The metastability of human UDP-galactose 4ʹ-epimerase (GALE) is increased by
variants associated with type III galactosemia but decreased by substrate and
cofactor binding.
Angel L. Pey1*, Esperanza Padín-Gonzalez1, Noel Mesa-Torres1 and David J. Timson2*.
1
Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av.
Fuentenueva s/n, 18071, Spain.
2
School of Biological Sciences, Queen´s University Belfast, Medical Biology Centre,
97 Lisburn Road, Belfast BT9 7BL, UK.
* correspondence to: angelpey@ugr.es (A.L.P.) and d.timson@qub.ac.uk (D.J.T.)
1
Abstract
Type III galactosemia is an inherited disease caused by mutations which affect
the activity of UDP-galactose 4ʹ-epimerase (GALE). We evaluated the impact of four
disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the
conformational stability and dynamics of GALE. Thermal denaturation studies showed
that wild-type GALE denatures at temperatures close to physiological, and diseaseassociated mutations often reduce GALE’s thermal stability. This denaturation is under
kinetic control and results partly from dimer dissociation. The natural ligands, NAD+
and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands
and disease-associated variations affect local dynamics in the N-terminal region of
GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact
protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD+
reduces the rate of the first step, increasing the amount of undigested protein whereas
UDP-glucose reduces the rate of the second step, increasing accumulation of the
intermediate. Disease-associated variants affect these rates and the amounts of protein
in each state. Our results also suggest communication between domains in GALE. We
hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and
that it should be possible to discover compounds which mimic the stabilising effects of
the natural ligands overcoming mutation-induced destabilization.
Keywords.- Protein conformational stability; Protein dynamics; Protein aggregation;
Proteolysis; Type III galactosemia; Ligand binding.
2
Introduction
UDP-galactose 4ʹ-epimerase (GALE; EC 5.1.3.2) catalyses the interconversion
of UDP-glucose (UDP-glc) and UDP-galactose (UDP-gal), a reaction which is required
for galactose metabolism [1]. Defects in GALE activity due to mutations in the
corresponding gene cause the inherited metabolic disease type III galactosemia (OMIM
#230350). To date, 22 disease-associated variants of the protein have been described in
the literature [2]. The symptoms of type III galactosemia are very varied. In the mildest
forms of the disease, altered blood chemistry is observed and no interventions are
recommended. In contrast, the most severe forms of the disease result in progressive
damage to key organs including the kidneys, liver and brain. In such cases, reduction in
dietary galactose intake is required. Currently this is the only available therapy for the
disease. However, while this slows down the development of symptoms, it does not
prevent or reverse them [3].
Human GALE functions as a dimer of two identical, 38 kDa subunits [4].
Sequence and structural analysis showed that GALE is a member of the short-chain
dehydrogenase/reductase (SDR) family of enzymes [5]. Each subunit contains one
active site with a tightly bound NAD+ cofactor which plays a key role in the catalytic
mechanism of the enzyme [6] . This cofactor transiently oxidises the C4-OH group on
the sugar moiety of UDP-galactose. Rotation of the sugar moiety, followed by its
reduction from the opposite face reverses the stereochemistry at C4 and produces the
product UDP-glucose [7]. Many GALE enzymes, including the human one, can also
catalyse
the
interconversion
acetylglucosamine [8].
of
UDP-N-acetylgalactosamine
and
UDP-N-
These compounds are precursors in the synthesis of
glycoproteins and glycolipids and disturbances in this metabolism are likely to
contribute to molecular pathology [9].
3
Many of the disease-causing mutations affect GALE catalytic properties (e.g.
p.G90E, p.V94M and p.K161N) and there is some degree of correlation between the
degree of impairment of the turnover number (kcat) and the severity of the disease
phenotype. In some cases, disease-associated variations in GALE affect its
conformational stability (e.g. p.N34S, p.G90E and p.K161N) or reduce the affinity for
the NAD+ cofactor [10-13]. Thus, disease-associated variants may result in altered
stability, reduced catalytic activity and/or cofactor binding. However, binding of the
substrate (UDP-gal), the product (UDP-glc) or its cofactor (NAD+) may enhance the
protein’s conformational stability [10, 12, 14]. Nevertheless, the effects of sequence
alterations on local and global GALE stability and the modulation by ligand binding on
the pathogenesis of type III galactosemia are not well explored or understood.
Here, we provide new insight on the conformation, stability, dynamics and
ligand binding to WT GALE and four disease-associated variants (p.N34S, p.G90E,
p.V94M and p.K161N), using a combination of spectroscopic analyses, thermal
denaturation and kinetics of proteolysis. These variants were chosen to represent a
diverse spectrum of GALE variants. p.N34S (c.101A>G; rs121908046)1 has near wildtype kinetics when assayed in excess NAD+, but has a much lower affinity for this
cofactor than the wild-type [10, 16]. Similarly, p.K161N (c.483G>T; no rs number) also
has reduced affinity for the cofactor, but has very low activity even in the presence of
excess NAD+ [12]. p.G90E (c.269G>A; rs28940882) is highly unstable towards limited
proteolysis studies and has a turnover number (kcat) reduced approximately 800-fold
compared to wild-type [11]. p.V94M (c.280G>A; rs121908047) is the variant most
commonly found in severely affected patients [10, 11]. It is also highly catalytically
impaired (30-fold reduction in kcat) [10, 11]. Interestingly, it has been reported that this
“p.” refers to the protein sequence and “c.” to the DNA coding sequence [15]. “rs” stands for
“reference SNP” and identifies these single nucleotide polymorphisms in databases such as NCBI
SNP and Ensembl.
1
4
variant is slightly more stable towards limited proteolysis than the wild-type [11]. A
recent computational study predicted little change in the global flexibility of p.V94M
compared to the wild-type [13]. Of particular significance are our findings on the
stabilizing effects towards partial denaturation and proteolysis upon ligand binding;
these suggest that long-range transmission of binding effects between domains leading
to significant changes in protein local flexibility and dynamics, and in dimer stability.
Therefore, our results provide a deep understanding of the mutational effects on protein
stability and dynamics in type III galactosemia which are important, first for unravelling
the fundamental links between sequence changes and disease, and second for the design
of small molecules to stabilise the disease-associated variants (i.e. pharmacological
chaperones) [17-19].
