(Received for publication, November 21, 1995; and in revised form, January 8, 1996)
From the
LytA amidase is the best known bacterial autolysin. It breaks
down the N-acetylmuramoyl-L-alanine bonds in the
peptidoglycan backbone of Streptococcus pneumoniae and
requires the presence of choline residues in the cell-wall teichoic
acids for activity. Genetic experiments have supported the hypothesis
that its 36-kDa chain has evolved by the fusion of two independent
modules: the NH-terminal module, responsible for the
catalytic activity, and the COOH-terminal module, involved in the
attachment to the cell wall. The structural organization of LytA
amidase and of its isolated COOH-terminal module (C-LytA) and the
variations induced by choline binding have been examined by
differential scanning calorimetry and analytical ultracentrifugation.
Deconvolution of calorimetric curves have revealed a folding of the
polypeptide chain in several independent or quasi-independent
cooperative domains. Elementary transitions in C-LytA are close but not
identical to those assigned to the COOH-terminal module in the complete
amidase, particularly in the absence of choline. These results indicate
that the NH
-terminal region of the protein is important for
attaining the native tertiary fold of the COOH terminus. Analytical
ultracentrifugation studies have shown that LytA exhibits a monomer
dimer association equilibrium, through the COOH-terminal part of
the molecule. Dimerization is regulated by choline interaction and
involves the preferential binding of two molecules of choline per
dimer. Sedimentation velocity experiments give frictional ratios of 1.1
for C-LytA monomer and 1.4 for C-LytA and LytA dimers; values that
deviated from that of globular rigid particles. When considered
together, present results give evidence that LytA amidase might be
described as an elongated molecule consisting of at least four domains
per subunit (two per module) designated here in as N1, N2, C1, and C2.
Intersubunit cooperative interactions through the C2 domain in LytA
dimer occur under all experimental conditions, while C-LytA requires
the saturation of low affinity choline binding sites. The relevance of
the structural features deduced here for LytA amidase is examined in
connection with its biological function.
Autolysins are enzymes that specifically degrade some bonds in
the peptidoglycan backbone of the bacterial cell walls and eventually
cause cell lysis(1) . These enzymes have been involved in many
important physiological functions(2) . The best known bacterial
autolysin is the N-acetylmuramoyl-L-alanine amidase
of Streptococcus pneumoniae of 36.5 kDa, referred to here as
LytA ()amidase(3) . S. pneumoniae is one of
the microorganisms that causes high mortality and morbidity in the
world and it has been proposed that the autolytic process might be one
of the factors responsible of its pathogenicity(4) . Moreover,
it has been shown that LytA amidase induces a protective response in
mice and can be a potential antigen to prepare a pneumococcal vaccine (5) . The pneumococcal lytA gene was the first
autolytic gene that was cloned, sequenced, and overexpressed in an
heterologous host (6) . This fact provided the tools for a more
detailed biochemical and genetical characterization of the
enzyme(7) . LytA amidase requires the presence of choline
residues in the cell wall teichoic acids for activity. In this sense, S. pneumoniae is one of the rare microorganisms that contain
choline in the cell envelope, and it does not autolyze when choline is
replaced by ethanolamine in the culture medium(8) . Genetic
experiments have provided solid evidence that the enzyme has evolved by
the fusion of two independent modules: the NH
-terminal
module, responsible for the catalytic activity, and the COOH-terminal
module, involved in the attachment to the cell wall(9) . It is
generally accepted that the binding of the autolysin to its insoluble
substrate (the cell wall) is an essential prerequisite for the
hydrolysis of the covalent bonds. The COOH-terminal module of LytA,
that is built by six repeated units of 20 amino acids, appears to play
a double function, fixing the enzyme to the choline residues of cell
wall and inducing its activation by a process called
conversion(10) . Interestingly, the COOH-terminal module of
LytA (C-LytA) can be expressed and folded independently(11) , a
finding that has allowed the construction of fusion proteins that can
be purified by affinity chromatography in a single-step process in
matrices derived with DEAE, an analogue of choline(12) .
