From the
In contrast to lysozymes, which undergo two-state thermal
denaturation, the Ca
Chicken-type (c-type) lysozymes and
Protein engineering
experiments mostly involve the introduction of point mutations at
specific sites into well characterized enzymes in order to investigate
the role of the corresponding amino acid residues either in binding and
catalysis or in molecular structure and stability. Since the stability
of protein molecules can be considerably promoted by the introduction
of metal ion binding sites, several attempts have been made to
introduce the Ca
In the
present contribution we describe a chimera, LYLA1, obtained by
transplanting the central segment of bovine
Natural
human lysozyme (HLY), isolated from human milk, was obtained from U. S.
Biochemical Corp. Recombinant (wild-type) human lysozyme (rHLY) was
expressed in Saccharomyces cerevisiae from a synthetic gene
and purified as described
(18, 19) . The HLY mutant, in
which a functional Ca
S. cerevisiae GRF182 (
Plasmid pTZAGLYSHR, carrying the hybrid
alcohol dehydrogenase-2-glyceraldehyde-3-phosphate dehydrogenase
promotor
(24) and the chicken lysozyme signal sequence followed
by the chemically synthesized human lysozyme gene
(18) , was
constructed starting from pAB24AGScLYSH
(16) . Plasmid
pGEMAGTLYSHR, also derived from pAB24AGScLYSH, carries the promotor,
the lysozyme gene, and the glyceraldehyde-3-phosphate
dehydrogenase-terminator. It was used as a source of the
glyceraldehyde-3-phosphate dehydrogenase-terminator sequence in the
final construct.
Plasmid pAB24 is a yeast shuttle vector that
contains the entire 2-µm circle needed for replication and the
leu2-d and ura3 genes for selection in yeast, as well
as the pBR322 sequences necessary for selection and replication in
E. coli(24) .
By using the newly created NdeI
restriction site in the hly gene and the NdeI site
already present in the bla gene at the corresponding amino
acid positions , and both newly created XhoII
restriction sites in the hly and bla genes, the
hly sequence corresponding to the amino acid sequence from
Ala-76 to Asp-102 was exchanged for that of bla, equivalent to
Ile-72 to Asp-97 (BLA numbering). Because the pTZAGLYSHR plasmid
contains several XhoII sites, it was cut by
XhoII/ SalI and SalI/ NdeI,
respectively. From each digest the appropriate fragment was isolated
and ligated to the NdeI- XhoII fragment of pTZs31T,
and the resulting plasmid was named pTZAGLYLA1.
After verifying the
sequence of the chimeric lyla1 gene, pTZAGLYLA1 was cut with
BamHI and SalI to isolate the DNA sequence
corresponding to the promotor, the signal sequence, and the lyla1 gene. The glyceraldehyde-3-phosphate dehydrogenase-terminator
sequence was isolated from pGEMAGTLYSHR as a
SalI- BamHI fragment. Both fragments
(promotor-signal-gene on the one hand and terminator on the other) were
ligated into the BamHI site of pTZ18U to obtain pTZAGLYLA1T.
The BamHI- BamHI fragment of the latter, containing
the promotor, signal, coding sequence, and terminator, were ligated
into the BamHI site of the pAB24 shuttle vector, resulting in
the pABAGLYLA1 expression plasmid.
The purity of the final product was controlled
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), by isoelectric focusing on precoated plates (Serva)
containing an ampholite gradient of pH 3-10, by electrospray-mass
spectrometry on a V. G. Biotech BIO-Q instrument, and by N-terminal
sequence analysis on a gas phase sequenator (Applied Biosystems model
477A) using Edman chemistry.
The binding of (GlcNAc)
We also checked the pH
dependence of the catalytic activity of LYLA1 and compared it with that
of HLY and of its mutant M4 (Fig. 3). The data show that the
chimera possesses optimal activity at a pH slightly lower than that of
HLY. The figure also indicates the virtual absence of a Ca
Another point of interest is the
wavelength of maximal intensity (
This conclusion is further
supported by analysis of the chemical shift data for the C
A difference between both T
When the degree of structural resemblance between HLY and
baboon
One way of building chimeric proteins is by
exchanging exons
(42, 43, 44) . Indeed,
chicken-type lysozymes and
The second approach, which is followed here, rests on the exchange
of complete secondary structure elements. These are expected to contain
sufficient autonomous structural capacity to confer stability to the
chimera. Joining of the fused parts thus occurs in loops that can adapt
to local tensions. Initially, we had planned to introduce into HLY only
the BLA sequence needed for a fully equipped Ca
Compared with the homologous HLY sequence, the
transplanted BLA segment shows a deletion at the penultimate position.
Consequently, the adjacent Pro-103 is moved one step in the direction
of helix C. This could cause the last turn of the transplanted helix to
unwind. Nevertheless, by applying the potential function of the BRUGEL
software
(34) the modeled chimera was indicated to possess
sufficient stability. In the contact zone between the HLY and BLA parts
a reasonable Van der Waals fit without cavities was observed.
Our
two-dimensional NMR analysis confirms these modeling predictions as
they show the secondary and tertiary structure of the HLY-derived part
of the chimera to be virtually identical to the parent HLY. Also the
BLA part of the chimera has a well defined structure related to the
native BLA and HLY. This is indicated not only by the NOE and chemical
shift data discussed above, but also by the protection from solvent
exchange of numerous amide hydrogens. For the region involved, a
comparison was made between the differences in residual chemical shifts
between LYLA1 and HLY on the one hand (Fig. 7 C) and
between LYLA1 and BLA on the other (data not shown). The latter had to
be restricted to helix C as for BLA only a few assignments are
available, mostly restricted to this helix
(12) . This analysis
shows that in the second half of the helix (residues 95-101;
LYLA1 numbering), there is a much better correlation with the residual
shifts of BLA than with those of HLY. The structure of LYLA1 is
therefore more likely to be akin to that of BLA in this region,
indicating that sequence is more important than micro-environment in
determining the local fold. Fig. 11shows LYLA1 with an
indication of the 20 residues possessing the highest residual C
The presence of the
Ca
The CD spectrum in the near-UV region of LYLA1 is very
similar to that of HLY. Taking into account that the hybrid and the
wild-type lysozymes have identical tryptophan and tyrosine residues,
these data suggest that the overall tertiary structure of both proteins
is very similar. Binding of Ca
The chimera also contains both residues
essential for the enzymatic activity of lysozyme (Glu-35 and Asp-53)
and thus LYLA1 should be an active muramidase. However, hydrolytic
activity also implies the correct positioning of the entire active
cleft. In LYLA1 most side chains lining the binding cleft are situated
in the lysozyme part of the hybrid. The possibility cannot be excluded,
however, that the insertion of the
One of the
most obvious manifestations of Ca
In
conclusion, by transplanting the Ca
Bold characters indicate amino acids
common to the three proteins. The transplanted BLA part in LYLA1 is
underlined. The sequence data for HLY are from Ref. 56 and data for BLA
from Ref. 23.
