thermostability of BLA, it was technically not
possible to record the entire unfolding curve. In order to obtain an
estimate of the relative stabilities for the mutants, we determined the
temperature where unfolding started. After normalizing the raw data for
sample concentration, this point was derived by calculating the
intersection between the CD baseline at low temperatures (between 20 and 60 °C) and a line drawn through the linear part of the
transition region.
Crystallization--
The BLA variant containing all five
stabilizing mutations was crystallized by vapor diffusion from drops
containing 4 µl of protein solution (10 mg/ml in 50 mM
Tris/HCl, pH 8.0) plus 4 µl of reservoir solution (50 mM
Hepes, 1 M ammonium sulfate, 1% (v/v) polyethylene glycol
(PEG) 500, pH 7.0) equilibrated against 1 ml of reservoir solution at
20 °C. Hexagonal bipyramids appeared after 3-10 days and grew to a
final size of 0.5 mm in length within 1-3 weeks. The crystals show the
symmetry of space group P61, with cell constants of
a = b = 91.3 Å, c = 137.7 Å, and contain one
molecule in the asymmetric unit.
X-ray Data Collection, Structure Solution, and
Refinement--
Diffraction data of up to 1.7 Å Bragg spacing were
collected on a MAR-research image plate system using CuK
The starting point for refinement was a crystal structure of another
BLA variant, which had previously been solved by Patterson search
techniques using the cleaved form of BLA (22). All refinement steps
were carried out with the program CNS version 1.0 (26). In order to
minimize model bias, ten residues around the individual mutation sites
were removed from the starting structure, which was then subjected to
simulated annealing at a starting temperature of 3000 K, followed by
cycles of energy-restrained positional refinement of the coordinates,
calculation of individual B factors, as well as visual inspection and
manual correction of the model using the program O (27). The missing
residues were built at the full resolution, and water molecules added
where stereochemically reasonable. Refinement statistics are
listed in Table II.
Selection of BLA Mutants--
For the present study, we have
selected five mutations that we had previously identified as retarding
the irreversible thermal inactivation of BLA. The mutation H133V was
identified while probing residues that are not conserved between BLA
and the highly homologous but much less stable Thermal Inactivation Behavior of the BLA Variants--
The
unfolding kinetics of wild type BLA and BLA variants at 85 °C was
followed by recording the CD signal at 222 nm (Fig. 1A). Highly destabilizing
conditions (low pH and calcium concentration) were used in order to
reduce the incubation time required for accurate measurements.
Experiments with different sample concentrations show that BLA
unfolding under these conditions is a monomolecular process (11).
Overall, the kinetics of BLA unfolding can be modeled with high
accuracy with a single exponential term as shown in Equation 1,
The half-life for the thermally induced denaturation of wild-type BLA
is about 14 min. The five mutations described above all prolong the
lifetime of BLA at 85 °C to a various extent (Table I). The stabilizing effects are
cumulative with the BLA variant that contains all five mutations
exhibiting a half-life of 447 min, which is 32 times longer than the
wild type.
The investigated variants not only unfold more slowly at 85 °C, but
the onset of unfolding is also shifted toward higher temperatures (Fig.
1B). From pseudomelting curves it can be estimated that the
most stabilized BLA variant has a melting temperature that is about
13 °C higher than that observed for wild type BLA (Table I).
Structure Determination--
We have determined the crystal
structure of the BLA variant containing the five stabilizing mutations
mentioned above to a resolution of 1.71 Å. Data collection and
refinement statistics are shown in Table
II. A representative part of the electron
density is shown in Fig. 2A.
The overall topology of BLA, shown in Fig. 2B,
is typical for
In the following, we present a structural interpretation of the
stabilizing effects exerted by the mutations. A general overview of the
location of these mutations is given in Fig. 2.