5
Materials and methods
Protein expression and purification
Human GALE proteins were expressed in, and purified from, E. coli essentially
as described previously [10]. E. coli BL21(DE3) cells were transformed with plasmids
containing WT and mutant GALE cDNAs. An overnight culture grown in Luria–Bertani
media supplemented with 0.1 mg/ml ampicillin was diluted 1:20 into 1 L of Luria–
Bertani media supplemented with 0.1 mg/ml ampicillin. The cells were grown at 37 ºC
to OD600 of 0.6 and then induced with 0.5 mM isopropyl--D-thiogalactopiranoside
(IPTG) for 8 h at 25 ºC. Cells were then harvested and frozen at -80 ºC for 16 h. Cells
were resuspended in binding buffer (20 mM Na-phosphate, 300 mM NaCl, 20 mM
imidazole pH 7.4 and COMPLETE EDTA-FREE protease inhibitor cocktail from
Roche) and disrupted by sonication. The supernatants obtained after ultracentrifugation
(70000 g, 30 min, 4 ºC) were loaded onto immobilized metal affinity chromatography
columns (GE Healthcare), washed with binding buffer (50 bed volumes) and eluted
using binding buffer supplemented with 250-500 mM imidazole. These eluates were
loaded onto a Superdex 200 prep grade column (GE Healthcare) running in 20 mM
Hepes-OH 200 mM NaCl pH 7.4 and calibrated using the following standards: blue
dextran (void volume), thryroglobulin (669 kDa), alcohol dehydrogenase (141 kDa),
bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa),
cytochrome C (12.3 kDa) and acetone (total volume). Those fractions corresponding to
GALE dimers (retention time of 88.00.2 ml for the five variants in two independent
purifications) were pooled, concentrated, flash frozen in liquid nitrogen and stored at 80 ºC. Protein concentration was measured spectrophotometrically using ε280 of 46215
M-1·cm-1, which is based on the primary sequence of GALE and using the procedure of
Pace and coworkers [20].
6
Spectroscopic and dynamic light scattering studies
Absorption spectra were measured in
an
Agilent
8453 diode-array
spectrophotomer, using 40 M GALE in monomer and 3 mm pathlength quartz
cuvettes.
Emission
fluorescence
spectra
were
acquired
in
Cary
Eclipse
spectrofluorimeter using 3 mm pathlength quartz cuvettes and 4 M GALE in
monomer, with an excitation wavelength of 280 or 295 nm, and slits for emission and
excitation of 5 nm and a scan rate of 200 nm/min. Circular dichroism was measured in a
Jasco J-710 spectropolarimeter, using 4 M (far UV) or 20 M (near UV-visible)
GALE, and scan rates of 50 (far UV) and 100 (near UV-visible) nm/min. Dynamic light
scattering (DLS) was carried out in a DynaPro MSX instrument (Wyatt) using 1.5 mm
path length cuvettes and 20 µM protein monomer at 25 ºC. 25 spectra were acquired for
each DLS analysis in three independent replicates, averaged and used to determine the
hydrodynamic radius and polydispersity using the average autocorrelation function and
assuming a spherical shape. These experiments were performed in 20 mM HEPES-OH
200 mM NaCl pH 7.4 at 25 ºC, except far UV CD which were acquired in 20 mM Kphosphate 200 mM KCl pH 7.4, and blanks in the absence of protein were routinely
measured and subtracted. Secondary structure content was determined using the K2D3
algorithm [21] (available online http://www. ogic.ca/projects/k2d3).
Differential scanning calorimetry (DSC)
DSC experiments were performed on a capillary VP-DSC differential scanning
calorimeter (GE Healthcare) with a cell volume of 0.135 mL. Thermal scans were
performed at a rate of 3°C·min-1 in a temperature range of 2−80 °C using 10 μM GALE
(in monomer) in 20 mM HEPES-OH, 200 mM NaCl pH 7.4, unless otherwise indicated.
In some experiments, NAD+ or UDP-glucose was added to a final concentration of 1
mM (unless otherwise indicated), and their concentration was measured using an ε259 of
7
16900 M-1·cm-1(NAD+) and ε262 of 10000 M-1·cm-1(UDP-glucose). To estimate the
apparent Tm and denaturation enthalpies (H), we applied a simple two-state
irreversible denaturation model as described [22-24].
Isothermal titration calorimetry (ITC)
ITC experiments were performed on an ITC200 titration microcalorimeter (GE
Healthcare) with an operating cell volume of 206 L. In the calorimetric cells, we
placed a solution of 25 M GALE (monomer) in 20 mM HEPES-OH 200 mM NaCl
pH 7.4, while in the titrating syringe we put 500M of ligand (NAD+ or UDPglucose) in 20 mM Hepes-OH 200 mM NaCl pH 7.4. Experiments were performed at
25 ºC by performing 1 × 0.5L plus 30 × 1.2L (NAD+) or 1 × 0.5L plus 60 × 0.6
L (UDP-glucose) injections upon continuous stirring at 1000 rpm. Binding heats were
integrated and corrected for dilution heats. Binding isotherms were analyzed using a
single type of independent and identical binding sites model found in the software
provided by the manufacturer. Briefly, the heat (Q) evolved from the non-ligated GALE
species to a given saturation fraction is expressed by the following equation:
𝑄=
𝑛𝑀𝑇 Δ𝐻𝑉0
2
𝐿
(1 + 𝑛𝑀𝑇 + 𝑛𝐾
𝑇
1
𝑎 𝑀𝑇
𝐿
− √(1 + 𝑛𝑀𝑇 + 𝑛𝐾
𝑇
1
𝑎 𝑀𝑇
2
4𝐿
) − 𝑛𝑀𝑇 )
𝑇
where n is the binding stoichiometry per GALE monomer, MT is the GALE
concentration in monomer, H is the binding enthalpy, V0 is the cell volume, LT is the
total ligand concentration and Ka is the association ligand binding constant. This
equation can be used to determined to total heat evolved after the ith injection [Q(i)].
