Structural and Folding Dynamic Properties of the T70N Variant of Human Lysozyme*,
Gennaro Esposito
,
Julian Garcia
,
Palma Mangione ¶,
Sofia Giorgetti ¶,
Alessandra Corazza
,
Paolo Viglino
,
Fabrizio Chiti ||,
Alessia Andreola ¶,
Pascal Dumy
,
David Booth **,
Philip N. Hawkins ** and
Vittorio Bellotti ¶ 
From the
Dipartimento di Scienze e Tecnologie
Biomediche, Università di Udine, 33100 Udine, Italy, the
Laboratoire d'Etudes Dynamiques et Structurales
de la Séléctivité, University Joseph Fourier of Grenoble,
38041 Grenoble Cedex 9, France, the ¶Dipartimento
di Biochimica, Università di Pavia, Centro Interdipartimentale di
Biologia Applicata, Laboratorio Biotecnologie Istituto di Ricovero e Cura a
Carattere Scientifico, Policlinico San Matteo Pavia, 27100 Pavia, Italy, the
||Dipartimento di Science Biochimiche,
Università di Firenze, 55100 Firenze, Italy, and the
**Centre for Amyloidosis and Acute Phase Proteins,
Department of Medicine, Royal Free and University College Medical School,
London NW3 2PF, United Kingdom
Received for publication, October 28, 2002
, and in revised form, April 22, 2003.
 |
ABSTRACT
|
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Definition of the transition mechanism from the native globular protein
into fibrillar polymer was greatly improved by the biochemical and biophysical
studies carried out on the two amyloidogenic variants of human lysozyme, I56T
and D67H. Here we report thermodynamic and kinetic data on folding as well as
structural features of a naturally occurring variant of human lysozyme, T70N,
which is present in the British population at an allele frequency of 5% and,
according to clinical and histopathological data, is not amyloidogenic. This
variant is less stable than the wild-type protein by 3.7 kcal/mol, but more
stable than the pathological, amyloidogenic variants. Unfolding kinetics in
guanidine are six times faster than in the wild-type, but three and twenty
times slower than in the amyloidogenic variants. Enzyme catalytic parameters,
such as maximal velocity and affinity, are reduced in comparison to the
wild-type. The solution structure, determined by 1H NMR and
modeling calculations, exhibits a more compact arrangement at the interface
between the
-sheet domain and the subsequent loop on one side and part
of the
domain on the other side, compared with the wild-type protein.
This is the opposite of the conformational variation shown by the
amyloidogenic variant D67H, but it accounts for the reduced stability and
catalytic performance of T70N.
 |
INTRODUCTION
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Amyloidosis is an emerging category of diseases characterized by the
extracellular accumulation of protein aggregates that share a common fibrillar
conformation. The 20 proteins that can generate amyloid deposits in humans are
extremely heterogeneous in function and structure, but, along the pathological
transformation leading to aggregation and precipitation, all of them exhibit
the same peculiar conformational pattern named cross-
structure,
irrespective of the parent-starting arrangement
(1). The lack of any sequence
similarity and folding analogy among the amyloid-forming proteins led Dobson
to conclude that the ability to form cross-
structure, wherein hydrogen
bonds are formed between polypeptide chains in directions parallel to the
fiber axis, is a generic property of polypeptide chains
(2). Investigations of
structure
(34),
folding dynamics
(57),
and fibrillogenesis (3,
8) of the initially reported
amyloidogenic variants of lysozyme have made important contributions to a
better understanding of the process involved in the conversion of globular
proteins into amyloid fibrils. Amyloidogenic lysozyme represents probably the
most convenient and informative model of fibrillogenesis from a globular
protein. Besides being, in fact, one of the best characterized enzymes, its
fibrillogenic mechanism is not influenced by protein fragmentation; nor, to
our knowledge, does the wild-type species generate amyloid deposits in
vivo, even in the elderly. Thorough analysis of several biochemical
properties of the amyloidogenic variants in comparison to the wild-type
species showed that pathogenic lysozymes are less stable than wild-type
(38).
This thermodynamic destabilization correlates with an increased concentration
of partly unfolded intermediates that self-aggregate into fibrillar polymers.
In this study we present the biochemical and structural characterization of a
new natural variant of human lysozyme, T70N
(9), that displays the general
properties of a less stable and less efficient enzyme in comparison to
wild-type but does not undergo pathological fibrillar conversion in
vivo. The T70N variant is present in the British population with an
allele frequency of 5% (9). The
comparison of the biochemical characteristics of this variant with those of
the amyloidogenic species can highlight the role of some of the folding
abnormalities identified in the pathogenic species.
 |
EXPERIMENTAL PROCEDURES
|
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Clinical StudiesGenotyping for lysozyme T70N was performed
as described previously (9) in
110 patients with systemic amyloidosis referred to the United Kingdom Centre
for Amyloidosis, whose amyloid fibril type was initially uncharacterized but
in whom the clinical phenotype was consistent with lysozyme amyloidosis. The
variant was also sought in 23 patients with amyloid A amyloidosis and
complicating rheumatoid arthritis and 60 patients with immunoglobulin light
chain (AL)1
amyloidosis.
Protein ExpressionMutagenesis, expression, and purification
of human lysozymes were performed as described previously
(3).
Equilibrium DenaturationGuanidine hydrochloride
(GdnHCl)-induced unfolding of lysozymes was monitored by intrinsic
fluorescence emission at 340 nm with excitation of 295 nm. Fluorescence
measurements were performed on a PerkinElmer LS50 spectrofluorometer at 20
°C using a 10-mm light path cell. The protein solution (0.01 mg/ml) was
incubated for1hat increasing concentrations of GdnHCl in sodium phosphate
buffer, pH 6.5. The change of fluorescence as a function of denaturant
concentration was analyzed according to the method described by Santoro and
Bolen (10) to determine the
main thermodynamic parameters of the unfolding reaction.
