Conformational dynamics and allostery in pyruvate kinase

Article abstract of DOI:10.1074/jbc.M115.676270

Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1, 6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1, 6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit This destabilizes the βstrands within the (β/α)₈-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1, 6-bisphosphate affects the active site.

Full text of DOI:10.1074/jbc.M115.676270

JBC Papers in Press. Published on February 15, 2016 as Manuscript M115.676270
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.676270
Conformational Dynamics and Allostery in Pyruvate Kinase
Conformational Dynamics and Allostery in Pyruvate Kinase
Katherine A. Donovan1, Shaolong Zhu2, Peter Liuni2, Fen Peng3, Sarah A. Kessans1, Derek J.
Wilson2,4, Renwick C.J. Dobson1,5
1 Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury,
Private Bag 4800, Christchurch 8041, New Zealand
2 Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada
3 Biology and Biochemistry, University of Houston, TX, 77204, USA
4 Centre for Research in Mass Spectrometry, Department of Chemistry, York University, Toronto,
Ontario, Canada
5 Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology
Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia
*Running title: Conformational Dynamics and Allostery in Pyruvate Kinase
To whom correspondence should be addressed: Renwick C.J. Dobson, Biomolecular Interaction
Centre, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch,
New Zealand, Tel.: +64 3 364 2987; Fax: +64 3 364 2590; E-mail: renwick.dobson@canterbury.ac.nz
or Derek J. Wilson, Centre for Research in Mass Spectrometry, York University, Chemistry
Department, 4700 Keele Street, Toronto, ON, Canada, Tel.: +1 (416) 736 2100 x20786; Fax: +1 (416)
736 5936; E-mail: dkwilson@yorku.ca
Keywords: pyruvate kinase; glycolysis; Escherichia coli; allosteric regulation; fructose-1,6-
bisphosphate; conformational flexibility; hydrogen-deuterium exchange
ABSTRACT
conformational flexibility. Based upon these
and mutagenic studies, we propose an
allosteric mechanism whereby the binding of
fructose-1,6-bisphosphate destabilises an α-
helix that bridges the allosteric and active site
domains within the monomeric unit. This
destabilises the β-strands within the (β/α)8-
barrel domain and the linked active site loops
that are responsible for substrate binding.
Our data is consistent with the ‘domain
rotation model’, but inconsistent with the
‘rigid-body reorientation model’, given the
increased flexibility at the inter-domain
interface, and we can for the first time
explain how fructose-1,6-bisphosphate affects
the active site.
Pyruvate kinase catalyses the final step in
glycolysis and is allosterically regulated to
control flux through the pathway. Two
models are proposed to explain how
Escherichia coli pyruvate kinase type 1 is
allosterically regulated: the ‘domain rotation
model’ suggests that both the domains within
the monomer and the monomers within the
tetramer reorient with respect to one another;
the ‘rigid-body reorientation model’ proposes
only a reorientation of the monomers within
the tetramer causing rigidification of the
active site. To test these hypotheses and
elucidate the conformational and dynamic
changes that drive allostery, we performed
time-resolved electrospray ionization mass
spectrometry coupled to hydrogen-deuterium
exchange studies, followed by mutagenic
analysis to test the activation mechanism.
Global exchange experiments, supported by
thermostability studies, demonstrate that
fructose-1,6-bisphosphate binding to the
allosteric domain causes a shift towards a
globally more dynamic ensemble of
conformations. Mapping deuterium exchange
to peptides within the enzyme highlight site-
specific regions with altered conformational
dynamics, many of which increase in
Allostery is the effect that ligand binding at
one site on a macromolecule has on the
physicochemical
properties
of
another,
topographically distinct site (1-5). Allostery
provides immediate enzymatic control of
reaction flux in response to cellular signals (e.g.
the feedback regulation of a branch point
enzyme by the concentration of the pathway
product). As such, allostery is critical for the
modulation of cellular metabolism and is
ubiquitous across all forms of life.
Pyruvate kinase (E.C. 2.7.1.40) catalyses the
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Conformational Dynamics and Allostery in Pyruvate Kinase
final step in the central energy production
To unpick the molecular mechanism(s) by
which pyruvate kinase achieves allosteric
control upon fructose-1,6-bisphosphate binding,
several groups have determined the three-
dimensional crystal structures of various
isozymes in both the allosteric activator bound
and unbound states (10,11,17,18).
pathway, glycolysis (6,7) and as such is a key
enzyme in cellular metabolism. It mediates a
phosphate transfer from phosphoenolpyruvate to
ADP, producing pyruvate and ATP (Fig. 1A).