The corresponding expression for the heat evolved [Q(i)] between two consecutive
injections i-1 and i is provided by the following equation:
8
Δ𝑄(𝑖) = 𝑄(𝑖) +
Δ𝑉𝑖 𝑄(𝑖) + 𝑄(𝑖 − 1)
[
] − 𝑄(𝑖 − 1)
𝑉0
2
Where Q(i) and Q(i-1) are the total heats evolved after i and i-1 injections, respectively,
and Vi is the volume of injection i. From this equation, the binding parameters (n, H
and Ka) are obtained by Marquadt methods and iteration.
Proteolysis by thermolysin
Thermolysin from Bacillus thermoproteolyticus rokko was purchased from
Sigma-Aldrich, buffer exchanged to 20 mM HEPES-OH, 200 mM NaCl pH 7.4 and
stored at -80 ºC (its protein concentration was measured using 280=66086 M-1·cm-1).
For SDS-PAGE analyses, thermolysin and GALE enzymes were customarily incubated
for 10 min at 25 ºC in 20 mM HEPES-OH, 200 mM NaCl pH 7.4, in the absence or
presence of 1 mM NAD+ or UDP-glucose, and then mixed to a final concentration of 1
M and 20 M, respectively, in a final volume of 200 L. The time-course of
proteolysis was monitored upon withdrawal of 20 L of the reaction mixture, mixed
with 5 L of EDTA 100 mM pH 8 and denaturated in Laemmli’s buffer. Samples were
analyzed in 12% acrylamide gels and the bands were scanned and their intensities
integrated using the ImageJ software (http://rsbweb.nih.gov/ij/).
To identify the primary cleavage site in GALE, thermolysin and p.G90E GALE
enzymes were incubated for 10 min at 25 ºC in 20 mM HEPES-OH, 200 mM NaCl pH
7.4, and then mixed to a final concentration of 1 M and 10 M, respectively, in a final
volume of 1 mL. The reaction was allowed to proceed for 5 min at 25 ºC, quenched
adding 250 L of EDTA 100 mM pH 8. This mixture was buffer exchanged to water
using VIVAspin 500 filters (10 kDa cut-off) and then concentrated to 200 L and
9
splitted in two aliquots: i) 100 L was denatured in Laemmli´s buffer and run in a 12%
SDS-PAGE followed by electrofransference to a PVDF membrane. The membrane was
stained with Coomasie blue G-250 and the band corresponding to the 35 kDa cleaved
form were cut, destained and equilibrated in water for N-terminal sequencing by the
Edman´s method (performed at the service of Protein Chemistry, Centro de
Investigaciones Biológicas, Madrid, Spain). ii) 100 L was submitted for High
performance
liquid
chromatography/electrospray ionization
mass
spectrometry
(HPLC/ESI-MS) analyses. HPLC/ESI-MS was performed in a Acquity UPLC system
(Waters), using a gradient of water/formic acid (0.1%) and acetonitrile/formic acid
(0.1%) in a Acquity UPLC® BEH300 C4 column (2.1x50 mm; Waters) coupled to a QTOF Synapt62 HDMS (Waters) (performed at the high-resolution mass spectrometry
unit, Centro de Instrumentacion Cientifica, Universidad de Granada).
Kinetic models for irreversible denaturation/proteolysis of GALE enzymes in the
absence or presence of a ligand
We considered a simple kinetic model to simulate the effect of ligands on the
rate of denaturation/proteolysis of GALE in which ligand-bound (GALE-L) and ligand
free (GALE) undergo irreversible denaturation/proteolysis with first-order rate
constants kF-L and kF respectively to yield the final state F:
𝐺𝐴𝐿𝐸 − 𝐿 ↔ 𝐺𝐴𝐿𝐸 + 𝐿
kE-L
𝐹+𝐿
kE
𝐹
In the presence of ligand the kinetic stability of GALE will shift from the stability of the
10
ligand-free protein (GALE) to the stability of the ligand-bound protein (GALE-L), and
the overall rate of denaturation will be determined by the rate constants of the ligandfree (kE) and ligand-bound protein (kE-L) to undergo denaturation or proteolysis as well
as the concentration of ligand-free and ligand-bound protein, according to this rate law:
𝑑[𝐺𝐴𝐿𝐸]𝑡𝑜𝑡𝑎𝑙
= −(𝑘𝐸−𝐿 [𝐺𝐴𝐿𝐸 − 𝐿] + 𝑘𝐸 [𝐺𝐴𝐿𝐸])
𝑑𝑡
For illustration, in these simulations the value of kE is arbitrarily fixed to 10 min-1, while
kE-L values used are 1, 0.1 or 0.01 min-1. Therefore, the ratio of kE/ kE-L but not the
absolute values of these constants, will determine the dependence of the kinetic
stabilization exerted by the ligands as a function of total ligand concentration.
The fraction of GALE protein as free and ligated protein as a function of free ligand
concentration ([Ligand]) are estimated from a binding polynomial considering one site
per GALE monomer, a Kd for the ligand of 1 M (Ka=106 M-1) and total GALE
concentration of 10 M in monomer. The binding polynomial (P) is given by:
P=1+Ka·[Ligand]. The protein concentration in each state is given by:
[𝐺𝐴𝐿𝐸] =
[𝐺𝐴𝐿𝐸 − 𝐿] =
1
𝑃
𝐾𝑎 · [𝐿𝑖𝑔𝑎𝑛𝑑]
𝑃
Results
The effect of disease-causing mutations on GALE conformation and ligand binding
Figure 1A shows the location of the altered residues in p.N34S, p.G90E,
p.V94M and p.K161N on the crystal structure of GALE in the presence of NADH and
11
UDP-glucose (PDB:1EK6; [25]). N34, G90 and K161 cluster around the NAD+ binding
site, while V94 is next to the UDP-glucose binding site. Interestingly, p.G90E, p.V94M
and p.K161N cause a dramatic decrease in catalytic efficiency, from 40-fold (p.V94M)
to 1000-2000-fold (p.G90E and p.K161N) [10, 12].