Although genetic approaches have provided substantial evidence on the modular design of the LytA amidase, few data concerning to its structure have been obtained by biochemical and physicochemical techniques. Hence, we have studied the structural organization in cooperative domains of the LytA amidase and its COOH-terminal module using differential scanning calorimetry (DSC). The oligomerization state and the molecular shape of both proteins have been characterized by analytical ultracentrifugation. In addition, we have determined the influence of choline as an analogue of the protein substrate on the thermal unfolding of LytA and C-LytA, as well as on their self-association equilibria. By combining results from thermal unfolding and ultracentrifugation a model for molecular organization of LytA is suggested. Finally, the structural characteristics of the amidase are examined in connection with its biological function.
Apparent weight-averaged molecular weights
were obtained by fitting individual data sets to a sedimentation
equilibrium model for single species, using the signal conservation
algorithm (17) from EQASSOC and XLAEQ programs. The equilibrium
dimerization constants, K, were calculated by
fitting experimental data to a monomer
dimer sedimentation
equilibrium model using the ORIGIN version of the NONLIN
algorithm(18) . Similar results were obtained using the signal
conservation algorithm(17) . The monomer molecular masses (in
daltons) were constrained to the following: 36,530 for LytA and 16,500
for C-LytA. The partial specific volumes for C-LytA and LytA calculated
from aminoacid composition were 0.725 and 0.722 ml
g
(19) . The specific volume of choline
measured with a DM2 Paar precision density meter was 0.885 ml
g
. The variations in the calculated specific volumes
of both proteins assuming 2 mol of bound ligand/monomer (see below)
were less than 0.3%.
Figure 1: Effect of choline concentration on the DSC transitions corresponding to the thermal unfolding of the LytA amidase. The choline concentrations are specified in the curve labels. Bold traces represent the experimental curves. Thin traces depict the results of the deconvolution analysis.
Considering the complexity of the endotherm
associated to LytA thermal denaturation, the interaction with choline,
a specific ligand of the COOH-terminal region, was studied (Fig. 1). Choline binding has a clear influence on the peak at
highest temperature, as reflected by the strong shift induced in the T value. This result suggests that at least that
peak should correspond to the melting of the COOH-terminal module of
LytA. On the other hand, above 80 mM choline, thermal
denaturation of LytA proceeds in two well resolved peaks. The
reversibility of the thermal-induced transitions was unaffected by
choline interaction. Increasing NaCl concentration up to 0.28 M did not modify the thermal denaturation profile of LytA,
discarding ionic strength effects.
The detailed analysis of the
calorimetric curves (see Fig. 1) shows that denaturation of LytA
can be resolved into three independent two-state transitions and a
final asymmetric peak characterized by a
H
/
H ratio of 1.4 ( Fig. 1and Table 1), suggesting the existence of
intermolecular interactions(23) . The asymmetry of the peak and
the value of
H
/
H would be consistent with a dimer which dissociates upon
denaturation. The transitions centered near 41 °C and 51 °C are
rather choline-independent up to about 80 mM ligand
concentration, supporting the notion that they should correspond to the
unfolding of the NH
-terminal region of LytA. However, above
such concentration, the low temperature calorimetric peak cannot be
deconvoluted into two elementary transitions, revealing a higher
interdependence. The final steps, 3 and 4, are stabilized by increasing
concentrations of ligand, suggesting that both transitions correspond
to the melting of the COOH-terminal region of the protein.
Figure 2: Calorimetric traces of C-LytA protein (curve A). Curves B and C show the rescan of the sample after stopping a first run at 43 °C and 85 °C, respectively.
Fig. 3shows the melting curves of C-LytA at increasing
concentrations of choline. The influence of ligand binding on the
thermal stability of C-LytA is complex and exhibits a biphasic
character. At the lowest ligand concentration assayed, C-LytA shows a
broad heat absorption curve and the total enthalpy change increases
from 162 kcal mol, in the absence of ligand, up to
220 kcal mol
, at 5 mM choline. Within this
ligand concentration range, the intensity of the small shoulder
observed in the free protein increases and its transition temperature
is shifted to higher values. The major peak is also shifted up to 63.6
°C, and its cooperativity becomes strongly protein-concentration
dependent (data not shown). Above 10 mM choline, a single
asymmetric peak is observed (see Fig. 3) and the calorimetric
profile is slightly dependent on protein concentration (data not
shown). Under these conditions, the ratio
H
/
H is 0.60,
indicating the existence of intermediates. Reversibility of C-LytA
thermal denaturation is dependent on choline concentration. Above 40
mM choline, 80% reversibility is achieved, while at lower
concentrations, renaturation never exceeds 50%.