Pyruvate,
and
to
a
lesser
extent
phosphoenolpyruvate, are common precursors in
metabolism, including the synthesis of glucose,
fatty acids, glycerol and amino acids (8). Thus,
The
fructose-1,6-bisphosphate
bound
structures reveal that the allosteric activator
binding site is located entirely within the C-
domain and must transmit an allosteric signal
over a 40 Å distance to the active site in the
same subunit. The binding site is located in a
pocket formed from an effector loop and the first
two turns of the Cα5’-helix in Saccharomyces
cerevisiae (11) and human liver (20) pyruvate
kinases. Fructose-1,6-diphosphate binds in the
equivalent pocket in Leishmania mexicana (17),
Trypanosoma cruzi (10) and Trypanosoma
brucei (21). Structural studies in L. mexicana
(17) and T. cruzi (10) suggest that the allosteric
transition involves a rigid-body rocking motion
of the AC-domains, reorienting these domains
within the tetramer.
Based largely of structural studies, two
models have been proposed by others to explain
how E. coli pyruvate kinase type 1 is regulated
by fructose-1,6-bisphosphate (Fig. 2) The first
suggests that the domains within the monomer
are reoriented along with changes in the
orientation of the monomers within the tetramer
(8,22,23) (Fig. 2A). The second competing
model puts forward that the domains are rigid
and that reorientation of the domains within the
tetramer causes the active site to rigidify,
presumably in a conformation that better binds
the substrate phosphoenolpyruvate (10,17) (Fig.
2B).
The structure of E. coli pyruvate kinase type
1 isozyme with fructose-1,6-bisphosphate bound
is lacking. Previous attempts by us, and others
(8), to soak fructose-1,6-bisphosphate into E.
coli pyruvate kinase type 1 crystals have caused
them to crack. This indicates that fructose-1,6-
bisphosphate binding causes conformational
changes that affect crystal packing. Moreover,
our attempts to co-crystallise fructose-1,6-
bisphosphate and E. coli pyruvate kinase type 1
have not yielded crystals, despite employing
large and sparse matrix screens that can trial
>1000 different conditions, and successfully
obtaining unliganded enzyme crystals in various
conditions. One explanation for this is that the
fructose-1,6-bisphosphate bound protein is
heterogeneous in some way.
it
is
essential
that
are
pyruvate
maintained
and
at
phosphoenolpyruvate
appropriate concentrations in response to the
needs of the cell (9).
Not surprisingly, pyruvate kinase enzymes
are almost always allosterically regulated.
Moreover, cells often express multiple pyruvate
kinase isozymes that are regulated by different
effectors. Pyruvate kinase isozymes are usually
homotropically activated by the substrate
phosphoenolpyruvate to allow regulation of the
glycolytic flux in response to elevated
phosphoenolpyruvate levels (10). In addition,
they are often heterotropically regulated by
various allosteric effectors, whose chemical
nature depends on the organism or tissue (8,11).
For example, Escherichia coli has two pyruvate
kinase enzymes that are regulated by different
allosteric activators. The type 1 pyruvate kinase,
the focus of this work, is heterotropically
activated by fructose-1,6-bisphosphate. The
binding of fructose-1,6-bisphosphate has a K-
type effect, whereby it increases the enzyme’s
affinity for the substrate, phosphoenolpyruvate
(12). In contrast, pyruvate kinase type 2 is
heterotropically activated by AMP (13,14).
Key to unravelling allosteric mechanisms are
the structures of the enzyme with and without
bound allosteric effectors. Atomic resolution X-
ray structures of pyruvate kinase enzymes from
mammalian, bacterial and parasitic organisms
have been reported (7,8,10,11,15-18). As is
common in allosterically controlled enzymes,
pyruvate kinase is oligomeric and is usually a
homotetramer. Each monomer comprises three
or four domains: A, B, C and N-terminal
domains (only present in mammalian enzymes)
(11) (Fig. 1B and 1C). The A-domain is a
(β/α)8-barrel (TIM-barrel), housing the active
site at the top of the barrel. The B-domain, also
known as the ‘lid domain’, is thought to close
over the active site upon substrate binding. The
active site is located in the cleft between the A-
and B-domains, and this is where the essential
mono- and divalent cations bind (19). The C-
domain consists of five α-helices and five
strands of mixed β-sheets, housing the allosteric
binding site close to the tetrameric interface
(8,17). Finally, the N-terminal domain is a short
α-helical extension preceding the (β/α)8-barrel
domain, whose role is less defined (11).