Thermal UnfoldingFar and near UV circular dichroism (CD)
spectra were recorded on a JASCO 710 spectropolarimeter equipped with a
temperature control system using 1- and 10-mm path length quartz cuvettes over
the wavelength ranges 200240 nm and 250310 nm, respectively. The
protein concentration was 200 µg ml1 in H20,
pH 5, and the CD data were expressed as mean residue ellipticity (
).
The measurements were collected at different constant temperatures from 20 to
90 °C. Equilibrium thermal unfolding of T70N lysozyme was monitored by
ellipticity values at 222 and 270 nm and then normalized to the fraction of
unfolded protein using fu =
(
N)/(
U-
N),
where
is the observed parameter and
N and
U are the ellipticities of the native and unfolded protein,
respectively, extrapolated from the pre- and post-transition baselines at the
corresponding temperatures.
Enzyme Kinetics and Inhibitor AffinityLysozyme enzyme
kinetics were determined with p-nitrophenol
penta-N-acetyl-
-chitopentaoside (PNP-(GlcNAc)5)
(11). Reaction mixtures (1 ml)
containing 10 µg of each type of lysozyme, 0.1 unit of
-N-acetyl-hexosaminidase (NAHase), and various concentrations
of PNP-(GlcNAc)5 (from 6 to 60 µM) in 0.1
M citrate buffer, pH 5.0, were incubated at 37 °C. The
enzymatic reaction was stopped after 25 min by adding 2 M
Na2CO3 (0.5 ml), and then the resulting free
p-nitrophenol was determined spectrophotometrically at 405 nm.
Affinity to the chitotriose (NAG)3 was estimated by fluorescence
measurements (12) on a
PerkinElmer LS50 spectrofluorometer at 30 °C in MacIlvaine's buffer (100
mM citric acid and 50 mM Na2HPO4,
pH 7.2). Protein concentration was adjusted to 3 µM. Excitation
and emission wavelengths were 285 and 325 nm, respectively. Affinity constants
were determined by plotting log (Fo
F)/(F F
) against log [S],
where Fo, F, and F
are
the fluorescence intensities of solutions of enzyme alone, enzyme in the
presence of a concentration [S] of (NAG)3, and enzyme saturated
with the inhibitor, respectively.
Unfolding-Refolding KineticsUnfolding and refolding
experiments were carried out with a Bio-Logic SFM3 stopped flow fluorometer by
using an excitation wavelength of 285 nm and monitoring the total fluorescence
emission change over 320 nm. All of the experiments were performed at 20
°C with a cell path length of 2.0 mm. For unfolding experiments, 1 volume
of each type of protein in 20 mM acetate at pH 5.0 was 10-fold
diluted with a solution containing 6 M GdnHCl at pH 5.0. The
refolding reactions were carried out by mixing 1 volume of enzyme in 6
M GdnHCl with 10 volumes of 20 mM acetate at pH 5.0.
NMR Spectroscopy and ModelingNMR spectra were obtained at
500.13 MHz and 37 °C with a Bruker Avance spectrometer on 0.70.8
mM protein samples dissolved in H2O/D2O
(95:5) or 99.9% D2O. No addition was done to adjust the uncorrected
pH meter reading that was 4.2 for all measurements except for the isotope
exchange and the conformational analysis experiments, where the values ranged
between 4.5 and 4.9. A number of two-dimensional TOCSY
(13), DQF-COSY
(14), and NOESY
(15) spectra were acquired
with the sculpting scheme for solvent suppression
(16) using selective pulses of
35 ms, 1.1 s steady state recovery time, mixing times
(tm) of 2050 ms for TOCSY and 100 ms for
NOESY, t1 quadrature detection by TPPI
(17), or States method
(18) or gradient-assisted
coherence selection (echo/anti-echo)
(19). The spin-lock mixing of
the TOCSY experiment was obtained with MLEV17
(20) or DIPSI-2
(21) pulse trains at
B2/2
= 910 kHz. The acquisitions were performed
over a spectral width of 7002.8 Hz in both dimensions, with matrix size of
10242048 points in t2 and 256512
points in t1 and 32128
scans/t1 free induction decay (FID). Selective
one-dimensional NOESY experiments for temperature coefficient measurements
were run by replacing the first two 90° pulses of the standard sequence
with selective Gaussian-shaped pulses of 6 ms centered on the side-chain amide
resonance of interest. The water resonance was suppressed by appending an
excitation-sculpting module
(16) to the nonselective
detection pulse. The acquisitions were performed between 33 and 40 °C by
collecting 512 scans over 213 data points to monitor the chemical
shift of the exchanging, unperturbed side-chain-amide resonance with
sufficient precision (
= ± 0.8 ppb). Isotope exchange
data were collected in either forward (D2O) and backward mode
(H2O) by consecutive acquisition of TOCSY spectra (2.5 h/spectrum
in D2O and 15 min/spectrum in H2O) over variable time
intervals from solvent addition (6, 12, or 24 h). For the fast acquisitions in
H2O, only 80 t1 increments were
collected, and the standard linear prediction routine of Bruker software was
employed to expand the indirect dimension to 200 data points with 40
coefficients prior to two-dimensional Fourier transform. Protection factors
were obtained from the ratio between intrinsic (calculated) and apparent
(experimental) isotope exchange rates. The intrinsic rates were computed
through the parameters and procedures reported by Bai et al.