Figure 3: Effect of choline concentration on the DSC transitions corresponding to the thermal unfolding of C-LytA. The choline concentrations are specified in the curve labels. Bold traces represent the experimental curves. Thin traces depict the results of the deconvolution analysis.
The analysis of the
calorimetric profiles at low ligand and protein concentrations shows
that C-LytA denaturation can be described by three independent
transitions (see Fig. 3and Table 2). The calorimetric
enthalpy changes of the two first endotherms increase with choline
concentration up to 52 and 75 kcal mol,
respectively, without changes in their van't Hoff enthalpies. On
the contrary, the highest temperature transition is characterized by a
constant enthalpy change of 115 kcal mol
and a
H*
/
H ratio of 0.95 (see Table 2). Above 10 mM choline, thermal denaturation of
C-LytA can be resolved into two transitions, stabilized by increasing
concentrations of ligand (Table 2). The former is a two-state
process with a calorimetric enthalpy change of 75 kcal
mol
, while
H
for the latter endotherm is about 1.4 times that of
H (150 kcal mol
), indicating, as in LytA amidase,
intermolecular interactions(23) .
The choline-induced variations observed in the thermal denaturation process of C-LytA suggest the existence of at least two types of binding sites with different affinity. At low choline concentration, ligand binding favors the refolding of the COOH-terminal module into a native-like tertiary structure, reducing the fraction of C-LytA molecules containing a region that does not undergo a cooperative unfolding transition in the free protein. Since calorimetric data are normalized to the total protein concentration, this effect would result in a reduced calorimetric enthalpy for the observed transition. At high choline concentrations, ligand binding seems to trigger intermolecular cooperativity.
On the other hand, the similarity of behavior of
C-LytA transitions 2 and 3 with with that of transitions 3 and 4 of
LytA agrees with their previous assignments to the unfolding of the
COOH-terminal module. Differences in T values and
cooperativity, particularly at low choline concentration, reveals the
influence of the NH
-terminal module on the final
organization and stability of the complete polypeptide chain.
Figure 4:
Sedimentation equilibrium data for choline
free C-LytA and LytA. Panels A and B represent the
data of C-LytA at pH 8 and 7 respectively. The data of LytA at pH 7 are
shown in panel C. Solid lines correspond to the best
fit of the experimental data to monomer dimer equilibrium. Dotted and dashed lines represent the theoretical fit
for monomer and dimer models, respectively.
Choline binding strongly enhances the dimerization of both proteins. Other oligomeric forms were not observed, even at the highest concentrations of protein and choline assayed. Table 3summarizes the dimerization equilibrium constants under different experimental conditions. The slopes of Wyman plots (24) of choline-induced dimerization are 1.93 and 1.96 for LytA and C-LytA, respectively, and essentially indicate the preferential interaction or binding of 2 choline molecules/formed dimer.
The sedimentation
coefficient of LytA dimer is 4.2 S (10 mM choline), which
corresponds to a f/f of 1.4. This value is
consistent with a prolate ellipsoid of 13
190 Å. The
impossibility of having 100% of LytA monomer hampered its hydrodynamic
characterization.
Figure 5: Schematic draw of C-LytA and LytA structural organization in cooperative domains.
The biphasic character of
choline effects brings about the existence of two different classes of
binding sites. The Wyman plot shows that a single molecule per monomer
is involved in the ligand-induced dimerization at low choline
concentration. However, the slope of the van't Hoff plot (26, 27) for the T values as a function of ligand concentration (data not shown)
results in a stoichiometry of 4.4 molecules of choline bound/dimer
(2.2/monomer). This second molecule of choline could be responsible for
the intermediate destabilization, the induction of intersubunit
cooperativity, and the reversibility increase.