Buffers used were either 10
mM Tris-HCl, pH 7.4, or 10 mM MES-KOH, pH 6.0, with
addition of either 1 mM CaCl
We thank Dr. Carol Robinson and Gary Howarth for
analysis of LYLA1 by electrospray mass spectroscopy, Dr. Lorna Smith
for advice on structural analysis by NMR spectroscopy, and Dr.
Christina Redfield and Christopher Penkett for valuable discussions.
The expert technical assistance of Sigried Vanryckeghem, Linda
Desender, Hilde Verhaeghe, Wim Noppe, and Frederik Coornaert is
gratefully acknowledged.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-free form of the homologous
-lactalbumins forms an intermediate ``molten globule''
state. To understand this difference, we have produced a chimera of
human lysozyme and bovine
-lactalbumin. In the synthetic gene of
the former the sequence coding for amino acid residues 76-102 was
replaced by that for bovine
-lactalbumin 72-97, which
represents the Ca
-binding loop and the central helix
C. The chimeric protein, LYLA1, expressed in Saccharomyces
cerevisiae was homogeneous on electrophoresis and mass
spectrometry. Its Ca
binding constant was 2.50
(±0.04)
10
M
,
and its muramidase activity 10% of that of human lysozyme.
One-dimensional NMR spectroscopy indicated the presence of a compact,
well structured protein. From two-dimensional NMR spectra, main chain
resonances for 118 of a total of 129 residues could be readily
assigned. Nuclear Overhauser effect analysis and hydrogen-deuterium
exchange measurements indicated the presence and persistence of all
expected secondary structure elements. Thermal denaturation, measured
by circular dichroism, showed a single transition temperature for the
Ca
form at 90 °C, whereas unfolding of the apo
form occurred at 73 °C in the near-UV and 81 °C in the far-UV
range. These observations illustrate that by transplanting the central
part of bovine
-lactalbumin, we have introduced into human
lysozyme two important properties of
-lactalbumins, i.e. stabilization through Ca
binding and molten
globule behavior.
-lactalbumins are
evolutionarily related proteins
(1) . Their comparative study
offers interesting possibilities in the fields of protein folding
(2, 3, 4) and enzymatic functioning. Although
their amino acid sequences
(5) and three-dimensional structures
(6, 7) are largely homologous, functionally they are
widely different. Lysozymes catalyze the hydrolysis of the
1-4 glycosidic linkage between N-acetylglucosamine
and N-acetylmuramic acid in the main polysaccharide
constituent of Gram-positive bacterial cell walls. Their hydrolytic
activity is carried by two essential carboxylate groups, contributed in
human lysozyme by residues Glu-35 and Asp-53.
(
)
On the other hand,
-lactalbumins regulate lactose
biosynthesis by modulating the specificity of
-galactosyltransferase. One of the best characterized functions of
-lactalbumins is their ability to bind Ca
strongly. This property resides in a typical
Ca
-binding loop, in which two peptide carbonyls and
three carboxylate groups (Asp-82, Asp-87 and Asp-88 in the bovine
species) act as ligands
(8) . The binding of Ca
to
-lactalbumin results in the formation of a stable
metalloprotein. Dissociation of Ca
, extremes of pH,
elevated temperatures, or moderate concentrations of denaturant lead to
the appearance of a typical partially unfolded state
(9) , which
has been designated a molten globule state. It has been characterized
by a variety of measurements and shown to exhibit a high content of
secondary structure, considerable compactness, nonspecific tertiary
structure and significant structural flexibility
(10, 11, 12) . As a result, upon denaturation
molecules of
-lactalbumin pass in a multistate process from a
native folded state through an equilibrium molten globule intermediate
into a completely unfolded state. By contrast, lysozymes generally
conform to the classical two-state model for cooperative unfolding
without showing an intermediate state with molten globule character. We
have recently observed a partially unfolded state comparable with the
molten globule state of
-lactalbumin in the case of equine
lysozyme
(13) . It is indicative that, although most lysozymes
are unable to bind Ca
, equine lysozyme possesses a
Ca
-binding loop
(14) .
binding function of
-lactalbumin in lysozyme by site-directed mutagenesis. Kuroki
et al.(15) have shown that introducing two of the
necessary Asp residues at the corresponding sites in the human lysozyme
molecule results in a functional Ca
-binding site.
More recently, we have been able to demonstrate, first, that a single
Asp (A92D) suffices for Ca
binding and, second, that
another mutant (M4
) (
)which contains a fully
equipped Ca
site is clearly stabilized against
thermal denaturation in its Ca
-bound form
(16) . In the meantime, the latter mutant was found to display
molten globule characteristics at very low pH
(17) .
-lactalbumin, which
comprises the Ca
-binding loop and the main helix C,
into the homologous position in human lysozyme. The result is a
compact, well structured molecule, which has the ability to undergo
Ca
-induced conformational changes and which shows the
typical multistate unfolding behavior of
-lactalbumin.
Materials
Restriction enzymes were purchased
from Boehringer Mannheim and from New England Biolabs. Oligonucleotides
needed for site-directed mutagenesis were obtained from Pharmacia
Biotech Inc. and from the Laboratory of Genetics (University of Ghent,
Belgium). Micrococcus luteus cells were from Boehringer;
N,N`, N"-triacetylchitotriose, (GlcNAc),
came from Sigma; Fura-2 was a product of Molecular Probes.