(22). All spectra were
referenced on the I106 C
H3 resonance at
0.61 ppm as reported previously
(23). Data processing and
analysis were performed using Felix (Accelrys, San Diego, CA) software with
shifted (6090°) square sinebell apodization and polynomial baseline
correction for NOESY data. Internuclear distances were quantified from the
cross-peak volumes of the NOESY spectra using some cross-peaks from Trp-64 as
calibrants. Tethered molecular dynamic (MD) simulations were performed by
using the Discover software (Accelrys), following the default protocol for
simulated annealing starting at a temperature of 600 K slowly lowered to 300 K
(time step = 1 fs, 8.6 ps MD, 500 conjugate-gradient minimization steps)
(24). The selected force field
was AMBER, and, to shorten the range of Coulomb interaction, a
distance-dependent relative dielectric constant,
r, was used
(
r(r) = 4r). Molecular structures were generated with Insight
II (Accelrys). To assess and improve the accuracy of the NOE restraint set,
the theoretical NOESY spectrum was back-calculated using the matrix doubling
module of Felix (total correlation time, 2 ns) and compared with the
experimental one. The restraints were subsequently refined, and a new MD was
performed until good concordance was reached between theoretical and
experimental NOESY spectra.
 |
RESULTS
|
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T70N Lysozyme Is Not Amyloidogenic in VivoSeveral clinical
and biochemical findings suggest that T70N lysozyme is not amyloidogenic. The
allele frequency of T70N is relatively high in the normal population (5/100)
(9). Both I56T and D67H are
very rare and have only been identified in kindreds with amyloidosis wherein
there is 100% penetrance (25).
If T70N was as amyloidogenic as these variants, 5% of the British population
might be expected to have amyloidosis. However, no individual with T70N
lysozyme amyloidosis has ever been identified. This has been the case despite
investigations designed to identify such individuals. Amyloid fibril type is
ultimately identified in all patients presenting at the United Kingdom Centre
for Amyloidosis. Prior to fibril identification, the prevalence of the T70N
lysozyme allele was determined in patients with systemic amyloidosis, the
clinical phenotype associated with I56T and D67H lysozyme amyloidosis. In
these 55 patients, the T70N allele frequency was 0.08 (9/110 alleles,
including one homozygote), similar to the allele frequency in the normal
population. In all other patients with amyloidosis seen at the clinic, the
fibril type had been characterized (except one, see below). In these eight
patients carrying at least one T70N allele, the amyloid fibril type was
subsequently identified as serum amyloid A (two patients), AL amyloid (four
patients), not amyloidosis (one patient, the homozygote), and hereditary
amyloidosis of unknown fibril type (one patient). In the one patient with
hereditary systemic amyloidosis, the fibril protein has yet to be
characterized. It is not lysozyme, and an individual in the kindred has the
T70N lysozyme but not amyloid. These data are consistent with the hypothesis
that T70N variant does not cause systemic amyloidosis in the British
population.
To test whether there was co-deposition of lysozyme within the amyloid
fibrils created by other proteins, we have isolated the natural amyloid
fibrils from amyloid deposits of one heterozygous T70N heterozygote patient
clearly affected by AL amyloidosis. No lysozyme was detected in the fibrils by
immunoblot. To test whether T70N lysozyme was interacting with other fibril
types, the allele frequency in rheumatoid arthritis patients (2 T70N from 56
alleles) and AL (4/120) amyloidosis groups was compared. Asn-70 was not over-
or under-represented, thus neither promoting nor inhibiting fibril formation.
Finally, in the one subject homozygous for the mutation, serum lysozyme
concentration was determined, and, according to the lysoplate method, the
circulating protein was 12 mg/liter (nv 413 mg/liter.)
Equilibrium Denaturation of T70N LysozymeThe unfolding of
the T70N variant was monitored at equilibrium by intrinsic fluorescence
emission at 340 nm as a function of denaturant concentration at pH 6.5 and 20
°C. The unfolding curves for the wild-type, T70N, I56T, and D67H,
normalized to the fraction of unfolded protein (fu), are
shown in Fig. 1. All
transitions are characterized by the presence of a single sharp change in the
fluorescence intensity that is typical of cooperative transition in a
two-state system. The transition midpoints are reduced, as compared with that
of the wild-type protein, by 0.5, 0.9, and 1.3 denaturant concentration units
for the T70N, D67H, and I56T, respectively. The data for the I56T and D67H
variants are in good agreement with previous data of Takano et al.
(6). The values of
GH2O of unfolding, calculated according to Santoro
and Bolen (10), indicate that
the three variants, T70N, D67H and I56T, are destabilized, in comparison to
the wild-type, by 3.7, 4.7, and 7.2 kcal/mol, respectively.
Thermal UnfoldingWe have used circular dichroism to monitor
T70N lysozyme unfolding behavior upon heating from 20 to 90 °C. The
measurements were performed in the far and near UV regions
(Fig. 2, a and
b) to estimate the equilibrium thermal unfolding of the
protein at pH 5. The coincidence of the two transition curves by the CD data
at 222 and 270 nm, normalized to the apparent fraction of unfolded species
(Fig. 2c), was
observed over the entire temperature range studied. Such behavior is quite
similar to the cooperative two-state unfolding displayed by the wild-type
protein under these conditions
(3), even if the midpoint of
thermal denaturation of T70N variant is 3 °C below that of the wild-type
lysozyme. From the analysis of CD measurements, there is no evidence of the
existence of an intermediate state with a helical secondary structure but
lacking tertiary interactions as was previously documented in the
amyloidogenic species (3).
Enzyme Catalysis PerformanceThe enzyme kinetics data
(Table I) suggest that T70N
lysozyme has a reduced enzymatic activity with respect to wild-type,
comparable with that reported for the D67H variant. This comparatively poor
performance is the combined result of both a lower substrate affinity and a
less efficient turnover. The reduced affinity of T70N for the chitotriose
(NAG)3, the trisaccharide inhibitor of lysozyme, which can be
inferred from the dissociation constant (Kd)
value of Table I, is also
confirmed in D67H and I56T, but the T70N variant has the lowest affinity for
(NAG)3 of the all natural human lysozyme variants.