We have expressed WT and the four disease-causing mutants in E. coli, and the
purified proteins were analyzed by spectroscopic methods under native conditions. We
first noticed that the UV-visible absorption spectra of WT and p.V94M differed from
the other GALE enzymes (Figure 1B, upper panel). These spectral changes are
compatible with the presence of cofactor/substrates bound to these two variants and not
released along the purification process. In the case of WT GALE, intrinsic fluorescence
spectra also support the presence of bound cofactor (Figure 1B, lower panel), but in all
cases, the fluorescence spectra show a maximum at 325 nm, consistent with similar
tertiary structures. The overall secondary structure of GALE enzymes was evaluated by
far-UV CD spectroscopy using the K2D3 algorithm, showing similar content for all
variants (Figure 1C; The average for the five GALE enzymes -helical and -sheet
content were 31.31.2% and 22.71.2%, respectively). The tertiary structure of the
GALE enzyme was further investigated by Near-UV CD spectroscopy (Figure 1D),
showing similar signals in the aromatic region for all enzymes (250-300 nm) and some
signals in the 300-400 nm region in WT and p.V94M which may arise from bound
substrate and/or cofactor. Finally, we studied the hydrodynamic behavior of GALE
enzymes by dynamic light scattering (Figure 1E). WT GALE displayed a hydrodynamic
radius of 3.80.1 nm, consistent with a molecular size of 765 kDa (i.e, a dimer), and
very similar to the size estimated along GALE purification by SEC (~70 kDa). None of
the variant GALE enzymes showed significant changes in the hydrodynamic size, and
the high monodispersity found for all them suggested that the GALE dimer is the main
12
species under native conditions.
We have evaluated the binding affinity of GALE enzymes for NAD+ and UDPglucose by isothermal titration calorimetry (see Figure 2A and 2C for representative
titrations). As isolated, WT GALE binds both ligands sub-stoichiometrically, suggesting
that the binding sites are already partially filled with these (or equivalent) ligands, in
agreement with the spectroscopic evidence shown in Figure 1B. Regarding GALE
variants, p.V94M and p.K161N show partially occupied binding sites (n0.4), while
ligand binding to p.N34S and p.G90E is almost stoichiometric (n0.8; Table 1). Thus,
the Kd values determined using these GALE enzymes must be considered as apparent
values, which enable comparison of the variants with a given ligand and both ligands
for a given enzyme. The binding affinity of WT for both substrates is moderate, with Kd
values around 1 M and 8 M, for NAD+ and UDP-glucose, respectively The diseaseassociated variants display different effects on the binding of ligands (Table 1). All
mutants show similar affinity for UDP-glucose to WT GALE, with the exception of
p.K161N, which binds this ligand with about 10-fold higher affinity. Regarding NAD+
binding, the most significant changes are observed for p.K161N, which decreases the
binding affinity by 10-fold, in agreement with previous evidence [12], and p.G90E,
which does not show a noticeable binding signal (which could be explained with a
markedly decreased binding affinity and/or binding enthalpy). The low response of this
mutant to NAD+ regarding thermal stability and proteolysis kinetics further support a
low binding affinity in this mutant (see below, Figures 4 and 5).
The presence of partially saturated binding sites in some GALE enzymes as purified
prompted us to attempt removal of these pre-bound ligands by dilution of the samples
to favour ligand release by dialysis, SEC and dilution-concentration cycles (see
13
Supplementary Information for a detailed description of these methods and associated
results). The success in removing bound ligands was tested by direct ITC titrations with
NAD+ and UDP-glucose (see Table S1). Unfortunately, none of these procedures
restored the full binding capacity of GALE enzymes, even though in some cases a slight
improvement is observed. The apparent Kd values obtained with or without using these
procedures were comparable, and support our conclusions regarding the mutational
effects on the apparent binding affinity for NAD+ and UDP-glucose (Table 1 and S1).
We must note all these procedures led to significant protein loss due to aggregation, and
SEC analyses suggested that dimer-monomer equilibrium is shifted towards the
monomer at very low protein concentrations (see Supplementary Information), which
implies that further dilution of GALE enzymes to facilitate ligand binding is leading to
irreversible denaturation of the enzyme due to the instability of monomeric GALE.
Thermal denaturation studies
We studied the thermal stability of GALE enzymes by differential scanning
calorimetry (DSC; Figure 3). Thermal scans of WT GALE showed an apparent single
transition with a Tm44 ºC and a denaturation enthalpy of 88 kcal·mol-1 (Figure 3A
and Table 2). In GALE variants, this apparent single transition seems to split into two
well resolved transitions, since the sum of their denaturation enthalpies (5410 and
368 kcal·mol-1, for the low and high temperature transitions; see Table 2) agree well
with the single transition observed for WT GALE. These results suggest that the single
transition in WT GALE may be composed of two overlapping transitions (with Tm
values of about 44 °C and 51 °C, see Figure 3A-C). The low temperature transition in
GALE mutants and the single transition in WT GALE are highly irreversible, scan-rate
and protein-concentration dependent (Figure 3B-G), indicating that this transition in
14
denaturation of GALE is under kinetic control and involves dimer dissociation [26]. The
high temperature transition may result from further denaturation of partially folded
monomers. p.N34S, p.G90E and p.K161N seem to decrease the Tm of the low
temperature transition by 4-7 ºC compared to WT GALE, while p.V94M shows little or
no effect (Figure 3A and Table 2). Regarding the high temperature transition, only
p.K161N shows an effect; there is a 5 ºC increase in the Tm of this transition (Figure
3A). We must note that denaturation enthalpies are known to scale with the protein size
and the degree of denaturation upon heating [27]. For a protein of GALE’s size (354
residues, including the his-tag), a denaturation enthalpy of about 168 kcal·mol-1 is
expected at the Tm of WT GALE which is almost double the experimental value. This
suggests that the thermally denatured GALE proteins retain a significant amount of
residual structure. The presence of residual structure in the thermally denatured state of
GALE is supported by comparison of far-UV CD and fluorescence spectra at low (25
°C) and high (60 °C, at which both denaturation transitions have fully developed)
temperatures and in the presence of 6M guanidium hydrochloride at low temperature
(Figure S1).
Addition of a large excess of either GALE cofactor (NAD+) or substrate (UDPglucose) has remarkable effects on the denaturation of GALE enzymes (Figure 4).
Addition of NAD+ increased the Tm of the low temperature transition, leading to an
apparent single peak for WT and p.V94M (with a Tm of 50 ºC), while in p.N34S and
p.K161N two peaks were still well resolved, but the low Tm transition was shifted
upwards by 5-6 ºC. In the case of p.G90E, NAD+ up-shifts the low Tm transition by only
1 ºC, supporting the strong defect in this enzyme for NAD+ binding shown by ITC
(Figure 2 and Table 1). Addition of UDP-glucose always led to a two-peak profile, with
an up-shift in the low temperature transition from 2-3 ºC (WT and p.V94M) to 6-8 ºC
15
(p.N34S, p.G90E and p.K161N) and no clear effect on the high-temperature transitions.