To build
a
detailed picture of the
conformational and dynamic changes that drive
E. coli pyruvate kinase type 1 heterotopic
allosteric
activation
by
fructose-1,6-
bisphosphate, we performed a time-resolved
2

Conformational Dynamics and Allostery in Pyruvate Kinase
electrospray ionization mass spectrometry
BL21 (DE3) cells for expression and
purification.
Purification of wild-type enzyme for
hydrogen-deuterium exchange studies was
adapted from the procedure used by Mattevi and
colleagues (29). Briefly, the procedure involved
(TRESI) coupled to hydrogen-deuterium
exchange study. The ‘new view’ of allostery
describes a dynamic process whereby a protein
fluctuates within an ensemble of conformations
(24-27). Although the concept of allostery has
evolved considerably in recent years, allosteric
mechanisms remain difficult to establish, since
defining changes in protein dynamics is
challenging even for small proteins (26). Here,
we present hydrogen-deuterium exchange data
that suggests the protein samples a larger
conformational ensemble upon fructose-1,6-
three
chromatography,
chromatography
purification
steps:
hydrophobic-interaction
and size-exclusion
anion-exchange
chromatography to obtain pure enzyme as
judged by SDS PAGE gel. The protein was
desalted using a 2 mL ZebaTM spin desalting
column (Thermo Scientific) followed by
overnight buffer exchange into a 200 mM
ammonium acetate buffer (pH 6.9) using a Slide-
A-Lyzer mini dialysis device (10 kDa MWCO;
Thermo Scientific).
bisphosphate binding.
Mapping deuterium
exchange to peptides within the protein reveal
regions that show significantly altered
conformational dynamics upon the binding of
fructose-1,6-bisphosphate.
Based on this
Recombinant wild-type and variant forms
were purified in the following way for the
mutagenic studies: crude lysate was loaded onto
a His-TRAP FF crude 5mL column (GE
Healthcare) pre-equilibrated with buffer A (20
mM Tris, 200 mM KCl, 20 mM imidazole, pH
8). The bound His-tagged protein was eluted
with an increasing imidazole concentration as
buffer A was replaced with buffer B (20 mM
Tris, 200 mM KCl, 500 mM imidazole, pH 8).
The fractions containing protein were pooled
and thrombin was added (5 U per mg protein),
for cleavage overnight on a rotation wheel at 4
°C. The cleaved protein was loaded onto a His-
TRAP FF column (GE Healthcare) and the flow
through was collected, concentrated and desalted
on a 16/600 Superdex 200 gel filtration column
(GE Healthcare) pre-equilibrated with buffer C
(10 mM Tris, 2 mM β-mercaptoethanol, 1 mM
EDTA, pH 7.5).
dynamic data, and further confirmed by
mutagenic experiments, we are able to propose a
new, detailed mechanism by which fructose-1,6-
bisphosphate promotes substrate binding, despite
the allosteric binding site being 40 Å away from
the active site.
EXPERIMENTAL PROCEDURES
Chemicals and supplies - Pepsin agarose
beads, deuterium oxide (D2O, 99.99%),
ammonium acetate (99.99%) and high purity
acetic acid (99.7%) were purchased from Sigma
Aldrich (St. Louis, MO).
Fructose-1,6-
bisphosphate (Sigma Aldrich, St Louis, MO)
was prepared in 200 mM ammonium acetate
buffer (pH 6.9). PTFE tubing (OD. 1/16”, ID.
400 mm and OD. 1/16”, ID. 205 mm) was
supplied by McMaster Plastics (Scarborough,
ON). Standard 1/16” fittings were purchased
from Upchurch (Oak Harbor, WA). Ultrapure
water was generated in-house on a Millipore
Milli-Q Advantage A10 system.
Thermal stability assays - The unfolding
temperatures of the wild-type pyruvate kinase
type 1 and four variants (Lys404Ala, Thr405Ala,
Gln408Ala, Leu411Glu) were determined using
differential scanning fluorometry, following
methods adapted from Morgan and colleagues
Cloning of pykF enzymes - The pykF gene
had been cloned into a pBluescript II KS+ vector
(23), and was kindly donated by Professor
Andrea Mattevi (University of Pavia).