Position 133--
Residue 133 is located at the surface of domain
B where it is the first residue of Position 190--
Residue 190 lies at one end of the central cleft
between domains A and B with the side chain fully solvent-exposed. It
is located in a long loop region that undergoes a disorder-order transition upon metal binding (Fig. 2). The structure of native BLA
harboring an asparagine at this position is not known, but the
mutational profile provides valuable information on possible thermostability determinants (19). Introducing large and mainly hydrophobic residues increases the stability of BLA by a factor of up
to four. Surprisingly though, tyrosine has no effect. On the other
hand, introducing small side chains, e.g. alanine, reduces the stability by a factor of up to four. Introducing a negatively charged residue in the form of a glutamate at position 190 is similarly
destabilizing, suggesting that a potential deamidation of Asn-190 is
deleterious to BLA stability.
Overall, the preference for hydrophobic residues indicates increased
hydrophobic packing with underlying aromatic residues. Indeed, the
mutation of Asn-190 to phenylalanine creates a triple aromatic
interaction where the ring system of Tyr-193 is oriented perpendicular
to the neighboring rings of Phe-190 and His-235 (Fig. 4). This
arrangement allows a favorable interaction of the partially positively
charged rims of Phe-190 and His-235 with the Position 209--
Residue 209 is located close to the N terminus
of
As previously observed (18), the introduction of small hydrophobic
residues in place of Ala-209 increases the half-life for the thermal
inactivation of BLA by a factor of up to three. In the absence of
structural information, it was argued that a favorable residue should
contribute to the hydrophobic packing at the bottom of the indentation
and should not disturb the pronounced water structure at the top (15).
For the mutant A209V, the first premise is true, but the water
structure is considerably diminished as indicated by the missing
central water molecule and significantly higher B factors for the rest
of the water molecules.
Positions 264 and 265--
Residues 264 and 265 have historically
been treated as a pair in mutational studies of BLA. They are located
on the surface of the interface between domains A and B (Fig. 2). In
wild-type BLA, residue 264 is a glutamine whose side chain is able to
form hydrogen bonds with the side chain of Glu-189 in domain B. A
possible deamidation of Gln-264 at high temperatures could place the
resulting negatively charged side chain in an electrostatically
unfavorable environment because of its close proximity to Glu-189 and
Asp-266 (Fig. 6). Yet, as for Asn-190,
there is no experimental proof that the removal of the amide side chain
at this position reduces BLA deamidation.
Residue 265 (asparagine in wild-type BLA) shields a hydrophobic region
composed of the underlying Pro-287 and Tyr-290. The crystal structure
of the BLA variant containing a mutation of asparagine to tyrosine at
this position shows that the tyrosine side chain participates in a
complex network of aromatic interactions with Tyr-290, Trp-263, and
His-289 (Fig. 6).
Surface Hydrophobicity and Protein Stability--
It is generally
assumed that amino acid substitutions on the surface do not affect the
stability of proteins by large amounts. This is because most surface
residues are involved in only a few often transient interactions. Yet,
this general rule suffers exceptions as exemplified here and in other
studies. Our engineering work on BLA (10) demonstrates that a few point
mutations at the surface are sufficient to drastically increase or
decrease the natural high resistance of this enzyme toward thermal
inactivation. Likewise, in some cases, it was shown that as few as one
or two solvent-exposed residues confer substantial thermostability to
natural or artificially engineered proteins (28-30).
Four of the stabilizing mutations examined in the present study replace
amino acids that are either small and hydrophobic, or hydrophilic with
larger, hydrophobic residues, resulting in an increased hydrophobic
surface area. Our results thus seem to contradict another general
concept, namely that, because of the energetic cost of solvating
non-polar side chains, the exposure of hydrophobic residues at the
protein surface is unfavorable for overall protein stability. Indeed
natural proteins usually attempt to bury hydrophobic residues in the
interior while placing hydrophilic residues at the surface. In line
with this observation, a reduction in the hydrophobic surface area has
often been recognized as an important factor contributing to the
enhancement of stability in thermophilic proteins compared with their
mesophilic counterparts (4). A similarly good correlation between
increasing stability and decreasing surface hydrophobicity has also
been found in mutagenesis studies involving solvent-exposed sites (28,
31, 32). However, radically different conclusions have been reached
from other studies on the effect of surface hydrophobicity on protein
folding and stability. It can indeed be argued that the introduction of
hydrophobic residues at the surface is not necessarily
thermodynamically unfavorable, if the non-polar side chains are more
partially buried in the folded state than in the unfolded state (33).