Unfolding-Refolding Kinetics of Mutant Human LysozymesTo
assess the effects of the T70N substitution on the folding and unfolding
kinetics, comparative stopped-flow kinetic studies of the reversible
unfolding-refolding process were performed. The unfolding-refolding reactions
were monitored by fluorescence intensity. The unfolding kinetics of T70N are
described by a single exponential function, as reported previously
(56).
The refolding reaction from the guanidine-denatured protein consists of two
phases in which the fast phase is predominant, in amplitude, over the slow
phase as shown previously
(56).
No significant differences were found between T70N variant and the wild-type
protein in the rates of the refolding phases (data not shown). On the
contrary, the unfolding process of the T70N is 34 times faster than
that of the wild-type protein. Fig.
3 depicts the unfolding kinetics of wild-type, T70N, D67H, and
I56T. The unfolding rates for the amyloidogenic mutants are consistent with
the data reported previously by Canet et al.
(5) and Takano et al.
(6) and are, respectively,
three and twenty times faster than that of the T70N variant.

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FIG. 3. Unfolding kinetics monitored by stopped flow fluorescence. The
kinetic traces of four human lysozyme variants were acquired at pH 5 with 5.4
M GdnHCl at 20 °C. The data have been scaled to unit amplitude
(a. u.) of the fluorescence of the unfolded species.
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1H NMR Chemical Shift ChangesBased on the
assignments of wild-type human lysozyme
(23) and amyloidogenic
variants (4), the 1H
chemical shifts for all residues of the T70N lysozyme were carefully
controlled (at least for the backbone resonances) by standard methodology
(27). The deviations of the
HN chemical shift values with respect to the wild-type protein are
shown in Fig. 4a.
Comparison with the corresponding histograms reported for the I56T and D67H
mutants (4) suggests that the
T70N variant has a 
HN pattern somehow intermediate,
i.e. whereas the extent of
HN deviations is
generally limited, similar to I56T, a number of 
HN
above the average are observed, as with D67H, for residues 52, 63, 64, 67, 72,
and 77 (upfield) and residues 68, 69, 73, 76, and 79 (downfield). The
interpretation of amide chemical shift changes may be complex and tricky, but
there is no doubt that all of the variations seen with T70N are spread over
the corresponding locations of the
-sheet, the subsequent loop, and the
following 310 helix segment of the wild-type structure. For
proteins, 1H NMR chemical shifts exhibit an established correlation
with secondary structure only for H
resonances
(28). When compared with D67H,
the
H
of T70N show meaningful differences only at
positions 51, 61, 66, 68, 73, and 80 (Fig.
4b). In general, the accepted threshold for meaningful
difference, i.e. 
Ha
±0.1 ppm,
refers to comparison with peptides in a statistically disordered conformation.
Thus, to ascertain meaningfulness, the deviations of
Fig. 4b were analyzed
against the corresponding parameters obtained from the comparison with the
wild-type protein data, which, in turn, were compared with the basic peptide
shifts. In particular, on moving from the D67H to the T70N mutant, the
downfield shifts at residues 51, 61, and 66 suggest that the conformation and
extension of the
strands of the wild-type tend to be restored.
Similarly, a recovery of the wild-type geometry at the end of the turn-like
segment 7073 is likely to be responsible of the opposite shift observed
for residue 73. An analogous interpretation applies also to the upfield shift
of residue 68 and the downfield shift of residue 80.

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FIG. 4. 1H NMR chemical shift changes. Chemical shift changes of
HN (a) and H (b) resonances of
T70N lysozyme with respect to the corresponding values in the wild-type
(a) and D67H (b) species.
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Restrained ModelingTo test the structural inference
obtained from the assignment and analysis of chemical shift changes in mutant
T70N with respect to D67H, I56T, and wild-type proteins, a number of
unambiguously classified NOEs, connecting hydrogens in the region 4192
of the investigated mutant, were quantified to extract the relative
internuclear separations. The substantial invariance of the chemical shifts of
T70N with respect to the wild-type protein in the fragments that flank the
region 4192 should grossly reflect a persistence of the local spatial
arrangement of the canonical structure. It appears therefore conceivable to
adopt the geometry of the original structure outside the fragment 4192.
To constrain part of the structure in a fixed arrangement, tethered MD
simulations were performed using the standard tools of Discover software
(Accelrys) (24). Because of
the high degree of signal overlap in the lysozyme spectrum, the collection of
the NOESY cross-peaks for quantitative purposes was necessarily limited to
resolved correlations of the selected region resonances. Overall, 125
internuclear distances, mostly medium and long-range ones, were estimated and
employed for the tethered MD calculations. Two simulation cycles were
performed with the same set of experimental distance restraints and
parameters, but with two different starting geometries in analogy to the
structural deviations of the amyloidogenic lysozyme variants with respect to
wild-type protein (3). The
wild-type lysozyme structure, in practice the same as that of I56T mutant or
the structure of D67H mutant (PDB coordinate codes 1REX
[PDB]
and 1LYY
[PDB]
,
respectively) (3), were
alternatively imposed on the T70N sequence. The starting conformations diverge
only in correspondence of the
-sheet and subsequent loop
(3), i.e. within the
region 4192 that was left untethered and restrained only by the
experimental distances in MD runs. The results are displayed in
Fig. 5, where the backbones of
the two final and the two starting structures are superimposed in the regions
of interest. It can be readily seen that both the MD-restrained structures
converge toward a conformation similar to the wild-type protein, irrespective
of the starting arrangement. More precisely, the calculated structures appear
to adopt an even more compact arrangement than the wild-type one, because the
loop 6674 and the turn 4750 consistently shift in the same
direction toward the helical domain, just the contrary of the shifts observed
in D67H. Although the tertiary structure resulting from a tethered MD
simulation should be regarded with some caution when the extent of fixed and
restrained molecular moieties are comparable, the outcome of the calculation
is in qualitative agreement with the conclusions that were reached from
chemical shift arguments, namely that, of the natural lysozyme variants that
carry a mutation in the loop region of the
-domain, the mutant T70N
tends to preserve the wild-type structure, in contrast to the D67H mutant that
diverges significantly from the wild-type even before undergoing the
pathologic amyloid transition.