Thus, the stabilizing effect of NAD+ and UDP-glucose on these GALE enzymes
correlates well with their corresponding ligand binding affinities. Since denaturation of
GALE is under kinetic control and involves dimer dissociation, these ligand effects
must translate at least into kinetic stabilization of the GALE dimer in the presence of
these natural ligands.
Proteolysis of GALE enzymes
Proteolysis have been proven to be an insightful tool to evaluate mutational
effects on protein folding, stability and dynamics (e.g. [10, 28]). We have measured the
proteolysis kinetics of GALE enzymes by thermolysin under native conditions (at 25
ºC, well below the denaturation temperature shown by DSC; Figure 5A). Proteolysis
rates linearly depended on the protease concentration (Figure 5B-C), with a 5.8-fold
increase in the proteolysis rate constant from 0.2 to 1 M protease, demonstrating that,
under these conditions, the proteolysis step is rate-limiting [28, 29]. All the diseaseassociated variants show enhanced sensitivity to proteolysis, with half-lives lower than
that of WT GALE, from 1.4-fold (p.K161N), 2.3-fold (p.V94M) to 15-fold (p.N34S and
p.G90E) (Figure 5C-G and Table 3). Interestingly, these results correlate well with the
Tm values for the low-temperature transition obtained from DSC (Figure S2). Since the
proteolysis step is rate-limiting, an interesting possibility is that thermal denaturation
and
proteolysis
experiments
are
correlated
because
native
state
local
flexibility/dynamics and stability towards partial unfolding are linked.
In the presence of NAD+, WT GALE is degraded 1.6-fold slower (Figure 6C and
Table 3). All the variants show stabilization upon NAD+ binding, ranging from 1.8-fold
(p.G90E) to 18-fold (p.N34S) (Table 3 and Figure 6D-G). In the presence of UDP-
16
glucose, WT GALE is degraded 1.9-fold slower (Figure 6C and Table 3). All the
mutants show stabilization upon UDP-glucose binding, ranging from 2.0-fold (p.V94M)
to 10-13-fold (p.K161N and p.N34S) (Table 3 and Figure 6-D). The lower stabilizing
effect of these ligands on WT GALE compared to some mutants might be explained the
partial saturation of these binding sites in the WT protein in the absence of exogenously
added NAD+ or UDP-glucose (Table 1).
Beyond the effects on the sensitivity of GALE native state towards proteolysis,
disease-associated variants and ligands have significant effects on the partial proteolysis
pattern of GALE (see Figure 5A and 6). Proteolysis of WT GALE showed the
accumulation of a 35 kDa band in the absence of ligand, which comigrates with the
thermolysin but its intensity is stronger and time-dependent, supporting the
accumulation of a partially proteolyzed form of GALE (Figure 5A and 6A).
Interestingly, the presence of NAD+ decreased the intensity of this band to values
corresponding to those of thermolysin (Figure 6B). In the presence of UDP-glucose, this
partially cleaved state is populated to a larger extent (Figure 5A and 6C), suggesting
that UDP-glucose binding stabilizes this partially cleaved state towards proteolytic
attack. All these results can be qualitatively explained by using a simple two-step
mechanism as the following:
2
N ¾k1¾
® I ¾k¾
®P
where proteolysis of native GALE (N) leads to the formation of a partially proteolyzed
state of 35 kDa (I, determined by a rate constant k1), and this state is further cleaved to
low molecular mass fragments (P, with a rate constant k2). In the absence of ligands, k2
~k1, leading to low population of the intermediate I state in WT GALE (Figure 6A). In
the presence of NAD+, the N state is kinetically stabilized to a larger extent than the I
17
state, leading to very low population of I (i.e. k2 >k1) (Figure 6B). In the presence of
UDP-glucose, the I state is stabilized to a larger extent than the N state (i.e. k2 <k1),
leading to accumulation of I state (indeed, the sum of N and I over two hours of
proteolysis almost equal the initial load of GALE WT; Figure 6C). This simple model
also explains the behavior found for variant GALE enzymes. For instance, p.N34S and
p.G90E strongly destabilize the N state towards proteolysis (i.e. k2 <k1) and show a
larger accumulation of the I state (Figure 6D and 6G). Addition of NAD+ stabilizes
largely p.N34S but not p.G90E, thus causing accumulation of the I state only for
p.G90E (Figure 6E and 6H). Consistently, addition of UDP-glucose stabilized the N
state of both p.N34S and p.G90E, thus leading to accumulation of the I state for both
GALE enzymes (Figure 6F and 6I) (i.e. k2 <k1). Although this model is likely to present
a simplified picture of GALE proteolysis kinetics, it nevertheless explains the
accumulation of the I state due to effects on the stability of the N state (e.g. by
mutations or NAD+ binding) or the stabilization of the I state (e.g. by UDP-glucose
binding).
To identify the primary cleavage site of GALE by thermolysin, we used GALE
p.G90E and a 5 min digestion time, in order to minimize proteolysis of secondary
cleavage sites and to form a significant amount of the 35 kDa cleavage product (about
20% of initial GALE). HPLC/ESI-MS analyses of this proteolysis mixture provided two
main forms, with a mass of 39116.4 Da (corresponding to intact GALE; theoretical
mass 39104.5) and 34329 Da (corresponding to the 35 kDa cleavage product identified
by SDS-PAGE). Among the 105 theoretical cleavage sites in GALE, these results are
consistent with cleavage between Ala38 and Phe39 cleavage, which would release a
fragment with a theoretical mass of 34300 Da. N-terminal sequencing of this cleavage
product showed the sequence Phe-Arg-Gly-Gly, confirming the cleavage between
18
Ala38 and Phe39. The primary cleavage site is located in a highly solvent exposed loop
(residues 34-45) at the N-terminal domain (Figure 7A and B). This loop seems to be
quite flexible based on the comparatively high B-factors determined from the crystal
structure (Figure 7C). Thus, it is likely that thermolysin cleaves at this site in the native
state without requiring a local or global unfolding event. Thus, the sensitivity of the
native state towards proteolysis and the mutational- and ligand-effects must be
explained by changes in native state conformational dynamics.