-binding site was introduced by
site-directed mutagenesis of four different amino acid residues (M4),
has been described before
(16) . Natural bovine
-lactalbumin (BLA) isolated from cows' milk was obtained
from Sigma.
Strains and Media
Escherichia coli cells
DH5 ( supE44
lacU169 (
80lacz
M15) hsdR17 endA1 recA1 gyrA96
thi-1 relA1) , CJ236 ( dut-1 ung-1 thi-1 relA1, pCJ105(Cm
)) , and MV1190 (
lac-proAB
thi supE (
scr1-recA )306::Tn10(tet
) (F`
traD36 proAB, lacI
Z
M15)) were used
as host strains for bacterial transformations and routine plasmid
preparations. They were grown in Luria broth containing 1%
Bacto-tryptone, 0.5% Bacto-yeast extract and 1% NaCl (optionally with
1.5% Bacto-agar).
,
leu2-3, leu2-112, his3-11, his3-15, ura3
,
pep4
, CAN, cir°), a derivative of GRF18
(20) was used
as the host strain and was cultivated either in uracil- or
leucine-selective medium (with 8% sucrose added) or in YPD medium (8%
sucrose) buffered with 50 mM MES, pH 7
(21) .
Plasmids
Plasmid pTZ18U was obtained from Bio-Rad.
Plasmid pTZs31T, which carries the synthetic bovine -lactalbumin
gene after the triose phosphate isomerase promotor, is a derivative of
pSCMFsBLA
(21) . In this construct the original codon at
position 30:GCG, coding for Ala due to a misinterpretation of the data
of Vilotte et al.(22) on the cDNA sequence of the
bla gene, was corrected by site-directed mutagenesis to ACG,
coding for Thr
(23) .
Construction of the Chimeric Gene
The purpose of
the present study was to transplant the structural element, which is
responsible for the Ca binding properties of BLA,
into the sterically homologous position in HLY and to study the effects
of the newly introduced Ca
binding capacity on the
properties of the hybrid. The transplanted structure is composed of a
short 3
helix, the Ca
-binding loop, and
the central helix C (Fig. 1), and corresponds to the amino acid
sequence 72-97 (BLA numbering) (). The construction
of the hybrid gene occurred in three steps. In the first, identical
restriction sites had to be introduced in register into the plasmids
pTZAGLYSHR and pTZs31T, that were used as the source material for the
hly and bla genes, respectively. In the second, the
homologous bla gene fragment was inserted into the hly gene. In the third, the hybrid gene was cloned into the
appropriate shuttle vector.
Figure 1:
Model of the constitutive parts of
LYLA1. Starting from the atomic coordinates of HLY (Brookhaven Data
Bank code 1LZ1) and those of BLA, derived from the baboon lactalbumin
data (BDB 1 ALC) by conservative on-screen mutation, the chimeric
protein was modeled. The transplanted BLA sequence (termed OA72-OA97
and shown in bold on the figure) was optimally fitted to the
homologous HLY sequence 76-102. As a result, the BLA-derived
residue OA72 is within binding distance of the HLY acceptor site 75.
However, between BLA residue OA97 and HLY residue 103 a small gap
exists, due to deletion of Arg-101. The possible consequences of this
deletion are discussed in the text.
In order to create an NdeI site
at the position of the hly gene corresponding to amino acids
75-77, oligonucleotide-directed mutagenesis according to Kunkel
(25) was carried out using the oligonucleotide
5`-CAAGTGACATATGTTAACAGC-3` as the primer (the NdeI site is
marked in italics). In order to create an XhoII site at codons
101-103 of the lysozyme gene, and to shift the XhoII
site from the corresponding amino acid position 94-96 to position
96-98 of the -lactalbumin gene (BLA numbering), we used two
oligonucleotides. The first one intended for pTZAGLYSHR was
5`-GATACCTTGTGGATCTAACAACTCT-3` and the second one for pTZs31T:
5`-GATACGACCGGATCTAGAATCTTCTTAAC-3` (the XhoII site in both is
marked in italics).
Transformation of the Yeast Cells and Expression in
Culture Medium
The newly constructed pABAGLYLA1 was used to
transform the S. cerevisiae GRF182 strain by the lithium
acetate method
(26) . Uracil-selective medium was used to screen
for plasmid-containing cells and Ura transformants
were grown in leucine-selective medium for 4 days. This culture was
used to inoculate (1/20) buffered YPD medium. Cells were grown at 28
°C in shaking culture or in a 10-liter fermentation batch supplied
with 4% ethanol after exhaustion of the carbon source. The culture was
harvested after 5-7 days of growth.
Purification
The LYLA1 protein was purified from
the pooled supernatant by repeated cation-exchange and size-exclusion
chromatography as follows. The supernatant was diluted with 4 volumes
of distilled water and its pH was adjusted to 6.5. It was loaded onto a
cation-exchange column Fractogel-SO (Merck; type EMD 650 M)
which was equilibrated with 20 mM phosphate buffer, pH 6.5.
After washing with the same buffer, the chimeric protein was eluted
with 1 M NaCl in buffer. Fractions positive for enzymatic or
antigenic activity were subjected to size-exclusion chromatography on a
Sephacryl HR-100 (Pharmacia) column, using 50 mM NaAc, pH 5.0,
0.2 M NaCl. The fractions containing the chimeric protein were
pooled, dialyzed against water, and rerun on the previously mentioned
cation exchanger, this time eluted with a linear NaCl gradient
(0-1.5 M). The final yield of LYLA1 was between 8 and 9
mg/liter of medium.
Immunochemical Analysis
The antigenic properties
of the chimeric LYLA1 were analyzed using affinity-purified polyclonal
antibodies to the parent proteins BLA and HLY. The latter were prepared
by injecting rabbits with recombinant HLY
(18) or with natural
BLA
(21) . Antibodies to HLY were affinity-purified on
CNBr-activated Sepharose (Pharmacia) to which a commercial preparation
of HLY was coupled. Antibodies to BLA were purified as described
(21) . The presence of antigenic determinants specific for the
Ca-binding loop and the helix C in LYLA1 was
confirmed using antibodies induced with a CAT-fusion protein in which
the C terminus of CAT was elongated by the BLA sequence 73-105
(16) . As both BLA- and HLY-specific epitopes are present on the
LYLA1 chimera, measurement of this protein was routinely done with a
sandwich immunoradiometric assay
(21) , using anti-BLA to coat
the wells and
I-labeled anti-HLY for detection.