In
addition to the wild-type, four variants of the
pykF gene were synthesized commercially by
GenScript and supplied in a pBluescript II KS+
cloning vector. The five genes were then
digested with SacI and HindIII (New England
Biolabs) restriction enzymes and subcloned into
the pET30ΔSE expression vector (28), purified
using an agarose DNA extraction kit (Roche)
and ligated with T4 DNA ligase at 20 °C for 30
minutes.
(17).
Specifically, 25 µL solutions were
prepared, containing: 10 × Sypro Orange
(diluted 1/500 in 10 mM Tris, 2 mM, β-
mercaptoethanol, 1 mM EDTA, pH 7.5), 0.25
mg/mL protein, and 0.25 mM fructose-1,6-
bisphosphate (dissolved in ddH2O) as
appropriate. The samples were heated in an iQ5
real-time PCR detection system, from 20 to 100
°C, with 10 second fluorescence measurements
taken in 0.5 °C increments.
Expression and purification
recombinant expression vectors
-
The
were
Kinetic analysis – Pyruvate kinase initial rate
data were measured using the lactate
dehydrogenase-coupled
transformed into chemically competent E. coli
spectrophotometric
assay, adapted from Valentini and colleagues
3

Conformational Dynamics and Allostery in Pyruvate Kinase
(30). Enzymatic activity was assayed at 30 °C
A second blank PMMA substrate was used as a
cover to seal the device. The chip and cover
were placed in a custom built clamp (LAC
Machine & Tooling Limited, ON) to pressure-
seal the microfluidic device. The reactants were
then supplied into the input channels of the
device using Harvard syringes through PTFE
tubing using automated syringe infusion pumps
(Harvard, Holliston, MA).
TRESI coupled proteolytic experiments - The
microfluidic chip was interfaced with a modified
QSTAR Elite hybrid quadrupole time-of-flight
(QqTOF) mass spectrometer (Sciex, MDS
Analytical Technologies, Concord, ON). The
instrument was operated in positive ion mode
with optimal running conditions of 4600–5000 V
source voltage, 10 V declustering potential, and
100 V focusing potential. Spectra were acquired
over the range of 400–1500 m/z with a scanning
rate of 1 s-1.
Hydrogen-deuterium exchange experiments
were carried out at 20 °C in triplicate. The
purified protein (38 µM, in 200 mM ammonium
acetate, pH 6.9) was introduced into the glass
capillary of the mixing device at a flow rate of 1
mL/min while the D2O was introduced into the
outer metal capillary at a flow rate of 3 mL/min
using Harvard 11+ infusion pumps (Holliston,
MA). Where appropriate, the enzyme was pre-
using varied concentrations of fructose-1,6-
bisphosphate, phosphoenolpyruvate, and ADP.
Kinetic parameters were determined as follows:
titration of phosphoenolpyruvate, at a fixed
concentration of 4 mM ADP in the absence or
presence of 4 mM fructose-1,6-bisphosphate;
titration of ADP, at a fixed concentration of 4
mM phosphoenolpyruvate in the absence or
presence of 4 mM fructose-1,6-bisphosphate; for
the fructose-1,6-bisphosphate titration, at 1 mM
phosphoenolpyruvate and 4 mM ADP. All
measurements were performed in duplicate and
the data were fit to the Hill cooperative model
(31) to derive Vmax, S0.5 and nH values.
Microfluidic device fabrication
-
The
microfluidic device was constructed as described
previously (32,33). Briefly, the microfluidic
channel design was generated in CorelDraw X3
(Corel Co., Ottawa, ON) and lasered onto blank
pre-cut polymethyl methacrylate (PMMA)
substrate (8.9 cm x 3.8 cm x 0.6 cm;
Professional Plastics, Fullerton, CA, USA). The
design included: two input channels for the
introduction of reactants, a pepsin digestion
chamber (31.5 mm, x 3 mm, 0.5 mm; vol. =
47.25 mL), and an output channel. The design
was engraved into the PMMA substrate using a
30
W CO2 Versa-Laser engraving device
(Universal Laser, Scottsdale, AZ).
incubated with
2
mM of fructose-1,6-
A protein microfluidic channel was created as
bisphosphate (10 times the KD of 0.2 mM
(30,35-37)), to ensure the saturation of the
allosteric binding sites. The fluids followed a
laminar flow pattern until they reached the notch
in the glass capillary, at which point they were
combined and mixed. The reaction time was
adjusted by changing the position of the mixing
capillary within the channel; labelling times of
59 ms to 1 s were used in the experiment.