Kinetic modeling supports that pre-bound ligands do not account for the
mutational and ligand effects on thermal stability and proteolysis sensitivity
In the previous section, we have shown that disease-causing GALE mutations affect
protein thermal stability and sensitivity to proteolysis, while NAD+ and UDP-glucose
binding stabilize GALE enzymes. However, WT GALE and to lesser extent p.V94M
and p.K161N, have significant amounts of pre-bound ligands as purified, and our
attempts to remove them were unsuccessful. Interestingly, the dependence of thermal
stability and proteolysis of purified GALE enzymes over a wide range of ligand
concentration show that the addition of stoichiometric amounts of ligand only slightly
affect thermal stability and sensitivity to proteolysis (Figure S3; note that the changes in
Tm exponentially translate into kinetic stabilization), while an large excess of ligand
stabilize to a larger extent. To test whether pre-bound ligands might explain the
differences in stability between GALE variants, we have evaluated the effect of ligands
(with the same binding affinity as NAD+) on the rate of denaturation/proteolysis by
kinetic model simulations.
To do so, we have evaluated the fraction of GALE monomer found in a
unligated- and ligated-state as a function of total ligand concentration (Kd=1 M; Figure
19
8A). Then, we determined the initial rate of denaturation/proteolysis using a model in
which ligated and unligated GALE undergo the irreversible process with different rate
constants kE-L and kE, respectively, and using different ratios between these two rate
constants (Figure 8B), observing two clearly different regimes: i) at total ligand
concentrations equal or lower than the monomer concentration, there is a sharp decrease
in the fraction of unligated species as total ligand concentration is raised, leading to a
several-fold kinetic stabilization. In this regime, the extent of kinetic stabilization
isquite insensitive to the ratio of the ratio kE/kE-L (Figure 8C); ii) at higher total ligand
concentrations, further kinetic stabilization is observed as the total ligand concentration
is raised. In this regime, a small change in the saturation fraction (for instance, from
0.99 to 0.999) causes a large kinetic stabilization (up to a 10-fold increase at very high
kE/kE-L ratios ) due to the decrease in the fraction of kinetically sensitive unligatedspecies (from 0.01 to 0.001, in this particular example). Therefore the dependence of the
kinetic stabilization on total ligand concentration clearly depends on the kE/kE-L value at
very high ligand concentrations (see the different saturation behaviour in Figure 8C).
Then, we considered saturation values consistent with the prebound-ligand
fraction determined for WT (0.8), p.V94M and p.K161N (0.5) and p.N34S and p.G90E
(0.2) (Figure 8B). The corresponding initial rates towards irreversible denaturation for
these saturation fractions are 2.590.08 (WT), 1.700.02 (p.V94M and p.K161N) and
1.240.01 (p.N34S and p.G90E) fold lower than the rate of the corresponding unligated
species (means.d. using three different kE/kE-L ratios, showing the rate is independent
of this ratio at the low ligand concentration regime). These analyses have two important
implications: i) prebound-ligands might exert up to a three-fold stabilization in WT vs.
GALE variants, which is much lower than fifteen-fold difference obtained
experimentally for p.N34S and p.G90E (Table 1). Thus, the pre-bound ligands can only
20
explain a small fraction of the higher stability of WT towards proteolysis (and possibly
thermal denaturation as judged by Tm(1) values) without added ligands; ii) the higher
stabilizing effect of added ligands towards proteolysis (and thermal stability) in some
GALE variants might imply different values of the ratio kE/kE-L from those of WT
GALE, since these experiments are performed with 1 mM ligand (the high ligand
concentration regime) and these ratios determine the saturation behaviour of the kinetic
stabilization vs. total free ligand concentration (Figure 8C). Thus, these simulations may
also explain how the different ligand concentration dependence of proteolysis rates
constants and Tm values for thermal denaturation between GALE variants (Figure S3)
together with changes in ligand binding affinities.
Discussion
The role of protein stability, conformational dynamics and ligand binding have
been investigated here for WT GALE and four variants associated with type III
galactosemia. Our results show that GALE dimer is marginally stable and partially
denatures at temperatures close to physiological. p.N34S, p.G90E and p.K161N may
further destabilize GALE dimer towards partial denaturation, thus rendering a
kinetically unstable protein at physiological temperature. Interestingly, p.N34S and
p.G90E are also much more sensitive to proteolysis than WT GALE, indicating greatly
altered local conformational dynamics in the N-terminal region of these two variants. In
contrast, p.V94M shows similar resistance to proteolysis compared to WT. This is
consistent with molecular dynamics (MD) simulations of this variant which predicted
little change in the global flexibility of the protein [13] and previous experimental
studies [10, 30]. In all cases, substrate and/or cofactor binding modulate the sensitivity
of GALE enzymes towards thermal denaturation and proteolysis, suggesting that
alterations in dimer stability and local dynamics by disease-causing mutants can be
21
efficiently modulated by binding to either natural or pharmacological ligands. These
effects were comparable between WT and p.V94M, again consistent with MD work
which predicted little or no change in binding affinity of this variant for the substrate
and the cofactor [13]. The results are also consistent with previous biochemical work
which showed that p.G90E is one of the most unstable of the currently known variants
[10]. This suggests that this variant could be associated with very severe forms of the
disease; however, to date, it has only been found in a heterozygous patient [31]. In a
diploid Saccharomyces cerevisiae model deleted for both copies of the yeast GALE
(GAL10) and heterozygous for human WT and p.G90E, GALE activity was
approximately 50% of that detected in strains homozygous for WT human GALE [11].
This suggests that the G90E allele is recessive to the wild-type one, possibly due to a
combination of largely impaired catalytic function, conformational instability and
altered local conformational dynamics.