Conformation of Relevant Side ChainsBecause a drastic
geometry change of a few side chains around the mutation site of D67H
accompanies the loss of the wild-type H-bond network that has been considered
to affect significantly the stability of the lysozyme
-domain
(3), a detailed conformational
analysis of some relevant side chains in variant T70N was attempted.
Additional independent evidence was necessary to characterize in detail these
structural features because of the resolution limits of the tethered MD
results. In the wild-type structure, the side chain of residue 67 is involved
in one or possibly two H-bonds to residue 70 (T70N-D67O
1 and
T70O
1-D67O
1)
(Fig. 6a). The number
of H-bonds of residue 70 is conserved in the D67H mutant, although none of the
original side chain H-bonds survives for the mutated residue
(Fig. 6b). The
examination of the NOESY and DQF-COSY pattern of T70N and wild-type lysozymes
in H2O and D2O enabled us to conclude that both
molecules possess the same conformation of the Asp-67 side chain with
1
60°. This geometry is consistent with all
coordinate sets available for the wild-type lysozyme from x-ray data and,
because it can accommodate the mentioned H-bonds to residue 70, the question
arises whether the mutation of threonyl into asparaginyl in T70N still
supports those H-bonds. Unfortunately the spectra of the T70N lysozyme showed
resonance degeneration for the two prochiral H
s of Asn-70,
which hinders extracting their diastereotopic assignments and the local
conformation, as was done with Asp-67. The chemical equivalence of Asn-70
H
resonances may be only fortuitous rather than due to
rotational averaging that would not support an H-bond involvement of Asn-70
side-chain carboxyamide. High resolution one-dimensional NOESY measurements
enabled us to measure precisely the chemical shifts of a few side-chain amides
in the range 3340 °C. The calculated chemical shift temperature
coefficients of Asn-70 side-chain amides were 3 ppb/deg and 6
ppb/deg for the anti (H
21) and syn (H
22)
amide resonances, respectively. Values between 6 and 8 ppb/deg
were obtained also for other primary amide pairs of the T70N lysozyme. Because
|
/
T| > 45 ppb/deg is typically
observed for solvent-exposed primary and secondary amides when not involved in
intramolecular H-bonds, our result is consistent with the occurrence of an
H-bond at the Asn-70 anti-amide hydrogen. According to the structure obtained
from the tethered MD calculation described previously, Asn-70
H
21 could form two simultaneous H-bonds with Asp-67 and
Ser-61 side-chain oxygen atoms, with the Asp-67 side chain adopting a
1 value close to 60 °. Thus two independent lines of
evidence converge toward the same conclusion, namely that in the T70N lysozyme
the spatial arrangement of the Asp-67 side chain is preserved along with some
H-bonds that are part of the interaction network within the large loop of the
-domain in the wild-type lysozyme.

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FIG. 6. Conformation of relevant side chains. H-bonding patterns within the
-domain large loop are shown as follows: wild-type, 69 N
··· 67 OD1 (0.437), 70 N ··· 67OD1
(0.175), 70 OG1 ··· 67 OD1 (0.661) (a); D67H, 66
ND2 ··· 70 O (0.59), 70 N ··· 66 OD1
(0.649), 70 OG1 ··· 67 O (0.503) (b); and the
T70N lysozyme, 70 ND2 ··· 61 O (0.459), 70 ND2
··· 67 OD1 (0.289), 73 N ··· 70 O
(0.045) (c). The occurrence of hydrogen bonds was ascertained using
the software WHATIF (38) with
a routine that positions polar hydrogen atoms by optimizing the total hydrogen
bond energy (26). A score
between 0 and 1, reported in brackets, is given for each possible hydrogen
bond and takes into account donor/acceptor types, the H-acceptor distance, the
donor-H-acceptor angle, and the position of the H with respect to the
acceptor. In all panels only the heavy atoms are shown, and the color code is
as follows: gray, carbon; blue, nitrogen; red,
oxygen; and yellow, sulfur.
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Isotope Exchange MeasurementsTo further confirm the
previous structural conclusions, 1H-2H amide exchange
rate determinations were performed on the mutant T70N. The protection factors
were calculated from the experimental apparent kinetic constants. The values
should be compared with the analogous data obtained for the wild-type and the
mutant D67H (7), as shown in
Fig. 7, for a specific
selection. Apart from a few limited local discrepancies, which are likely to
arise from the specific experimental conditions of each determination, the
most important difference among our and previous data sets is observed in the
segment 6590, comprising the last stretch of the four-stranded sheet,
the loop, the 310 helix, and the connection turn to helix C
(according to the wild-type protein geometry). In particular, in T70N the NH
of Cys-65 recovers the extremely high protection factor that is lost in mutant
D67H, whereas for residue 66 a protection factor >104 is
obtained. The 310 helix appears more protected in mutant T70N than
even in the wild-type protein. Four residues in T70N (83, 84, 85, and 86)
exhibit slow or moderately slow amide exchange, with protection factors
ranging between 200 and 5000, against only two residues (84 and 85) in the
wild-type protein. A similar degree of protection is observed also for the
amides of residues 74, 77, 79, and 90 of T70N. The same amides, instead, were
reported to exhibit no exchange protection in the D67H variant in the
comparative study with the wild-type species
(7). Previous qualitative
1H-2H exchange data on human lysozyme at pH 3.8 had
shown slow amide exchange rates for residues 65, 66, 76, and 77 and
intermediate rates for residues 79 and 86
(23). Therefore the amide
exchange pattern of the T70N lysozyme can be considered quite related to that
of the wild-type protein.