In the cold shock protein CspB from B. subtilis, three
surface-exposed phenylalanines participate both in activity and
stability, and their replacement by alanine is highly destabilizing
(34). Similarly, aromatic side chains that form clusters or are
embedded in hydrophobic pockets at the protein surface have been shown
to be critical for the thermal stability of a thermophilic protease
(29, 35) and xylanase (36). In all these cases, the energy penalty
associated with the partial exposure of the non-polar side chains in
the folded protein must be overcompensated by the positive effect resulting from their partial burial and the stabilizing interactions they establish upon folding.
In the following we discuss the nature of the interactions that may
account for the beneficial impact of the surface hydrophobic residues
we introduced in our 5-fold mutant BLA.
Hydrophobic Packing in Surface Indentations--
A close packing
of the atoms in the interior of a protein is considered to be essential
for stability. Reducing the number and volume of internal cavities
through improved hydrophobic packing is an established strategy to
stabilize its core and the protein overall (37-40). In contrast,
because of the general belief that surface hydrophobicity is
destabilizing, increasing the hydrophobic packing within surface
indentations has rarely been considered in mutagenesis studies. In the
case of our hyperthermostable BLA variants, there are at least two
mutations, A209V and H133V, for which the stabilizing effects are
likely a result of increased hydrophobic packing of surface
indentations. Both mutation sites lie in a small surface groove or
cavity formed between secondary structure elements: between three
strands of the large Stabilization of Aromatic-Aromatic Interactions--
The interaction between the
side chains of aromatic residues can contribute about 1.3 to 6 kcal/mol
to protein stability (45). Wild-type BLA naturally contains a large
number of aromatic clusters at the surface, and their contribution to
the overall stability of BLA is expected to be substantial. Note that
hydrophobic residues at the surface are expected to be less
thermodynamically unfavorable when clustered than when isolated
since the energetic cost of solvating non-polar side chains is shared
between the interacting residues. Interestingly, aromatic clusters are
also observed at the surface of other glycoside hydrolases such as
xylanase and cyclodextrin glucanotransferase (CGTase) from thermophilic
organisms (36, 46). In starch-degrading enzymes, aromatic side chains serve as a stacking platform for the sugar moieties of the substrate. Their abundance at the surface may thus contribute to increased thermostability in the presence of starch, by allowing amylose chains
to bind non-specifically to the protein (46).
In the hyperthermostable mutant amylase that is described here, two of
the stabilizing substitutions (N190F and N265Y) introduce additional
aromatic side chains at the protein surface. In both cases, the
introduced side chain extends a network of aromatic-aromatic interactions of underlying residues to the outside of the protein. The
aromatic ring of Tyr-265 is involved in a rather complex system of
hydrophobic interactions, and its hydroxyl group is hydrogen bonded to
water molecules. The aromatic interaction network involving Phe-190 is
less complex, and its side chain is completely solvent-exposed. Despite
those seemingly unfavorable characteristics, the N190F mutation is by
far the most effective of all mutations characterized in BLA to date.
The fact that leucine, but not tyrosine, is also beneficial at position
190 indicates that it is the pronounced hydrophobic character of the
inserted residue that is the primary stability determinant at this
surface site.