Our proteolysis analyses provide an insight into the mutational and ligand
effects on GALE protein dynamics. p.N34S and p.G90E locally alter flexibility or
dynamics in the N-terminal domain most likely due to repulsive/destabilizing
interactions with the loop 34-45. In the crystal structure of human GALE (PDB:1EK6),
Asn-34 lies adjacent to the adenine moiety of NAD+ [25]. Asn-34 is predicted to
hydrogen bond with NAD+ and is close in sequence to other residues (i.e. Asp33 and
Asn37) which interact with the cofactor [19, 25] which may explain the 4-fold
decreased affinity for NAD+ (Table 1). In general, serine residues tend increase the
local backbone flexibility of the polypeptide chain and it would be expected that
increased flexibility in this region would also weaken these protein-cofactor interactions
[32]. G90 is located close to N34 (<1.5 Å) in the structure of human GALE and
adjacent to the phosphate groups in NAD+ [25]. All together, these structural analyses
22
may explain the very low affinity of p.G90E for NAD+ (indeed, no binding signal was
detected by ITC; Figure 2A and B). The replacement of glycine by the much larger
glutamate side chain will require considerable rearrangement and, probably,
destabilisation and distortion of the local structure, especially of the sequence
connecting -sheet 4 and helix-4 where G90 is located. This may be exacerbated by
repulsive interactions between the negatively charged side chain and the phosphate
moieties in NAD+. Thus, both p.N34S and p.G90E are predicted to result in local
changes to the structure and flexibility of the protein. This may also explain why these
two mutations cause accumulation of the partially cleaved state I (lacking the Nterminal 38 residues) due to selective destabilization of the native state, probably
because the effects of these two mutations are weakened or eliminated when the Nterminal 38 residues are removed (Figure 6A, D and G).
NAD+ and UDP-glucose binding increase the resistance of the native state
towards proteolysis and this effect is mutant-selective, corresponding fairly well with
the affinity of their native state for these ligands (Tables 1 and 3), which also supports
the proposition that binding of a ligand (UDP-glucose) far from the cleavage site have
long-range effects that propagate between domains in the native state. Interestingly, the
two ligands affect differently the accumulation of the partially cleaved state (Figure 6),
which can be reasonably explained based on the structure of GALE in the presence of
substrate and cofactor [25]. The cofactor binds to the N-terminal domain and establishes
hydrogen bonds with residues close to the 34-45 loop (such as Asp33 and Asn37 and
Lys161). However, upon proteolytic attack on this primary site, most of these
favourable interactions are removed, thus leading to the release of the bound cofactor or
substantially decreased binding affinity for it, but not affecting the stability of the
cleavage intermediate I state. Accordingly, cleavage of the loop 34-45 might not greatly
23
affect binding of UDP-glucose which binds to the C-terminal domain, and thus,
resulting in the stabilizing effects of UDP-glucose on the cleavage kinetics of the
intermediate and its accumulation along the proteolysis reaction (Figure 6C, F and I).
To our knowledge, we provide the first evidence for long-range communication
between domains in GALE structure (upon mutation and ligand binding), which allow
further understanding on the conformational consequences of disease-associated
variants and might be therapeutically exploited to correct mutation-induced
destabilization in type III galactosemia.
We must note that GALE enzymes as purified seem to have partially occupied
binding sites for NAD+ and UDP-glucose (this work and [12]), especially for WT,
p.V94M and p.K161N enzymes (Table 1). Attempts to remove by different procedure
were unsuccessful, probably due to a shift in the dimer-monomer equilibrium towards
monomers causing GALE destabilization and aggregation (see Supplementary
Information and Table S1). The presence of pre-bound ligands will influence thermal
stability and proteolysis results beyond the effects of mutations. However, we provide
experimental (Figure S3) and theoretical (Figure 8) evidence strongly supporting a
minor role of pre-bound ligands in the mutational and NAD+ and UDP-glucose effects
described here. This is a logical consequence of the kinetic control of thermal
denaturation and proteolysis and the comparatively low affinity for the ligands (see [22,
26, 33]). In conclusion, the differences observed in stability for GALE variants must
reflect to a large extent true effects of the disease-causing mutations.
Taken together, our data strongly support the conclusion that the sequence
alteration results in destabilization and altered conformational dynamics of GALE
dimer at physiological conditions (temperature and pH) which are linked to loss of
enzyme function in type III galactosemia. We also demonstrate that ligand and cofactor
24
binding trigger large changes in global dimer stability and local conformational
dynamics. We hypothesise that these changes might modulate GALE intracellular
protein turnover and functionality, as shown for other conformational diseases [17, 3436]. These results further support the previous proposal for the use of ligands, such as
cofactor analogues or pharmacological chaperones, as potential approaches to treat type
III galactosemia [19]. Critically, our finding that the natural ligands can stabilise
different states of the protein supports the idea that pharmacological ligands which
reduce the rate of the N→I transition are likely to be effective in rescuing GALE
activity. Furthermore, the approaches taken here suggest methods by which such
compounds could be identified through high-throughput screening. After initial
identification of protein binders, these hits could be used in a limited proteolysis assay
similar to that used in this work in order to identify the subset that reduces the rate
constant of the first transition.
Acknowledgements.- We thank Dr. Jose Manuel Sanchez-Ruiz for support. This work
was supported by grants from MINECO (BIO2012-34937 and CSD2009-00088), Junta
de Andalucia (P11-CTS-07187), The Royal Society (2004/R1) and FEDER Funds. A.L.P.
is supported by a Ramón y Cajal research contract from MINECO (RyC-2009-04147).
N.M-T. is supported by a FPI predoctoral fellowship from MINECO.
Abbreviations:
GALE:
UDP-galactose
4´-epimerase;
UDP-glc:
UDP-glucose;
HPLC/ESI-MS: high performance liquid chromatography/electrospray ionization mass
spectrometry; MD: molecular dynamics; CD.- circular dichroism; DSC.- differential
scanning calorimetry.
25
26
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28
Table 1. Binding properties of NAD+ and UDP-glucose to GALE enzymes
determined by ITC. Data are best-fit values and their corresponding fitting errors.
NAD+
GALE
enzyme
n
UDP-glucose
Kd
H
(M)
(kcal/mol)
n
Kd
H
(M)
(kcal/mol)
WT
0.230.01
1.00.1
-9.60.4
0.200.05
7.91.1
-17.45.2
p.N34S
0.780.01
3.80.2
-11.70.3
0.660.02
3.90.4
-21.00.9
0.830.03
9.30.6
-14.90.7
Not detected
p.G90E
p.V94M
0.360.01
0.350.01
-11.60.1
0.440.08
10.31.6
-8.91.9
p.K161N
0.420.03
121
-10.91.0
0.490.01
0.750.06
-22.30.3
29
Table 2. Apparent Tm values and denaturation enthalpies (H) for thermal
denaturation of GALE enzymes. Experiments performed at 3oC·min-1 and 10 M
protein in monomer. Parameters are obtained from fittings to a two-state irreversible
model and errors are those from fittings.