Pulling the mixing glass capillary back increased
the dead space after mixing, thus increasing the
described previously (34), by passing
a
polyimide coated glass capillary (OD. 153 mM,
ID. 75 mM; Polymicro Technologies, Phoenix,
AZ, USA) through a 12 cm long metal capillary
(OD. 318 mM, ID. 158.75 mM). The distal end
of the glass capillary was sealed with fused silica
by cutting at high laser power and a notch was
cut 2 mm from the sealed end. The proximal
end of the mixing channel was pressure-fit into
PTFE tubing and connected to a three-way T-
union. The distal end of the glass capillary was
then pulled back until it was flush with the metal
capillary and soldered into one of the input
channels on the PMMA substrate, which created
the entrance for both the protein and D2O. Two
metal capillaries (OD. 400 mM, ID. 200 mM;
Small Parts, Inc., Miramar, FL, USA) were
soldered into the second input channel, for
supplying the acid quenching solution (12.5%
acetic acid, pH 2.3); and the output channel,
which served as the ESI source for direct
coupling to the MS. The engraved PMMA
substrate was lined with a piece of silicon-rubber
to create a liquid-tight seal around the reaction
well. Pepsin agarose (~15 mg) was evenly
spread over the digestion ‘well’ using 1 M HCl.
reaction time.
Reaction labelling was
immediately quenched by reduction of pH to
~2.6 by using a 12.5% acetic acid solution (8
mL/min; pH 2.3) creating a 75:25 H2O/D2O
mixture. The protein-D2O-acid mixture was
digested across the pepsin agarose beads of the
digestion well for rapid digestion with minimal
back exchange. Voltage (4600–5000 V) was
applied to the ESI source capillary creating a gas
‘spray’ of peptides which was detected on the
QqTOF-MS. The samples were scanned over
the 400–1500 m/z range.
Data analysis - All MS spectra analyses were
carried out as previously described (38) on the
mMass software, version 5.5 (39). Peptide
identification was performed using a FindPept
4

Conformational Dynamics and Allostery in Pyruvate Kinase
result from stable hydrogen bond formation
within secondary structure elements and
decreased solvent accessibility.
tool on the ExPASy proteomic server (Swiss
Institute of Bioinformatics, Basel, Switzerland)
in
conjunction
with
collision-induced
The global hydrogen-deuterium exchange
kinetic plot (Fig. 3) reveals that when fructose-
1,6-bisphosphate is present, the enzyme has a
higher amplitude of deuterium exchange
(~Δ1966 Da), which is evident in the increased
change in mass seen at 1−1.5 s. In the absence
of fructose-1,6-bisphosphate, the amplitude is
~Δ1884 Da. Moreover, we note that there is a
difference in the burst phase (0−14 ms) between
dissociation (CID) to identify any ambiguous
peptides. The amount of deuterium exchange
was computed using software for isotropic
distribution analysis developed in-house, and
normalised to the maximum deuterium exchange
of 75%. Hydrogen-deuterium exchange kinetic
data was fitted using single exponential non-
linear regression analysis [y=y0 + a(1-e-kt)] and
normalised in OriginPro (v8.5.1).
Protein
the differently treated proteins.
This is
structures were rendered using PyMOL (The
PyMOL Molecular Graphics System, Version
1.5.0.4 Schrödinger, LLC).
consistent with a globally more dynamic protein
that can exchange deuterium more readily.
From this, we infer that the binding event causes
a
shift towards
a
more unstructured,
RESULTS AND DISCUSSION
heterogeneous protein.
Proteins exist as an ensemble of conformers
and conformational sampling allows proteins to
fluctuate between different conformers (40-42).
The lowest energy state is the most favourable;
thus, a greater proportion of the population will
exist in this state (24,43). In theory, all possible
conformational states of a protein can be
mapped and described by an energy landscape
(24,44), although in practice detecting high
energy conformers is more challenging owing to
their small number in the population and
Not only was there a change in the amount of
deuterium taken up by the protein on the TRESI
timescale (milliseconds to seconds), but the rate
of exchange was also augmented upon fructose-
1,6-bisphosphate binding, corresponding to an
increase from 1.9 ± 0.5 s-1 to 2.9 ± 0.8 s-1. This
is also consistent with activator binding
increasing the globally averaged dynamic
conformational sampling the protein is
undergoing over time: that is, fructose-1,6-
bisphosphate increases not only the extent of
conformational space being sampled, but also
how ‘fast’ the protein explores conformational
space (46).
transient nature.