The Importance of Metal Binding Sites--
Many mutations that
influence the stability of BLA are located near the unique Ca-Na-Ca
metal triad. The most stabilizing single site mutation observed in BLA
so far is the N190F mutation. Phe-190 is located at the tip of a long
loop of domain B that folds over domain A and forms part of the cage
that entraps the metal ions. Although Phe-190 does not participate
directly in metal binding, the triple aromatic interaction it forms
together with Tyr-193 and His-235 acts as a clamp that may reduce the
flexibility of the cage, thereby hindering the metal ions from
diffusing out of the binding pocket at high temperatures where
structural breathing is increased. The cage is composed of six
aspartate residues that provide neutralizing negative charges for the
metal ions. Once the metal ions diffuse out of their pockets, the high
density of negative charges from the aspartates must severely
destabilize this region. The N-terminal ends of the nearby
How sensitive the triadic metal binding array is with respect to the
overall stability of BLA is demonstrated by the thermosensitive mutant
N192A. Asn-192 stabilizes the metal binding cage by forming two
hydrogen bonds between its polar side chain atoms and the main chain
atoms of two direct metal ligands (Fig. 4). Removal of these hydrogen
bonds leads to a BLA variant that can be expressed, but that is
instantaneously inactivated when subjected to higher temperatures
(19).
These results point to the central ternary metal binding site in BLA as
one of the major nucleation sites for unfolding. Because of the high
charge density in the ternary Ca-Na-Ca binding site, small changes in
the stability of the metal binding cage are expected to lead to sharply
cooperative, amplified effects on the overall stability of BLA.
Furthermore, a cis-peptide bond between Trp-184 and Glu-185 is vital
for maintaining the integrity of the metal binding cage. It is likely
that this cis-peptide bond isomerizes at high temperatures once the
metal ions have been removed from their binding pocket. Since the
reverse process, the formation of non-proline cis-peptide bonds, is
energetically extremely unfavorable (47), this isomerization could be
the cause of the irreversibility of BLA unfolding. In good agreement
with this model, a two-stage unfolding transition has recently been
proposed for the thermal inactivation of B. amyloliquefaciens
The detailed analysis of the structural aspects of metal
binding in BLA as a result of various mutations is expected to be generally valuable for our understanding of the correlation between metal binding and protein stability. The introduction of metal binding
sites has long been considered a promising general strategy to increase
the stability of a protein (51). However, because of the structural
complexity of metal binding sites, with contributions coming from
sequentially separated regions, de novo engineering is a
formidable task. On the other hand, favorably modifying existing metal
binding sites in metallo-proteins appears to be a feasible challenge
(52).
Kinetic Stability and the Theory of Helix-Coil
Transitions--
Examination of wild-type and mutant crystal
structures at high resolution permits evaluation of the relative
contribution of attractive forces to the overall thermodynamic
stability of a protein. Using this approach, we partly explained the
stabilizing effect of the seemingly unfavorable hydrophobic
substitutions we introduced at the surface of BLA. Yet, the
compensating local effects discussed above do not seem to fully account
for the remarkable stabilizing effects observed with some of our BLA
mutations, particularly N190F. Similarly, the dramatic destabilizing
effect of the N192A mutation cannot be merely due to the energetic loss
associated with the removal of two hydrogen bonds.
Protein folding and unfolding following first-order kinetics are highly
cooperative processes that can be described by the theory of helix-coil
transitions (53). In this context, "helix" denotes the ordered
state of any kind of cooperatively formed structure, and "coil"
denotes its disordered state. Helix-coil transitions can be separated
into two steps, nucleation and propagation. In a protein, any region
that tends to unravel upon external influences can act as a nucleation
site for unfolding. However, because changes in the environment have
their greatest impact on the surface of a protein, unfolding is
expected to start at solvent accessible regions. There is a large
energy barrier associated with nucleation, which renders this step the
energetically most expensive part in a helix-coil transition. Once this
barrier is overcome, less energy is required to drive the transition,
which then progresses rapidly in a zipper-like fashion. Helix-coil
transition theory accounts for the fact that relatively moderate
changes in the local stability can lead to unproportionately large
overall effects, provided these changes occur at nucleation sites. We
believe that the effects of the mutations in BLA that lead to
kinetically stabilized variants can be explained in this light. All
mutated residues are involved in forming complex, cooperative
substructures, namely metal binding, aromatic clusters, and regular
secondary structural elements. They are exposed to bulk solvent and are
located at the edge of these cooperatively formed substructures. These
residues are therefore candidates for "weak points" where unfolding
is initiated, e.g. through the effects of temperature.