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FIG. 7. Comparison of the protection factors. Protection factors within the
fragment 6590 of T70N (black bars), wild-type (gray
bars), and D67H (white bars) lysozyme. The bar is not reported
on the scale when the corresponding protection factor value was below
102. The values for wild-type and D67H are taken from Canet et
al. (7).
|
|
 |
DISCUSSION
|
---|
Linking Functional and Structural Properties of T70N
VariantCompared with the structure of wild-type lysozyme, the T70N
replacement generates a more compact arrangement at the interface between the
-sheet and subsequent loop from one side and part of the
domain
from the other side. In particular, the large loop 6674 moves toward
helix C, followed by
-sheet 4255 that extends in a plane nearly
perpendicular to the former loop and thus shifts away from helix D. This is
the opposite of the conformational variation observed in mutant D67H
(3). Based on the deuterium
exchange results, some additional structural deviations of T70N lysozyme
should occur in regions further apart from the mutation site. The differences
should concern the Ser-80 capping role, the actual extension of the
310 helix, and the angle between the latter and helix C as imposed
by the intervening turn. However, the extent of divergence of T70N from the
wild-type structure is much more limited than the deviations observed with the
D67H mutant, and, overall, when compared with the latter, T70N resembles the
wild-type species much more closely (Fig.
5). The network of H-bonds that stabilizes the
-domain of
lysozyme (3) appears preserved
in the variant containing asparagine at position 70, as demonstrated by direct
determination of conformation and H-bond involvement for the side chains of
Asp-67 and Asn-70, respectively (Fig.
6). In the wild-type structure, that network connects Thr-52,
Tyr-54, Ser-61, Asp-67, and Thr-70, and its rupture in the D67H mutant
determines extensive structural modifications and pathological destabilization
(3). Similar effects have been
documented also for mutation at residue 70 with Ala and Val
(29). The T70A substitution
results in local disorder that prevents structure determination. The analogous
mutation into Val, however, induces a significant rearrangement in the region
6878 that preserves the local geometry and packing through the
involvement of the Lys-69 side chain for the H-bonds and that of Arg-62 and
Tyr-63 for interaction with the isopropyl group. Indeed, at 64.9 °C and pH
2.7, mutant T70V is destabilized by 2.9 kJ/mol with respect to wild-type,
whereas T70A loses 6.2 kJ/mol of stability
(29). The structural elements
obtained by our 1H NMR study qualitatively account for the
stability properties of T70N. The wild-type packing geometry between the
and
-domains is largely preserved along with the H-bonding
pattern within the
-domain loop. Thus, the corresponding contributions
to the folding free energy should be conserved as well. Based on the analogy
of the comparison between T70V and T70A or the T70N and D67H mutants, one may
be tempted to link, in any lysozyme, the conservation of the H-bond network of
the
-domain or the whole structure with folding stability. The
significant destabilization of mutant I56T, despite substantial invariance
with respect to wild-type fold
(3), suggests that such a
generalization may be too simplistic and risky. The features of the H-bond
network of the
-domain should be explicitly addressed. In addition, the
surface hydration structure should also be considered
(30) before drawing stability
conclusions from structural invariance.
The 1H NMR results qualitatively account for the activity loss
of T70N also (Table I). Because
of the shift of
-sheet 4255, the width of the catalytic cleft is
increased compared with that of the wild-type protein
(Fig. 8), and, hence, a
decrease of affinity should ensue that is expected to become more evident the
smaller the substrates or inhibitors are. A decreased affinity should be
responsible for the significant increase of the equilibrium dissociation
constant of T70N and the (NAG)3 inhibitor. Along the same lines,
one can also explain the data obtained with the chitin oligosaccharide used
for testing the catalytic activities. The Km of
T70N increases and reaches a similar value to that seen for the pathologic
mutant D67H, for which the activity loss results from a more complex
conformational rearrangement. Overall however, the affinity loss is reduced
with a substrate larger than the (NAG)3 inhibitor as argued from
comparison of Km and
Kd values
(Table I).

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FIG. 8. Best fit superposition of wild-type lysozyme-NAG complex (PDB ID 1LZR
[PDB]
)
and the T70N lysozyme in the region of the catalytic site. The wild-type
protein structures with and without substrate do not show any major difference
in practice. A single ribbon trace is drawn outside the region 4290.
The C traces are red for wild-type and
blue for the T70N variant. The presence of the substrate, shown as a
green space-filling model, highlights the increased width of the
catalytic cleft in the variant protein.
|
|
The T70N Variant Is Less Destabilized Compared with the I56T and D67H
Amyloidogenic VariantsThe equilibrium unfolding experiments show
that the T70N variant is less stable than the wild-type lysozyme.
Nevertheless, the free energy destabilization resulting from mutation is less
pronounced than that resulting from the previously characterized amyloidogenic
variants I56T and D67H. This "intermediate" behavior of the T70N
variant is also observed in its unfolding rate, which is faster than that of
the wild-type but slower than those of the two amyloidogenic variants.
Finally, as discussed above, the structural perturbation derived from the T70N
substitution is not as marked as that resulting from the two established
amyloidogenic mutations. These three observations are all correlated. The
minor structural perturbation of the T70N variant, compared with those of the
amyloidogenic variants, indeed results in a lower free energy destabilization
and a less important acceleration of the unfolding reaction. The ultimate
result is that the partially denatured state that is thought to promote
fibrillogenesis of lysozyme in vivo
(3,
7) is not as significantly
populated as in patients in which one of the two amyloidogenic variants is
present. Fully consistent with these data are the results obtained by
monitoring thermodenaturation in the near and far UV CD. By this analysis, one
of the main differences in the unfolding pathways of normal and pathological
species was previously highlighted
(3). Temperature-dependent
unfolding of the amyloidogenic variant appeared to be not cooperative, and the
partially unfolded state was highly populated near the midpoint of unfolding.