In the dynamic view of
allostery, the binding of the allosteric modulator
remodels the energy landscape and triggers a
shift in the relative occupancy of states in the
To
corroborate
this
global
TRESI
conformational
ensemble
(24,26,42)—the
experiment, we measured the thermal stability of
the wild-type enzyme in the absence and
conformation that represents the lowest energy
state is changed, although many conformers may
still be sampled. Importantly, this shift increases
how often a given (activated or inhibited) state is
sampled (24,26,44). For example, the binding of
reduced nicotinamide adenine dinucleotide
phosphate increases how often dihydrofolate
reductase samples the active ‘R-state’ (27,45).
Fructose-1,6-bisphosphate binding causes an
increase in global protein flexibility – To gauge
changes in global conformational dynamics,
global hydrogen-deuterium exchange was
measured as a time course (14 ms to 1.5 s) for E.
coli pyruvate kinase type 1 with and without its
allosteric activator, fructose-1,6-bisphosphate.
The data were fitted to an exponential function,
generating a kinetic plot of overall protein
deuterium exchange (Fig. 3). Increases in
deuterium exchange characteristically result
from intermittent loss of hydrogen bonds within
and between secondary structure elements, due
to changes in structure or conformational
flexibility, leading to increased solvent access
associated with destabilisation of secondary
structure and tertiary structure. By the same
token, decreases in deuterium exchange typically
presence
bisphosphate.
(0.25
mM)
of
fructose-1,6-
We found that fructose-1,6-
bisphosphate decreases the thermal stability by
~5 °C, from 54.3±0.3 (S.D.) to 49.7±0.3 °C.
This had been previously observed for the E.
coli pyruvate kinase type 1 enzyme (30), but is
in contrast to the Trypanosoma cruzi and T.
brucei pyruvate kinase enzymes, where fructose-
1,6-bisphosphate increases the thermal stability
(10). Taken together, these data suggest that
fructose-1,6-bisphosphate binding to the
allosteric domain causes a shift towards a
globally more unstructured and dynamic
ensemble of conformations. This may also
explain our (and others’ (8)) observation that
treating crystals of E. coli pyruvate kinase type 1
with fructose-1,6-bisphosphate results in crystal
cracking and the lack of conditions for co-
crystallisation.
Localised changes in deuterium exchange
reveal regions with altered conformational
flexibility – To provide detailed information on
regions of the protein where conformational
flexibility significantly changes upon fructose-
1,6-bisphsphate binding, enzyme that was
5

Conformational Dynamics and Allostery in Pyruvate Kinase
premixed with deuterium (59 ms to 1 s) was
regions that show significant changes, which we
address in the sections below.
digested using pepsin and we determined the
amplitude of deuterium exchange on individual
peptides over this time period. Digestion of
pyruvate kinase type 1 was effective, given the
size of the protein, resulting in a 64% sequence
coverage corresponding to 36 distinctive
peptides, and an average spatial resolution of 10
amino acid residues. Typical raw hydrogen-
deuterium exchange mass spectrometry data of
representative peptides (Fig. 4A) demonstrates
the shift in the mass envelope caused by
differences in deuterium exchange of unbound
and bound pyruvate kinase peptides. Kinetic
hydrogen-deuterium exchange profiles of the
representative peptides were fit to a single
exponential expression to illustrate a change in
peptide deuterium exchange as a function of
labelling time (Fig. 4B).
Dynamic changes to the allosteric domain
upon fructose-1,6-bisphosphate binding – We
first examined the allosteric binding domain to
assess how fructose-1,6-bisphosphate binding
affects its conformational flexibility. From Fig.