GALE
Parameter
enzyme
Tm1 (°C)
H1 (kcal·mol-1)
WT
44.10.1
883
p.N34S
39.60.1
451
49.60.1
422
p.G90E
38.80.1
501
52.00.1
301
p.V94M
42.30.1
542
49.10.1
456
p.K161N
37.00.1
681
55.80.1
291
Tm2 (°C)
H2 (kcal·mol-1)
Not applicable
30
Table 3. Half-lives for proteolysis of native GALE enzymes in the absence or
presence of ligands (1 mM). The concentration of GALE enzymes and thermolysin
was 20 M and 1 M, respectively. Experiments were performed at 25 ºC.
GALE enzyme
Half-life (min)
No ligand
NAD+
UDP-glucose
WT
595
947
11411
p.N34S
3.90.3
726
515
p.G90E
4.00.2
7.30.3
211
p.V94M
264
13917
537
p.K161N
8.02.0
334
796
31
Figure 1. Mutational effects on GALE conformation. A) Location of the altered
residues in the crystal structure of GALE (PDB code: 1EK6; [25]) indicated as red
sticks. UDP-glucose is shown in black and NADH in magenta. GALE monomers are
displayed in different colors (green and cyan). B) UV-visible absorption spectra (lower
panel; inset show a magnified view of the absorption spectra between 300 and 400 nm)
and intrinsic fluorescence spectra (upper panel, excitation at 280 nm; inset: excitation at
295 nm) at 4 M protein in GALE monomer; C) Far-UV CD spectra at 4 M; D)
Near-UV CD at 20 M; E) DLS at 20 M.
32
Figure 2. Ligand binding to GALE enzymes by ITC. Thermograms and binding
isotherms for the binding of NAD+ (panel A and B) and UDP-glucose (panel C and D)
to GALE enzymes. Symbols in panels B and D are as follows: WT (circles), p.N34S
(up-triangles), p.G90E (down-triangles), p.V94M (squares), p.K161N (diamonds).
33
Figure 3. Thermal denaturation of GALE enzymes by differential scanning
calorimetry (DSC). A) Denaturation profiles of WT and variant GALE enzymes at 3
ºC·min-1 and 10 M protein (monomer equivalent). B and E) Reversibility tests on WT
(B) and p.N34S (E) GALE. Protein samples (10 M protein monomer equivalent) were
heated up to 57 ºC or 47-48 ºC, cooled down to 2 ºC and rescanned. A scan rate of 3
ºC/min was used. C and F) scan rate dependence of thermal denaturation of WT (C) and
p.N34S (F) GALE (10 M protein monomer equivalent); Scan rates were 3 °C min-1
(closed circles), 2 °C ·min-1 (open down triangles) or 1 °C·min-1 (closed squares); D and
G) protein concentration dependence of thermal denaturation of WT (D) and p.N34S
(G) GALE (3 ºC/min scan rate). Protein concentrations were 20 M (closed circles), 10
M (open down triangles) and 5 M (closed squares).
34
35
Figure 4. Effect of ligands (NAD+ or UDP-glucose, 1 mM) on the thermal
denaturation of GALE variants by DSC. Protein concentration was 10 M, and
experiments were performed in 20 mM HEPES-OH, 200 mM NaCl pH 7.4 at a scan
rate of 3 ºC·min-1.
36
Figure 5. Kinetics of proteolysis of GALE enzymes by thermolysin. A)
Representative SDS-PAGE gels for WT (upper panel) and p.G90E (lower panel). B-G)
Kinetics of proteolysis of GALE enzymes in the absence (closed circles) or presence of
1 mM ligands (NAD+, open circles; UDP-glucose, closed down-triangles). GALE
enzymes were at 20 M and thermolysin at 1 M (panels C-G) or 0.2 M (panel B).
Experiments were performed at 25 ºC. Lines in panels B-G are fits to a single
exponential function.
37
38
Figure 6. Time dependent population of the full-length GALE (closed circles), the
35 kDa band (open circles) and sum of them (open stars) from SDS-PAGE
analyses. The horizontal dashed line shows the fraction that corresponds to the
contribution of thermolysin to the 35 kDa. Fractions were determined by
normalization using the band intensity of full-length GALE without thermolysin (t=0
min).
39
Figure 7.
Conformation of the primary cleavage site of GALE enzymes by
thermolysin (loop 34-45). A) Structure of GALE dimer (PDB code: 1EK6)
highlighting the solvent exposed loop 34-45 and the primary cleavage site between
Ala38 and Phe39 (in black); B) Side-chain accessibility of the loop 34-45 and adjacent
residues calculated using the Shrake-Rupley algorithm [37] with a radius of 1.4 Å for
the solvent probe and the Chothia set for the protein atoms [38]. The asterisks indicate
glycine (no side-chain, i.e. 0% accessibility). C) Average-B factors calculated from the
GALE crystal structure (PDB code: 1EK6) using the program Baverage from CCP4
Suite [39].
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Figure 8. Kinetic modeling of GALE stability in the absence or presence of bound
ligands. A) Dependence of the concentration of unligated (GALE) and ligated-GALE
(GALE-L) on total ligand concentration; B) Dependence of the initial rate for
irreversible denaturation/proteolysis as a function of total ligand concentration using a
rate constant kE=10 min-1and different values of kE-L (from 1 to 0.01 min-1). The
symbols indicate the initial rates considering saturation fractions of 0.8 (WT), 0.5
(p.V94M and p.K161N) and 0.2 (p.N34S and p.G90E) derived from pre-bound ligand
values estimated from ITC titrations; C) Dependence of kinetic stabilization (rate with
ligand/rate without ligand) on total ligand concentration using a rate constant kE=10
min-1 and different values of kE-L (from 1 to 0.01 min-1). All calculation are performed
using 10 M GALE monomer and Kd of 1 M (similar to NAD+).
41