Ca
The standard enthalpy for CaBinding
Measurements
binding (
H
) was measured using a batch
microcalorimeter (LKB type 2107)
(27) and the binding constant
( K
) by competition titration with Fura-2
(14) . Decalcification of LYLA1 was performed as described
before
(28) , and the final Ca
content of the
apo form, measured by atomic absorption spectroscopy, was always lower
than 0.05 mol/mol of protein.
Enzymatic Activity
The lytic activity was measured
using M. luteus as substrate
(29) , with the exception
that the buffer used was 0.1 M MES, pH 6.2. In order to
monitor the effect of Ca on the enzymatic activity,
the assay mixture was adjusted to either 1 mM CaCl
or 1 mM EDTA. In a study of the pH dependence of the
hydrolytic activity, different buffers were used as indicated. Protein
concentrations were determined by absorbance measurements at 280 nm,
based on the following values for A (1%): for BLA =
20.1; HLY, A92D, and M4 = 25.5; and LYLA1 = 25.0,
computed on the basis of the calculated molecular weight and Tyr and
Trp content. Absorbance measurements were done on a Beckman DU-70
spectrophotometer.
to these
proteins was monitored by the Trp fluorescence enhancement that is
induced upon substrate binding to the active site cleft
(30) .
In practice, to 2.0 ml of 0.1 mg/ml protein in 10 mM Tris
buffer, pH 7.4, containing either 1 mM Ca
or
1 mM EDTA, small aliquots of a suitably diluted (GlcNAc)
solution in water were added. After each addition, the
temperature was re-equilibrated to 25 °C and the Trp fluorescence
emission spectrum recorded (
= 285 nm). The
enhanced fluorescence intensity at 325 nm was corrected for dilution
and plotted as a function of the logarithm of the total (GlcNAc)
concentration. From the sigmoidal saturation curves the
pEC
(the logarithm of the concentration of (GlcNAc)
needed to obtain 50% of the maximally enhanced fluorescence
intensity) values, that are representative for the relative binding
constants, were deduced by the point of inflection. All fluorescence
measurements were performed on an Aminco SPF-500 spectrofluorometer,
and only corrected spectra are shown.
Circular Dichroism
Circular dichroism (CD)
measurements were carried out on a Jasco J-600A spectropolarimeter
using cuvettes of 1-cm path length in the near-UV region and of 1 or
0.1 mm in the far-UV. Base-line normalization was done at 250 (far-UV)
and 320 nm (near-UV). The data were expressed as residual ellipticity
[]
(deg
cm
dmol
) using 115.1 and
113.1 as the mean residue weight for LYLA1 and HLY, respectively.
NMR Spectroscopy
NMR spectra were recorded using a
GE Omega 500 MHz spectrometer. One-dimensional spectra were measured
with a sweep width of 7042 Hz using 4096 points, giving a digital
resolution of 1.71 Hz/point. The following two-dimensional experiments
were performed for the purposes of assignment and structural analysis:
phase-sensitive J-correlated spectroscopy (COSY), single and double
relayed coherence transfer spectroscopy, and nuclear Overhauser
enhancement spectroscopy
(31, 32, 33) . Data
sets were acquired as 512 t increments of 2048 data points
and 48-96 transients. Several mixing times were used in the
nuclear Overhauser enhancement spectroscopy experiment ranging between
150 and 250 ms. Samples were internally referenced using 1,4-dioxane
which resonates at 3.743 ppm. All NMR spectra were recorded at pH 4.5,
35 °C, and were processed using the program Felix 2.1 (Hare
Research Inc.) on a Sun computer.
Computer Modeling
Computer modeling was done on a
Silicon Graphics IRIS Indigo computer. The software used for
interactive modeling and energy calculations was the BRUGEL program
(34) . Atomic coordinates used were those of the Brookhaven
Protein Data Bank
(35) for HLY (code: 1 LZ1) and for baboon
-lactalbumin (code: 1 ALC).
Purity Control
SDS-PAGE of the purified protein
indicated that neither aggregation nor fragmentation had occurred (data
not shown). On isoelectric focusing, the chimera migrates to a position
close to that of HLY. By extrapolation from the reference mixture, the
pI of the hybrid is estimated to be 9.6. In comparison with HLY (pI
around 11), this figure is consistent with expectations since the
hybrid carries four additional Asp residues and one additional Lys.
Again, the presence of a single band illustrates the relative purity of
the chimera. When measured by electrospray-mass spectrometry, the
molecular weight of the peak corresponded to the Ca form (14,878 versus a predicted value of 14,879).
N-terminal sequencing confirmed correct processing by the yeast cells.
Immunological Properties
In analogy with previous
work on mutant M4
(16) , using affinity-purified polyclonal
antibodies against the whole BLA molecule and also against a CAT-fusion
protein that carries the BLA sequence 73-105, we proved that
LYLA1 clearly possesses BLA epitopes (data not shown).
Ca
The binding of
Ca Binding
to LYLA1 was examined by competition titrations
with the Ca
binding dye, Fura-2
(14) in 0.01
M Hepes, 0.1 M KCl at pH 7.1 and 20 °C. The
titration data are consistent with a single, strong binding site with a
K
of 2.50 (± 0.04)
10
M
(Fig. 2). In ,
thermodynamic data obtained from microcalorimetric titrations are
compared with those for BLA, HLY, and the Ca
-binding
HLY mutant, M4.
Figure 2:
Titration of Fura-2 and apoLYLA1 with
Ca. A mixture of Fura-2 (70.88 µM) and
apoLYLA1 (63.43 µM) in 10 mM Hepes, 0.1
M KCl, pH 7.1, was titrated with Ca
at 20
°C. Inset, microcalorimetric titration of apoLYLA1 with
Ca
in 10 mM Tris, pH 7.15, at 25
°C.