6, fructose-1,6-bisphosphate binding results in
clear changes in deuterium exchange in the
allosteric C-domain. Since a structure of E. coli
pyruvate kinase type 1 with bound fructose-1,6-
bisphosphate is not available, we have modelled
fructose-1,6-bisphosphate binding to the
allosteric binding domain using overlays with
the S. cerevisiae pyruvate kinase structure (11)
(Fig. 7A and 7B). For S. cerevisiae pyruvate
kinase, fructose-1,6-bisphosphate binding in the
allosteric site is facilitated by residue Arg459,
forming a strong electrostatic interaction with
the 1’-phosphate group of fructose-1,6-
bisphosphate. In addition, the 6’-phosphate
makes a series of hydrogen bonds with side
chains from the sequence Ser402-Thr-Ser-Gly-
Thr-Thr407 (Thr378-Gln-Gly-Gly-Lys382 are
the equivalent residues in E. coli pyruvate kinase
type 1) and the sugar ring of the fructose-1,6-
bisphosphate forms interactions with residues
Gln483 and His491. Moreover, chemical
modification of E. coli pyruvate kinase type 1
identifies Lys382 (equivalent to Thr406 in S.
cerevisiae pyruvate kinase) as important for
fructose-1,6-bisphosphate binding, providing
some evidence that the allosteric binding site is
located in a similar pocket to that of S.
cerevisiae pyruvate kinase (11).
We then mapped the percentage of deuterium
exchange for each peptide onto the pyruvate
kinase structure to illustrate regions that have
significant changes in local backbone dynamics
(Fig. 5, the colour spectrum indicates the level of
exchange: blue to red; low to high exchange,
respectively). Qualitatively, the protein in the
absence of fructose-1,6-bisphosphate shows
generally low exchange of deuterium (20−60%),
commensurate with a stable, globular structure.
In particular, the peptides close to the tetrameric
interfaces (A/A’, peptide 291−294; and C/C’,
peptide 452−467) show low exchange, which is
consistent with our previous work demonstrating
that pyruvate kinase type 1 is a very stable
4-1
tetramer (KD < 10 nM) (35). Moreover, the
peptides at the A/C inter-domain interface
(peptides 398−408 and 409-419, circled in Fig.
5) have relatively low deuterium exchange (47%
and 56%) compared to the same peptide in the
presence of fructose-1,6-bisphosphate (87% and
95%).
When mapping the degree of exchange in
peptides derived from protein pre-treated with
fructose-1,6-bisphosphate and comparing this
with the unbound peptides, it is evident that the
pattern of exchange is different (Fig. 5A and
5B). Figure 6A shows only those peptides that
exhibited a significant change in absolute
deuterium exchange (>15% absolute difference)
when comparing peptides derived from unbound
enzyme with those from fructose-1,6-
The
binding
site
for
fructose-1,6-
bisphosphate in the allosteric domain shows key
changes in conformational flexibility. Assuming
fructose-1,6-bisphosphate binds in the same
pocket and in a similar orientation, ligand
binding requires a displacement of surface
accessible water molecules and a change in the
hydrogen bonding network of the pocket,
including residues of the Cα3’ and Cα4’-helices
and the effector loop (Fig. 7C), so we expected
to find changes to the backbone flexibility in
these regions. We note firstly that the surface
that binds fructose-1,6-bisphosphate has
decreased exchange, consistent with fructose-
1,6-bisphosphate binding decreasing the
availability of amides and preventing exchange.
In addition, there is a large increase in deuterium
exchange for the peptide containing the effector
loop, residues 452 to 467 (Fig. 6C). The effector
loop is at the suspected activator-binding site
and it shows a 38% increase in deuterium
bisphosphate bound enzyme.
To permit
straightforward interpretation of the data, the
absolute changes are noted only as an increase or
decrease in exchange (Fig. 6B). The percentage
change in deuterium exchange upon fructose-
1,6-bisphosphate binding was then mapped to
the structure to highlight regions of the protein
that exhibited significant changes in backbone
deuterium exchange (Fig. 6C). Clearly, there are
exchange.
It has been implicated as the
fructose-1,6-bisphosphate binding loop (10,17),
and in the unbound state it is located very near
the position of suspected fructose-1,6-
6

Conformational Dynamics and Allostery in Pyruvate Kinase
bisphosphate binding. One interpretation of our
(10,47), which show a rigid-body rocking of the
A/C-core domains, and temperature factor
analysis of these structures does not suggest that
fructose-1,6-bisphosphate binding affects the
mobility of the A/C inter-domain interface. Our
result is, however, consistent with the model put
forward by Mattevi et al. (8), who reasoned that
the A/C-domains were altered based on the non-
allosteric rabbit M1 structure.