For the present structural work, we have focused on those mutations
that have particularly strong positive influences on the kinetic
stability of BLA. By doing so, we believe in retrospect that we have
serendipitously selected for nucleation sites of BLA unfolding.
-amylase (BLA) that result from the
incorporation of hydrophobic residues at the surface. Under highly
destabilizing conditions, a variant combining five stabilizing
mutations unfolds 32 times more slowly and at a temperature 13 °C
higher than the wild-type. Crystal structure analysis at 1.7 Å resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic
packing, usually applied to cavities, to surface indentations,
(b) introduction of favorable aromatic-aromatic
interactions on the surface, (c) specific stabilization of
intrinsic metal binding sites, and (d) stabilization of a
-sheet by introducing a residue with high
-sheet forming
propensity. All mutated residues are involved in forming complex,
cooperative interaction networks that extend from the interior of the
protein to its surface and which may therefore constitute "weak
points" where BLA unfolding is initiated. This might explain the
unexpectedly large effect induced by some of the substitutions on the
kinetic stability of BLA. Our study shows that substantial protein
stabilization can be achieved by stabilizing surface positions that
participate in underlying cooperatively formed substructures. At
such positions, even the apparently thermodynamically unfavorable
introduction of hydrophobic residues should be explored.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase
(BLA)1 is widely used in
biotechnology for the initial steps of starch degradation at
temperatures up to 110 °C (5). The primary substrate for BLA is
starch, but similar compounds, such as glycogen or smaller
oligosaccharides, are readily converted as well. Furthermore, BLA, like
other
-amylases, shows pronounced transglycosylation activity and is
therefore also used in synthetic chemistry for the enzymatic synthesis
of oligosaccharides. For this purpose, attempts are being made to
modify the enzymatic activity of BLA in order to accommodate a wider
variety of substrates with varying specificity (6). The main focus,
however, is the search for
-amylases that are less prone to
oxidative effects, highly stable at temperature and pH extremes and
still enzymatically active. Although thermophilic and acidophilic
-amylases have been found in extremophiles (3, 7), BLA remains a
prime target for extensive protein engineering projects (8-10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
radiation
and a beam-focusing mirror system (MSC). Data were processed and
reduced with the DENZO/SCALEPACK package version 1.9.0 (25). Details of
the data collection statistics are presented in Table II.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase from
B. amyloliquefaciens (17, 18). The mutation A209V was
obtained by in vivo screening of suppressive mutations in a
thermosensitive BLA variant (20). The double mutant Q264S/N265Y was
found when replacing residues that potentially undergo deamidation in
BLA by non-deamidating amino acids found at the same positions in the
homologous
-amylase from B. stearothermophilus.2
These four mutants were identified before detailed experimentally obtained structural information about BLA was available. Recently, we
have carried out a broad study based on the first reported crystal
structure of BLA (14) where we probed 15 positions and characterized
more than 175 mutations in terms of their influence on the kinetic
thermostability of BLA (19). During this study, we identified the
mutation N190F as the most stabilizing single site mutation found for
BLA so far.
View larger version (27K):
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Fig. 1.
Thermally induced BLA unfolding.
A, unfolding kinetics of BLA variants at 85 °C (see
"Experimental Procedures" for details). The solid
lines show fits to a formula representing a monomolecular
unfolding reaction (see text). B, pseudomelting curves of
BLA variants. The numbers in the graphs describe the mutations
present in the different variants: 133, H133V;
190, N190F; 209, A209V; 264, N264S;
265, Q265Y.
where Nt specifies the concentration of
native molecules at time t, N0
specifies the concentration of native molecules at time 0, and
kap is the apparent rate constant for BLA unfolding.
(Eq. 1)
Kinetic stability and onset of unfolding of BLA variants
Crystal parameters, data collection and structure refinement statistics
View larger version (52K):
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Fig. 2.
Crystal structure of kinetically stabilized
BLA. A, representative 2Fo Fc simulated annealing omit electron density map
for a region in the core of the kinetically stabilized BLA variant.