T70N, to the contrary, presents a cooperative unfolding transition in which
the loss of tertiary and secondary structures are concomitant. These data
prompt us to exclude the presence of the partially unfolded structure at the
midpoint of denaturation for this new variant, and, even if the melting point
is lower, the mode of denaturation appears to be quite similar to that
reported for the wild-type lysozyme.
Concluding RemarksGlobular protein stability has been
inversely correlated with the propensity to form amyloid fibrils in
vitro
(3133).
The T70N lysozyme variant represents, in the category of amyloidogenic
proteins of medical interest, the first globular protein in which the mutation
destabilizes the molecule but does not cause, in vivo, the
pathological conversion of the globular protein into amyloid fibrils. The
comparison of the properties shared by wild-type and T70N, on one side, with
those shared by I56T and D67H, on the other, allows one to highlight the
abnormalities involved in vivo in the genesis of amyloid disease. The
biochemical effects of the mutation make this new lysozyme variant less
active, less stable, and less protected than the wild-type form against a
denaturing environment, but, notwithstanding the destabilizing mutation, the
level of amide exchange protection is not affected or is even increased, as
for helix 310. This means that the local stability, which contributes
to the overall folding free energy, is preserved, i.e. structures,
with unfolded deviation beyond a certain level to allow competitive formation
of the amyloid aggregate, are poorly represented within the statistical
ensemble of the native folded state
(34). Therefore the
conformational modifications induced by the replacement of Thr in position 70
with Asn minimize fluctuations from the folded to a partially folded or an
unfolded state, which appears to be one of the key pathogenic properties of
amyloidogenic variants (7).
Thus the hypothesis that amyloid transition involves those fluctuations is
consistent with our observations on T70N, wherein such fluctuations should be
much less extensive than in the pathological variants. In the field of new
therapeutic strategies against conformational diseases
(35) in which pathogenic point
mutations of target proteins affect their stability, the discovery of
destabilized variants devoid of pathological implication bears the relevance
of a finely adjusted model. As a matter of fact, stabilization through
synthetic ligands is becoming a promising approach against these diseases, and
encouraging experimental results have been provided for the amyloidogenic
variants of transthyretin (36)
or, for a completely different type of disease, the oncogenic variants of the
p53 protein (37). The
demonstration of the existence of thermodynamic and kinetic thresholds that
are lower than those of wild-type proteins but still adequate to protect from
the pathological conversion suggests that conformational diseases could be
prevented even with a partial stabilization of the pathological proteins.
 |
FOOTNOTES
|
---|
* The work of the Centre for Amyloidosis and Acute Phase Proteins is
supported by grants from Medical Research Council, the Wellcome Trust, the
Wolfson Foundation and National Health Service Research and Development Funds.
The National Health Service National Amyloidosis Centre is funded by the
United Kingdom Department of Health. This work was also supported by the
financial contribution of "Ministero della Sanità Italia"
(Ricerca finalizzata sulla malattia di Alzheimer, 020ALZ00/01), DSTB, Telethon
(grant number GP0186Y/01), "Ministero della Università e Ricerca
Scientifica Italy (through Fondo per gli Investimenti della Ricerca di Base
2002-2005 and COFIN 2002058218_003 projects) and by Fondazione CARIPLO. The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
The on-line version of this article (available at
http://www.jbc.org)
contains Table S1 (1H chemical shifts and amide protection
factors), Table S2 (NOE restraint list from NMR data of T70N lysozyme), Fig.
S1 (distance restraint histograms), Fig. S2 (TOCSY and NOESY fingerprints),
and Fig. S3 (overview contour maps). 

To whom correspondence should be addressed: Dipartimento di
Biochimica-Università di Pavia, Via Taramelli 3/b, 27100 Pavia, Italy.
Tel.: 39-0382-507783; Fax: 39-0382-423108; E-mail:
vbellot{at}unipv.it.
1 The abbreviations used are: AL, immunoglobulin light chain amyloidosis; CD,
circular dichroism; DQF-COSY, double quantum-filtered correlation
spectroscopy; GdnHCl, guanidine hydrochloride; MD, molecular dynamic;
(NAG)3,
-1,4-linked trimer of
N-acetyl-D-glucosamine; NOE, nuclear Overhauser effect; NOESY, NOE
spectroscopy; TOCSY, total correlation spectroscopy. 
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Prof. Mark Pepys for continuous support and valuable
discussion. We thank Dr. Christina Redfield for providing the NMR data of I56T
and D67H mutants. G. E. thanks Dr. A. Makek for assistance.
 |
REFERENCES
|
---|
- Sunde, M., Serpell, L. C., Bartlam, M., Fraser, P. E., Pepys, M.
B., and Blake, C. C. F. (1997) J. Mol.
Biol. 273,
729739[CrossRef][Medline]
[Order article via Infotrieve]
- Dobson C. M. (1999) Trends Biochem.
Sci. 24,
329332[CrossRef][Medline]
[Order article via Infotrieve]
- Booth, D. R., Sunde, M., Bellotti, V., Robinson, C. V., Hutchinson,
W. L., Fraser. P. E., Hawkins, P. N., Dobson, C. M., Radford, S. E., Blake, C.
C. F., and Pepys, M. B. (1997) Nature
385,
787793[CrossRef][Medline]
[Order article via Infotrieve]
- Chamberlain, A. K., Receveur, V., Spencer, A., Redfield, C., and
Dobson, C. M. (2001) Protein Sci.