Enzymatic Activity
Since the chimera contains the
two residues essential for catalytic activity, Glu-35 and Asp-53, one
might expect LYLA1 to possess muramidase activity. Also, the
introduction of the Ca site derived from BLA raises
the question whether its lytic activity can be modulated by
Ca
binding. LYLA1 possesses about 10% of the
hydrolytic activity of HLY. In the presence of Ca
,
the specific activity of HLY (506 enzyme units/mg) is reduced to 46 for
LYLA1. In 1 mM EDTA, the corresponding figures are 342 and 53.
Thus, the specific activity of the chimera is only slightly dependent
on the presence of Ca
.
effect at all pH values. In contrast, although no strong
Ca
site has been found on HLY, Ca
seems to improve its muramidase activity, whereas in the case of
the Ca
-binding mutant M4, Ca
slightly suppresses the catalytic activity.
Figure 3:
Muramidase activity of LYLA1 and HLY
toward M. Luteus cell walls at different pH values measured at
25 °C in the presence of 1 mM EDTA ( open symbols)
or 1 mM Ca ( filled symbols).
A, LYLA1; B, HLY; and C, HLY mutant M4.
Different buffer solutions were used: 50 mM glycine, pH
2-3; 50 mM potassium acetate, pH 4-5; 50
mM MES, pH 6-7; 50 mM Tris, pH 8-9; and
50 mM KHCO
, pH
10-11.
The binding
affinity of the competitive inhibitor (GlcNAc) was also
evaluated. Binding of this oligosaccharide can conveniently be followed
by its enhancing effect on tryptophan fluorescence
(30) . In
I the binding data for LYLA1 are compared with those for
HLY and its mutant M4. Titrations were performed at two different pH
values: 7.4 and 6.0, corresponding to the activity maxima of HLY and
LYLA1, respectively. Taken together these data indicate that, first,
(GlcNAc)
binding constants for LYLA1 are 2-3-fold
lower than those for HLY and its mutant M4 and, second, that in nearly
all cases the presence of Ca
seems to improve the
binding affinity. The slope of the linearized binding curve turns out
to be lower for LYLA1 than for the other two proteins (data not shown),
suggesting that there is probably more than one binding site for the
inhibitor on this hybrid.
) of tryptophan
fluorescence and its shift upon saturation with the ligand. The three
proteins have a
of about 330 nm, independent of the
presence of Ca
. Upon inhibitor binding, the maximum
shows a small blue shift (about 3 nm) in the case of HLY and M4 and a
small red shift in the case of LYLA1 (data not shown). More important
is the relative increase in quantum yield (I). The ratio
of the emission intensities measured at saturation with inhibitor
versus those in its absence
(I
/I
) increases in the presence of
Ca
for the three proteins. The difference in the
fluorescence increase induced in the different proteins upon saturation
with (GlcNAc)
is large, ranging from 1.9-fold for HLY to
1.3-fold for LYLA1.
Circular Dichroism
In order to study the
conformational state of the chimeric protein, CD spectra were recorded
in the near- and far-UV regions. Fig. 4shows these spectra for
LYLA1 in the presence and absence of Ca and compares
them with the spectra of wild-type HLY, which are independent of
Ca
. The near-UV spectrum of HLY is characterized by a
positive band at 292 nm ascribed to tryptophan residues, and a trough
near 270 nm, mostly due to tyrosine groups. Apart from intensity
differences, especially at 270 nm, these features are also present in
the spectrum of the chimera.
Figure 4:
CD spectra of LYLA1 at 25 °C in the
far- and near-UV. --, LYLA1 in 5 mM Tris, 1
mM Ca, pH 7.55; - - -
-, LYLA1 in 5 mM Tris, 1 mM EDTA, pH 7.6; and
-
-) recombinant human lysozyme in 5 mM Tris,
pH 7.55.
In the far-UV region, the spectrum of
the apo form of LYLA1 differs markedly from that of wild-type HLY,
especially around 190 nm, indicating that the secondary structure of
the apo form is affected by the presence of the transplanted BLA
sequence. In the presence of Ca these differences are
minimal.
Tryptophan Fluorescence
In Fig. 5the
fluorescence emission spectra of LYLA1 measured in the presence and
absence of Ca are compared with those of natural HLY
and BLA. As the spectra for apo- and Ca
-HLY
practically coincide, only that of the Ca
form is
shown here. The fluorescence intensity of the chimera is only slightly
lower than that observed for HLY. The emission maximum occurs at 328.4
and 327.9 nm for the apo and Ca
forms of LYLA1,
respectively, compared with 330.7 nm for HLY.
Figure 5:
Trp
fluorescence of LYLA1 at 25 °C. --, LYLA1 in 5
mM Tris, 1 mM Ca, pH 7.5; -
- - -, LYLA1 in 5 mM Tris, 1 mM
EDTA, pH 7.5; +++, human lysozyme in 5 mM Tris,
1 mM Ca
, pH 7.5; -
-,
bovine
-lactalbumin in 5 mM Tris, 1 mM
Ca
, pH 7.5; and - -, bovine
-lactalbumin in 5 mM Tris, 1 mM EDTA, pH
7.5.
No spectral shift and
only a small increase in intensity is observed upon Ca binding to LYLA1. For BLA by comparison, the binding of
Ca
provokes a large shift in the position of the
emission maximum (from 340 to 327 nm) and a significant decrease of
fluorescence intensity
(36) .
NMR Spectroscopy
In order to gain information
about the secondary and tertiary structure of LYLA1 at the individual
residue level, NMR spectroscopy was undertaken. The one-dimensional
H spectrum of LYLA1 recorded in D
O is shown in
Fig. 6
. It is well dispersed and is fully characteristic of a
completely folded globular protein
(37) . The existence of many
resolved resonances in the methyl and aromatic regions indicates that
the molecule has ordered tertiary structure. Furthermore, in the region
between 4.8 and 5.5 ppm several resonances are evident. These arise
from C
-hydrogens whose downfield shift is indicative of
-sheet structure. Furthermore, the presence of many amide
resonances downfield of 7.0 ppm indicates that there must be extensive
hydrogen-bonded structure, which protects these protons from exchange
with the D
O solvent. Both NMR and electrospray-mass
spectrometry show the presence of approximately 55 protected amide
hydrogens, very similar to the number found in human lysozyme
(38, 39) .