B, stereoview of a schematic representation of the overall
structure of BLA with the mutation sites labeled. Calcium ions are
shown in cyan, and sodium is in yellow. All
figures were created using the programs Bobscript (54) and POVRray
(Persistence of Vision, v3.02, POV-Team, www.povray.org) and GL_RENDER
(L. Esser, University of Texas Southwestern Medical Center).
-amylases with a central A domain containing a
(
)8-barrel forming the core of the enzyme. Attached
to this core is a C-terminal domain that invariably comprises a Greek
key motif. A third domain (B domain) is established as a loop-rich
protrusion from the A domain. This domain is the most dissimilar among
the known
-amylase structures and varies widely in size, with BLA
exhibiting the largest and most complex structure (14, 22).
-strand B
6 (secondary
structure nomenclature described in Ref. 14), which is involved in
forming the large central
-sheet in this domain (Figs. 2 and
3). The wild-type histidine at this
position is part of a weak hydrogen-bonding network (Tyr-175OH
Wat
His-133NE2 and His-133ND1
Gly-131O) involving a well-ordered
water molecule at the bottom of a small surface indentation in between
-strands B
4, B
5, and B
6. The histidine side chain partly
fills this cavity and shields the hydrophobic interior formed by
residues Ala-117, Ile-135, and Tyr-175. It was observed that the
introduction of hydrophobic residues at position 133 is particularly
effective in increasing BLA stability (18), with isoleucine, tyrosine,
and valine being the most stabilizing residues. As a consequence of the
H133V mutation, the hydrogen-bonding network that originally involved
the histidyl side chain is disrupted, and the hydrophobic nature of the
surface indentation is increased. The crystal structure of the mutant reveals that there is a main chain displacement around residue 133 with
the
/
angles of residue 133 changing from
150/163° for the
histidine in wild-type BLA to
135/145° for the valine in the mutant
(Fig. 3A). The latter values are more favorable for forming
the hydrogen-bonding patterns observed in
-sheets. As a result, the
hydrogen bonds between the two strands are slightly shorter and
consequently the entire
-sheet presumably more stable when compared
with wild-type BLA (Fig. 3B). The interactions of the
wild-type histidine side chain with its neighbors prevent the side
chain from turning more into the hydrophobic indentation causing the
main chain to slightly tilt backwards. A detailed analysis of the
deviation of geometric parameters, such as bond lengths, dihedral, and
improper angles from ideal values (carried out with the program CNS
(26)) shows that there is considerable strain around position 133 in
wild-type BLA (Fig. 4C). This
strain is diminished and spread out over a wider range in the H133V
mutant.
View larger version (25K):
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Fig. 3.
Region around position 133. Wild-type
carbon atoms are in green, the carbon atoms of the H133V
mutant are in gray, and other atoms are in standard colors.
A, stereoview of the wild-type structure superimposed on the
structure of the H133V variant emphasizing the main-chain conformation
of residue 133. B, stereoview of the differences in the
backbone conformation of -strands B
5 and B
6 in domain B
between the wild-type and the thermostable variant. C,
wild-type structure indicating the strain at each atom from 0 kcal/mol
(blue) to 69 kcal/mol (red) relative to the
equilibrium energies for bond distances, bond angles, dihedral angles,
and improper angles (26). D, same representation as in
C, for the H133V mutant.
View larger version (32K):
[in a new window]
Fig. 4.
Stereoview of the region around the Ca-Na-Ca
metal triad containing the mutation N190F. Calcium ions are shown
in cyan and the sodium ion is in yellow; other
atoms are in standard colors.
-electron cloud of the
central Tyr-193.
-helix A
3 in domain A of BLA (Fig. 2). In wild-type BLA, there
is a shallow hydrophobic indentation around the C
atom
of Ala-209 (Fig. 5). The base of this
indentation is composed of residues Tyr-203 and Phe-240, whereas the
rather hydrophilic rim is formed by the side chains of Lys-213,
Asp-243, and Lys-237 as well as by carbonyl oxygen atoms from residues
203 through 206. Phe-240 is located in the opposing
-helix A
4,
which, together with A
3, points toward the Ca-Na-Ca metal triad.