The comparison of the thermodynamic parameters of
transitions 3 and 4 of LytA with those of transitions 2 and 3 in C-LytA
establishes a dependence between the NH- and the
COOH-terminal modules. In this sense, suppression of the
NH
-terminal region results in a loosening of the structure
of the isolated COOH-terminal module. In addition, cooperativity
between monomers through the C2 domain is always observed in LytA,
while in C-LytA saturation of the lower affinity binding sites is
required. Communication between LytA modules is also derived from the
observed interdependence between N1 and N2 domains at high choline
concentration, despite the ligand specificity for the COOH-terminal
module. On the other hand, the lower reversibility of LytA thermal
denaturation suggests that the unfolded NH
-terminal region
blocks the renaturation of the whole polypeptide.
In spite of the homology in the sequences of the COOH-terminal modules of LytA amidase and CPL1 lysozyme(28) , there are great differences between their structural organization in cooperative domains(29) . Although both choline-binding modules are designed to improve the attachment of the enzyme to the cell wall, it is obvious that a host-encoded lytic enzyme should exhibit a more efficient regulatory mechanism. In this sense, CPL1 lysozyme is encoded by the bacteriophage Cp1 and its biological regulation should be restricted to the phage cycle. In contrast, LytA amidase is a host-encoded protein involved in cell division of the S. pneumoniae whose activity should be under time and spatial regulation.
Scheme I: Scheme I.
LytA dimer can be modeled as a prolate
ellipsoid of about 13 190 Å, which can be visualized as a
stalk, made of the COOH-terminal modules, bearing at each end a
catalytic site (Fig. 5). This shape would facilitate the
diffusion of the molecule through the highly cross-linked framework of
the cell wall and increase the number of accessible hydrolyzable bonds
per attachment site. On the other hand, dimerization can also
facilitate the concomitant interaction with two teichoic acids and the
motion of the amidase along the septum by means of a step
binding-release mechanism, using the COOH-terminal modules of both
subunits, without completely detaching from the cell wall.
The
finding of two classes of binding sites, with different affinity, per
monomer of LytA or C-LytA is in agreement with previous COOH-terminal
deletion studies(33, 34) . Deletion of the terminal
tail of 11 amino acids, responsible for the conversion process,
dramatically reduces the catalytic activity. The loss of choline
recognition occurs upon additional suppression of P6, P5, and P4
motives, while intermediate deletions have no significant effect.
Recently, Markiewicz and Tomasz (35) have reported the presence
of a single mole of choline bound per mole of LytA activated by
affinity chromatography purification, which dissociates very slowly.
This observation is not incompatible with the existence of other types
of binding sites, since any molecule of choline bound in fast
equilibrium would dissociate after choline depletion of the sample
buffer(35) . The retained choline molecule might be that
involved in LytA dimerization, explaining the higher degree of
dimerization found in LytA when compared with C-LytA. Nevertheless,
there is no experimental evidence supporting a slow monomer
dimer equilibrium. Moreover, the enzyme purified by the procedure
described under ``Experimental Procedures'' is retained again
in DEAE-cellulose, after exhaustive dialysis. The previous finding that
the activated enzyme was no longer retained by choline-Sepharose
columns(35) , even after extensive dialysis, may be ascribed to
a different local concentration of binding residues in both matrices.
The results here reported provide new insights on the molecular
structure of pneumococcal autolysins. They establish the absence of a
straight forward correspondence between module and structural
cooperative domain, as demonstrated by the complex organization of LytA
amidase. The NH- and COOH-terminal modules are organized
into independent domains but the influence of choline on LytA stability
argues in favor of the existence of communication between modules. The
stepwise acquisition of repeating units of the COOH-terminal module may
represent an evolution-dictated enzyme advantage related to the
existence of several choline-binding sites, which in turn would improve
the affinity toward the substrate, as also does the protein
dimerization. Finally, the molecular shape of the tail-to-tail dimer
would also confer on the system notable properties, by means of a
facilitated diffusion and a catalytic site distance constraint.