Figure 6:
One-dimensional H NMR spectrum
(500 MHz) of LYLA1 in D
O at 35 °C, pH
4.5.
In order to analyze the secondary
structure of LYLA1 in more detail, two-dimensional NMR methods were
employed. Using standard assignment techniques
(37) as
discussed previously for human lysozyme
(38) , the main chain
resonances of 118 of the 129 residues of LYLA1 were assigned (data
available on request). The NOE data were then analyzed to identify the
regions of the sequence involved in helical and sheet structure
(). Clear evidence for four helices was obtained,
corresponding closely to helices A-D in the structure of the
parent human lysozyme
(38, 40) . Importantly, the
C-helix is clearly evident; this corresponds to a region of the
inserted
-lactalbumin sequence. The NOE analysis is also
indicative of a
-sheet region between residues 42 and 61, exactly
as found in the human protein. Similarly, NOEs characteristic of the
3
helices in the parent structure were identified; one of
these (residues 121-126) corresponds to a region in the sequence
of human lysozyme and one (residues 79-85) to a part of the
inserted BLA sequence. Overall, therefore, marked similarity between
the secondary structure of LYLA1 and human lysozyme is evident from
these data, even in the inserted region.
-H
resonances. Residues involved in helices mostly have C
-H shifts
upfield of the corresponding random coil values, whereas those in
-sheets are shifted downfield. This approach is simplified in the
chemical shift index approach
(41) used in .
Comparing the C
-H chemical shifts of HLY and LYLA1, not only are
the overall patterns identical, but also the individual amplitudes are
closely similar (Fig. 7, A and B). This is
particularly true for the first 72 and the last 18 residues
(Fig. 7 C), showing that their environment in nearly all
of the HLY-derived part of the chimera is essentially identical to that
of the parent HLY. There are two isolated exceptions, Lys-13 and
Ala-32. The latter residue is found in the B-helix, very close to the
interface with the C-helix of the inserted section. Lys-13 is found on
the final turn of the A-helix, and it is this part of the helix which
makes contact with the N terminus of the C-helix in the inserted
section.
Figure 7:
Residual
C-H shift comparison. Residual chemical shifts are calculated by
subtracting the random coil shift (37, 41) from the measured shift.
A, human lysozyme residual chemical shifts. Elements of
secondary structure are indicated. B, LYLA1 residual chemical
shifts. Residues 75-78 and 87-89 have not been assigned.
C, difference in residual chemical shift between LYLA1 and
HLY. The 15 most perturbed resonances are
labeled.
The assignment of the two-dimensional H
spectrum of LYLA1 has also enabled the regions of structure where
backbone amide hydrogens are protected from exchange with solvent
hydrogens to be identified. In Fig. 8, the fingerprint region of
a two-dimensional COSY spectrum of LYLA1, recorded after the protein
was exposed to D
O at pH 4.5 and 35 °C for several
hours, is shown. The resonances in this spectrum arise only from those
amide hydrogens whose exchange is slow on the time scale of this
experiment and show that amides in all major regions of secondary
structure are protected, including at least four of the six helices and
both regions of
-sheet structure. The pattern of protection is
broadly consistent with that of human lysozyme
(38) .
Importantly, it includes protection in the substituted region of the
sequence, notably in the 3
helix (residues 80-85)
and the C-helix (residues 90-101) and the region of the sequence
where Ca
binding occurs.
Figure 8:
500
MHz COSY spectrum of LYLA1 at pH 4.5 and 35 °C showing the
fingerprint region where correlations between C-H and amide NH
protons are observed. The sample was dissolved 1 h prior to the
recording of the spectrum, which took a total of 12 h. The resonances
in the spectrum, therefore, all arise from amide hydrogens which are
significantly protected from exchange with the D
O solvent.
The amides are largely located in regions of secondary structure in the
protein, indicated in Table IV. The prominent peak at 5.61, 8.03 ppm is
unassigned but correlates closely with a similar resonance in the COSY
spectrum of BLA ( C. M. Dobson, unpublished data); it must therefore
arise from the amide of a residue in the inserted region of LYLA1. The
unlabeled peak at the bottom right-hand corner of the figure
is due to aromatic proton correlations.
Thermal Denaturation
Fig. 9
shows the
thermal transition curves of LYLA1 at pH 4.5, measured in the absence
( A) and in the presence of 10 mM Ca ( B), respectively. In Fig. 10 A, the same
unfolding data are presented in terms of the apparent fractional extent
of unfolding ( f
) versus temperature.
This fraction was calculated from the ellipticity changes at 270 nm
(near-UV) and 222 nm (far-UV) and also from the red shift of the
emission maximum of tryptophan fluorescence upon increasing
temperature. In all cases, the transition is fully reversible at this
pH. In the presence of 10 mM Ca
, the
unfolding curves obtained from CD measurements in the far- and near-UV
coincide and indicate a T
(temperature at the
midpoint of a conformational transition) value of 90 °C. Under
these conditions, both the secondary (222 nm) and tertiary structure
(270 nm) unfold simultaneously in a highly cooperative way.
Figure 9:
Thermal unfolding of apo- and
Ca-LYLA1 followed with CD at 270 nm ( left
axes) and 222 nm ( right axes). A, residual
ellipticity at 270 nm ( open symbols) and 222 nm ( filled
symbols) for apoLYLA1 in 10 mM NaAc, 90 mM NaCl,
pH 4.5; B, at 270 nm ( open symbols) and 222 nm
( filled symbols) for LYLA1 in 10 mM NaAc, 90
mM NaCl, 10 mM Ca
, pH
4.5.
Figure 10:
Thermal
unfolding of LYLA1 ( A), mutant M4 ( B), and HLY
( C) in the presence or absence of Ca.