There are five ordered water molecules covering the
indentation.
View larger version (85K):
[in a new window]
Fig. 5.
Stereoviews of position 209. A, wild-type BLA; B, A209V mutant. Atoms
belonging to residue 209 are held in yellow; other atoms are
in standard colors.
View larger version (21K):
[in a new window]
Fig. 6.
Stereoview of the region around the double
mutation Q264S/N265Y with the wild-type structure for residues 264 and
265 (14) superimposed. Carbon atoms belonging to wild-type
residues are held in green; other atoms are in
standard colors.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet of domain B (position 133) and between
two helices of domain A (position 209). Hydrophobic contacts between
these secondary structure elements are reinforced as a result of the
mutations. In addition, substantial changes in the hydration shell of
these indentations are likely to occur, as indicated by the exclusion
of one ordered water molecule in the immediate vicinity of the mutation
sites. The shielding from solvent of buried hydrophobic clusters and backbone structures may prevent the potential disruptive action of
water molecules, as water molecules generally compete with main-chain
atoms for hydrogen bonds and thereby disrupt secondary structures. The
creation of a hydrophobic shield protecting
-sheet structures
against invading water molecules has also been proposed for explaining
the stabilizing effect of surface-located residues in the thermophilic
protease and cold shock protein mentioned above (30, 35).
-Sheets--
At position 133, a good
correlation was observed between the stabilizing effects of the 20 different amino acids and their
-sheet-forming propensity (18). As
the first residue of an edge
-strand, residue 133 is expected to be
key for the formation of the
-sheet structure within domain B. Analysis of our high resolution crystal structures shows that the
dihedral angles at position 133 are more favorable for
-sheet
formation in the mutant than in the wild-type structure. The structural
changes are a consequence of reduced conformational stress around
position 133 with the mutant side chain, thus allowing improved
main-chain hydrogen bonding. The same conformational relaxation could
be achieved by substitutions to other small amino acids,
e.g. glycine or alanine. However, such substitutions do not
increase the stability of BLA, supporting the idea that good
sheet
formers are preferred at this site. How the side chain of amino acids
favor particular dihedral angles and thereby
-sheet formation, and
to what extent the intrinsic
-sheet propensity is modulated by the
local environment remains debatable (41-44). In the case of position
133 in BLA it is clear that the side chain determinants of
-sheet
stability do not fully describe the effects of mutations; packing and
solvation effects at the surface have to be taken into account as well.
-helices
A
3 and A
4 point toward the metal-binding cage. Their helix
dipoles therefore contribute partial positive charges for stabilizing
the negatively charged aspartate residues in the metal-binding cage. In
this respect, the mutation A209V, in addition to its groove-filling effect discussed above, also contributes to the stability of the triadic metal array as it lies in the middle of
-helix A
3,
therefore likely increasing the overall stability of the helix.
-amylase in which metal ion dissociation
occurs in the first step immediately followed by irreversible unfolding
(48). The loss of stabilizing bivalent ions has also been invoked as
the first step of the unfolding reaction for other metallo-enzymes such
as subtilisin (49) and xylose isomerase (50).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Ken Usher, Alexander Pertsemlidis, and Phil Thomas for critical reviews of the paper.
![]() |
FOOTNOTES |
---|
* This research was supported by grants from the Deutsche Forschungsgemeinschaft (Wi 1100/1).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.
The atomic coordinates and the structure factors (code 1OB0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ To whom correspondence should be addressed. Fax: 1-214-648-8954; E-mail: Mischa.Machius@UTSouthwestern.edu.
Present address: Centre de Biochimie Structurale,
CNRS-UMR5048, INSERM-U554, 29 Rue de Navacelles, F-34090 Montpellier
Cedex, France.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M212618200
2 N. Declerck, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BLA, B.
licheniformis -amylase;
nd, not determined;
r.m.s., root mean
square.
![]() |
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