10,
25252530[Abstract/Free Full Text]
- Canet, D., Sunde, M., Last, A. M., Miranker, A., Spencer, A.,
Robinson, C. V., and Dobson, C. M. (1999)
Biochemistry 38,
64196427[CrossRef][Medline]
[Order article via Infotrieve]
- Takano, K., Funahashi, J., and Yutani, K. (2001)
Eur. J. Biochem. 268,
155159[Abstract/Free Full Text]
- Canet, D., Last, A. M., Tito, P., Sunde, M., Spencer, A., Archer,
D. B., Redfield, C., Robinson, C. V., and Dobson C. M. (2002)
Nat. Struct. Biol. 9,
308314[CrossRef][Medline]
[Order article via Infotrieve]
- Morozova-Roche, L. A., Zurdo, J., Spencer, A., Noppe, W., Receveur,
V., Archer, D. B., Joniau, M., and Dobson, C. M. (2000)
J. Struct. Biol. 130,
339351[CrossRef][Medline]
[Order article via Infotrieve]
- Booth, D. R., Pepys, M. B., and Hawkins, P. N. (2000)
Hum. Mutat. 16,
180
- Santoro, M. M., and Bolen, D. W. (1988)
Biochemistry 27,
80638068[Medline]
[Order article via Infotrieve]
- Nanjo, F., Sakai, K., and Usui, T. (1988)
J. Biochem. 104,
255258[Abstract]
- Muraki, M., Harata, K., and Jigami, Y. (1992)
Biochemistry 31,
92129219[Medline]
[Order article via Infotrieve]
- Braunschweiler, L., and Ernst, R. R. (1983)
J. Magn. Reson. 53,
521528
- Piantini, U., Sørensen, O. W., and Ernst, R. R.
(1982) J. Am. Chem. Soc.
104,
68006801
- Jeener, J., Meier, B. H., Bachmann, P., and Ernst, R. R.
(1979) J. Chem. Phys.
71,
286292
- Hwang, T. L., and Shaka, A. J. (1995) J.
Magn. Reson. 112,
275279[CrossRef]
- Marion, D., and Wüthrich, K. (1983)
Biochem. Biophys. Res. Commun.
113,
967974[Medline]
[Order article via Infotrieve]
- States, D. J., Haberkorn, R. A., and Ruben, D. J.
(1982) J. Magn. Reson.
48,
286292
- Keeler, J., Clowes, R. T., Davis, A. L., and Laue, E. D.
(1994) Methods Enzymol.
239,
145207[Medline]
[Order article via Infotrieve]
- Bax, A., and Davis, D. G. (1985) J. Magn.
Reson. 65,
355360
- Shaka, A. J., Lee, C. J., and Pines, A. (1988)
J. Magn. Reson. 77,
274293
- Bai, Y., Milne, J. S., Mayne, L., and Englander, S. W.
(1993) Proteins Struct. Funct. Genet.
17,
7586[Medline]
[Order article via Infotrieve]
- Redfield, C., and Dobson, C. M. (1990)
Biochemistry 29,
72017214[Medline]
[Order article via Infotrieve]
- Dayringer, H. E., Tramontano, A., Sprang, S. R., and Fletterick, R.
J. (1986) J. Mol. Graph.
6,
8287
- Gillmore, J. D., Booth, D. R., Madhoo, S., Pepys, M. B., and
Hawkins, P. N. (1999) Nephrol. Dial.
Transplant. 14,
26392644[Abstract/Free Full Text]
- Hooft, R. W. (1996) Proteins
26,
363376[CrossRef][Medline]
[Order article via Infotrieve]
- Wüthrich, K. (1986) NMR Spectroscopy
of Proteins and Nucleic Acids, John Wiley & Sons, Inc., New
York
- Wishart, D. S., and Sykes, B. D. (1994)
Methods Enzymol. 239,
363392[Medline]
[Order article via Infotrieve]
- Takano, K., Yamagata, Y., Funahashi, J., Hioki, Y., Kuramitsu, S.,
and Yutani, K. (1999) Biochemistry,
38,
1269812708[CrossRef][Medline]
[Order article via Infotrieve]
- Funahashi, J., Takano, K., Yamagata, Y., and Yutani, K.
(2002) J. Biol. Chem.,
277,
2179221800[Abstract/Free Full Text]
- Chiti, F., Taddei, N., Bucciantini, M., White, P., Ramponi, G., and
Dobson, C. M. (2000) EMBO J.
7,
14411449[CrossRef]
- McCutchen, S. L., Lai, Z. H., Miroy, G. J., Kelly, J. W., and
Colon, W. (1995) Biochemistry
34,
1352713536[Medline]
[Order article via Infotrieve]
- Hurle, M. R., Helms, L. R., Li, L., Chan, W., and Wetzel, R. A.
(1994) Proc. Natl. Acad. Sci. U. S. A.
91,
54465450[Abstract]
- Luque, I., Leavitt, S. A., and Freire, E. (2002)
Annu. Rev. Biophys. Biomol. Struct.
31,
235256[CrossRef][Medline]
[Order article via Infotrieve]
- Carrell, R. W., and Lomas, D. A. (2002) N.
Engl. J. Med. 346,
4553[Free Full Text]
- Peterson, S. A., Klabunde, T., Lashuel, H. A., Purkey, H.,
Sacchettini, J. C., and Kelly, J. W. (1998) Proc.
Natl. Acad. Sci. U. S. A. 95,
1295612960[Abstract/Free Full Text]
- Bullock, A. N., and Fersht, A. R. (2001)
Nat. Rev. Cancer 1,
6876[CrossRef][Medline]
[Order article via Infotrieve]
- Vriend, G. (1990) J. Mol.
Graph. 8,
5256[CrossRef][Medline]
[Order article via Infotrieve]