Upper part, apparent fractions were calculated from residual
ellipticities measured for the apo forms ( squares) in 10
mM NaAc, 90 mM NaCl, pH 4.5, for the Ca
forms ( circles) in the same buffer with 10 mM
Ca
added. In the case of HLY, only the data in the
absence of Ca
were shown as they coincide with those
in the presence of Ca
. Residual ellipticities were
measured in the near-UV at 270 nm ( open symbols) and in the
far-UV at 222 nm ( filled symbols) or at 228 nm in the case of
HLY. Thermal unfolding of apoLYLA1 was also followed by tryptophan
fluorescence ( crosses) measured as the ratio of emission
intensities I
nm/I
nm. Lower part,
population of the N, I, and U states is shown for the apo ( dashed
lines) and the Ca
forms ( full lines),
respectively.
The
thermal unfolding of the apo form of LYLA1, followed by CD at 270 nm
and by tryptophan fluorescence, also shows a single cooperative
transition, with a T of 72.6 °C. Thus, the
thermal stability of the chimera is considerably lower in the
Ca
-depleted form than in the
Ca
-bound form. The unfolding process of the apo form
starts at 65 °C and is complete by about 80 °C. In contrast,
the transition begins only at 70 °C when followed by CD at 222 nm,
resulting in a T
value of 80.9 °C. Thus, the
unfolding of apoLYLA1 does not follow a simple two-state mechanism, but
involves population of an intermediate state, since the tertiary
structure denatures at lower temperatures than the secondary structure.
On the assumption that the residual ellipticity at 222 nm is the same
for the native ( N) and the intermediate ( I) state,
calculations of the fractional amounts of these and of the unfolded
( U) state were carried out (Fig. 10 A). These
indicate that as much as 65% of the protein is in the I state at 77
°C.
values (270
versus 222 nm) was also observed in the case of the apo form
of M4, a Ca
-binding mutant of HLY
(Fig. 10 B). Here, however, the difference amounts only
to 3 °C. In contrast, in the case of HLY, whether Ca
was present or not, a simple two-state mechanism was found to
occur under these conditions, since unfolding measured both at 270 and
228 nm coincides (Fig. 10 C). The resulting
T
value (78 °C) is far below that of the
Ca
form of LYLA1.
-lactalbumin is calculated using the spatial coordinates of
the Brookhaven Protein Data Bank
(35) , a root mean square value
of 1.167 Å is obtained for the main chain atoms, indicating a
very pronounced three-dimensional homology. Slight deviations are
localized in the loop region between residues 62 and 75 and in the
region of the C terminus, similar to the differences between HLY and
BLA, the latter deduced by homology modeling from the baboon
equivalent. This formed the basis for the rationale followed in the
present work, i.e. to transplant parts of BLA into homologous
regions of the HLY molecule and vice versa with the aim of
obtaining chimeric molecules possessing hybrid functions but with
conserved conformations.
-lactalbumins might have evolved from a
common ancestor through exon shuffling
(22, 45) . The
basic function of lysozyme resides in a single exon
(45, 46) , which has led Kumagai et al.(47) to transplant this exon of hen lysozyme into goat
-lactalbumin. Objections to this rationale
(48, 49) are the fact that the complete muramidase function of
lysozyme requires residues from different exons and that frequently
exon boundaries arise in the middle of secondary structure elements.
site.
However, as two of the essential aspartate ligands of the
Ca
ion (Asp-87 and Asp-88; BLA numbering) are located
on the central
-helix (helix C) adjacent to the Ca
loop, we finally chose to co-transplant this whole helix together
with the authentic BLA-derived Ca
-binding loop and
the appending N-terminal arm which contains a 2-turn
3
-helix.
-H
shift differences (Fig. 7 C). It is immediately obvious
that most of them are either part of the substituted region or, when
belonging to the HLY part, are in direct contact with this segment. The
latter probably experience different packing interactions than when
present in the parent HLY. Of particular interest is the subset of
residues 98, 99, 100, 101, 108, and 111 (LYLA1 numbering). These
residues form a cluster between helices C and D. The former set of four
constitutes the last turn of the inserted helix C. In view of the
abovementioned single residue deletion, a conformational difference
with the homologous turn in HLY is expected.
Figure 11:
Position of the C-H resonances
showing the largest differences in residual chemical shift between
LYLA1 and HLY. The gray shaded area represents the inserted
BLA sequence, filled circles represent the position of the
carbons with a difference in residual chemical shift of >0.2
ppm. This figure was produced using Molscript software
(55).
The hybrid was
expressed in substantial amounts in the supernatant of yeast cultures,
although yields were markedly lower than for wild-type HLY. This may be
due to the presence of the BLA part of the molecule, since we have
observed before that using the same promotor recombinant BLA is
recovered with a yield of only 1-2 mg/liter
(21) . In any
case, the lower yield of LYLA1 cannot be attributed to a lower
stability of the hybrid molecule since in the presence of
Ca its T
is even higher than
that of the parent HLY. The occurrence of proteolysis can also be ruled
out as no fragments were found by radioimmunoassay after each
chromatographic purification step.
-binding loop was indicated by the pronounced
reactivity of the chimera with polyclonal anti-loop antibodies. The
binding constant K
= 2.50 (± 0.04)
10
M
determined by
Fura-2 titration, indicates a strong site intermediate in binding
strength between that of BLA
(27) and the HLY mutant M4
(). Although in the latter all the necessary
Ca
ligands have been introduced, their conformation
seems less optimal for binding than in the case of BLA. The fact that
the K
of BLA is still about four times higher
than that of LYLA1 may have to do with the freedom of the former to
undergo pronounced conformational changes upon Ca
binding. Furthermore, Ca
binding to LYLA1 is
characterized by a small negative
H° value and a
large positive
S°. From similar measurements of
Ca
binding to goat
-lactalbumin Desmet et
al.(50) estimated the fraction of the total
H° and
S° values that is due to
``pure'' binding, i.e. binding not accompanied by
conformational changes, to amount to -36 (± 4) kJ
mol
and +54 (± 12) J mol
K
, respectively. Whereas the thermodynamic
data obtained for the M4 mutant come very close to these values, as
discussed elsewhere
(16) , those for LYLA1 are quite different.
These deviations could be due to small conformational alterations upon
Ca
binding occurring in the chimera but not in the M4
mutant.
to LYLA1 causes only
small changes in conformation as indicated by the CD spectra in both
the far- and near-UV range (Fig. 4). By analogy with the CD
spectra, virtually no change in the tryptophan fluorescence spectrum is
observed upon Ca
binding (Fig. 5). The spectra
of both the apo and the Ca
form of LYLA1 closely
resemble that of HLY, in which exactly the same tryptophan residues are
present. Thus, the newly implanted BLA sequence barely affects the
fluorescence behavior of these residues. The
of the
emission spectrum is very slightly blue-shifted, indicating that in the
hybrid the Trp residues are somewhat more shielded from the solvent
than in HLY. In contrast with BLA, upon withdrawal of the
Ca
ion the fluorescence spectrum does not undergo a
drastic red shift, indicating that Trp residues do not become more
exposed to the solvent.
-lactalbumin sequence causes
small but critical structural changes in the active site region which
affect its lytic activity. An indicator of conformational changes that
might have occurred in the active cleft is the affinity for inhibitor
molecules like (GlcNAc)
. This molecule is able to bind to
lysozymes, but it is not susceptible to hydrolysis. In the presence of
1 mM Ca
at pH 7.4, the chimera shows a
rather high relative affinity for (GlcNAc)
(pEC
= 3.84), although this is 2-fold lower than in the case of
natural human lysozyme (pEC
= 4.12). These data
suggest that the active cleft of the chimera is less suitable for
(GlcNAc)
binding than that of human lysozyme. It has
already been pointed out from NMR analysis that residues 98-101
(LYLA1 numbering) show conformational or environmental differences
compared to the equivalent HLY residues. Also, as discussed above the
deletion of a single residue at the C-terminal end of helix C could
cause the two subsequent residues to adopt a BLA-like conformation.
This region is likely to constitute part of the (GlcNAc)
binding site if binding can be conceived to occur as in HLY.
Therefore, these considerations can also, at least partially, explain
the reduced lytic activity toward bacterial cell walls.
binding is its
effect on the thermal stability of the chimera. Upon increasing the
temperature, the Ca
form denatures cooperatively
according to a simple two-state mechanism as is the case for wild type
human lysozyme under similar conditions. The observed T
is even higher than that of HLY. The unfolding of apoLYLA1 at pH
4.5, however, followed by ellipticity measurements at 222 and 270 nm,
respectively, does not obey a simple two-state mechanism involving only
the native (N) and the fully denatured state (U). The noncoincidence of
both unfolding curves can be explained assuming a three-state unfolding
mechanism with a partially unfolded intermediate state (I), referred to
as a ``molten globule'' state. In the temperature zone from
65 to 90 °C a fraction (f
) of the chimera is present in
the intermediate state (Fig. 10 A). Similarly, for BLA a
molten globule state is found under various experimental conditions
(51, 52, 53) . Up to now no such intermediate
state has been reported for HLY under such conditions, although we
recently found evidence for a partially folded state at very low pH
(17) . Therefore, it is extremely interesting to note that in
the hybrid, LYLA1, the capacity to form a molten globule state at pH
4.5 has been transferred together with the implantation of a restricted
part of BLA. Apparently this part of the molecule is a key element for
molten globule formation. Recently it was shown that the helical domain
of human
-lactalbumin in isolation forms a molten globule with the
same overall fold of the intact molecule
(54) . Nevertheless,
with the present work the question remains whether it is the
Ca
-binding loop or helix C that determines this
character. On the one hand, the apo form of the
Ca
-binding mutant M4
(16) shows an
intermediate state during thermal denaturation at pH 4.5
(Fig. 10 B). This suggests that the peculiar
aspartate-rich sequence of the Ca
site is at least
one of the contributions to the stability of the molten globule state.
In accord with this we have described the existence of an intermediate
state for the apo form of the Ca
-binding equine
lysozyme
(13) . On the other hand, the fractional population of
intermediate state molecules is very limited in M4, amounting only to a
maximum of about 32%, compared with 65% in LYLA1. This suggests that
helix C of the BLA molecule itself carries important features that
determine molten globule propensity. In accord with this conclusion,
this helix has been found to have a high level of persistence within
the molten globule state of
-lactalbumins
(12) .
-binding loop and
helix C of BLA we have been able to introduce into HLY one of the most
striking properties of BLA, i.e. the ability to form a stable
molten globule state under relatively mild conditions. By optimization
of the length of the transplanted BLA segment we hope to define further
the structural basis for this interesting characteristic of
-lactalbumins and hence to understand the factors stabilizing
intermediate states of proteins in general.
Table:
Comparison of the amino acid sequences of the
chimeric protein LYLA1 and its two parental proteins, human lysozyme
and bovine -lactalbumin
Table:
Thermodynamic parameters of Ca binding to LYLA1, compared with those for BLA, HLY, and the HLY
mutant M4 at 25 °C
Table:
Binding of
(GlcNAc) to LYLA1, HLY, and mutant M4 measured from
enhancement of tryptophan fluorescence
or 1 mM EDTA. The logarithm of the (GlcNAc)
concentration,
needed to obtain half-maximal enhancement of fluorescence at 325 nm
(pEC
), was considered to be a measure of relative binding
affinity. Also the ratio of the maximally enhanced versus basic fluorescence intensity at 325 nm
(I
/I
) was calculated. At pH 6.0, in the
presence of EDTA, HLY solutions were slightly opaque, prohibiting
spectral measurements.
Table:
Secondary structure of LYLA1 derived
from chemical shift index analysis (41) and from NOE spectroscopy,
compared with HLY (38, 40)
-lactalbumin; LYLA1, chimera obtained from
human lysozyme by substituting the central part for the homologous
sequence of bovine
-lactalbumin as described in the text; CAT,
chloramphenicol acetyltransferase; (GlcNAc)
=
N,N`, N"-triacetylchitotriose; Fura-2,
1-[2-(5`-carboxyoxazol-2`-yl)-6-aminobenzofuran-5-oxy]-2-(2`-amino-5`-methylphenoxy)-ethane- N,N,N`, N`-tetraacetic
acid; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.