Mutational analysis of residues at the A/C
inter-domain interface is consistent with the
dynamic changes that we observe. Firstly,
Valentini and colleagues created variant
enzymes to perturb salt bridges between the A-
and C-domains. They created the substitutions
Lys413Gln, which is at the end of the Cα4’-
helix, and Arg271Leu, which sits above the
Cα3’-helix, resulting in severely altered
functional behaviours compared to the wild-type
enzyme (30). Lys413 is positioned on a peptide
that has increased deuterium uptake in the
result is that fructose-1,6-bisphosphate binding
pushes the enzyme towards a conformational
state that favours the effector loop positioned to
the side. Figure 7A shows the position of the
effector loop when fructose-1,6-bisphosphate is
bound in the S. cerevisiae structure, while Fig.
7B shows the effector loop position in the
absence of fructose-1,6-bisphosphate (E. coli
pyruvate kinase with fructose-1,6-bisphosphate
modelled into the binding site from 1A3W),
illustrating the change in conformation for this
loop that may occur.
The neighbouring Cα3’-helix (residues
370−391) has a 17% decrease in deuterium
exchange (Fig. 6B and 6C). The decrease in
dynamics for the Cα3’-helix may be due to
hydrogen
bonding
with
fructose-1,6-
bisphosphate, decreasing the number of amide
hydrogen atoms available for exchange. In the
S. cerevisiae structure (PDB ID 1A3W), the 6’-
phosphate
of
fructose-1,6-bisphosphate
presence
of
fructose-1,6-bisphosphate.
hydrogen bonds with Thr407 of the Cα3’-helix
(11). That the 382 variant, which precedes the
Cα3’-helix of E. coli pyruvate kinase type 1,
results in a 70-fold decrease in fructose-1,6-
bisphosphate binding affinity (30) supports the
assumption that this helix also binds the
allosteric activator in the E. coli enzyme.
Hydrogen bond formation with fructose-1,6-
bisphosphate may cause a reorientation of
residue Gln379, weakening its interactions with
residues Asn402 and Thr405 of the Cα4’-helix
(Fig. 7D). The mutagenic study at Lys382 and
our significant decrease in conformational
flexibility collectively support the concept that
the Cα3’-helix is directly involved in
interactions with fructose-1,6-bisphosphate and
important for communicating the allosteric
signal to the Cα4’-helix.
Importantly, the Lys413Gln amino acid
substitution induced activation of the enzyme in
the absence of fructose-1,6-bisphosphate, shown
by a left-shift of the phosphoenolpyruvate
saturation curve (S0.5 for phosphoenolpyruvate
binding decreased in Lys413Gln compared to
wild-type) (30).
Similar work in Bacillus
stearothermophilus pyruvate kinase, where the
equivalent residue is substituted (Trp466Tyr),
also demonstrated activation of the variant
enzyme in the absence of the allosteric activator
(48). In contrast, the Arg271Leu amino acid
substitution resulted in a 5-fold increase in S0.5
for fructose-1,6-bisphosphate binding, as well as
a
right-shift of the phosphoenolpyruvate
saturation curve in the absence of fructose-1,6-
bisphosphate, suggesting that it does affect the
Cα3’-helix,
which
binds
fructose-1,5-
Significantly,
fructose-1,6-bisphosphate
bisphosphate. Thus, these two variants validate
the significance of the A/C inter-domain salt
bridges for allosteric signal transfer between
allosteric and catalytic domains, consistent with
our hydrogen-deuterium exchange studies.
Next, we created four E. coli pyruvate kinase
variants at the Cα4’-helix to probe the role of
hydrogen bonds within the region that bridges
the A- and C-domains on the allosteric
mechanism. Thr405 interacts with the end of the
Cα3’-helix, while Lys404 and Gln408 form
hydrogen bonds with residues at the bottom of
the A-domain, and Leu411 sits in a hydrophobic
pocket at the N-terminal end of the (β/α)8-barrel.
Kinetic analysis of these variants is presented in
Table 1.
binding also causes destabilisation of the inter-
domain interface that links the allosteric domain
(C-domain) and the (β/α)8-barrel (A-domain)
within the monomer (Fig. 7). Our dynamic data,
when mapped onto the structure, suggests that
the interactions between the allosteric domain
and the A-domain are largely mediated by the
Cα4’-helix of the allosteric domain (circled in
Fig. 5B and 7C) and the N-terminal loops of the
(β/α)8-barrel.
bisphosphate causes
The binding of fructose-1,6-
40% increase in
a
deuterium exchange of an α-helix (Cα4’-helix)
motif that forms the interface between the
(β/α)8-barrel (A-domain) and the allosteric
domain (C-domain). This strongly suggests it
may play an important part in the transition
between inactive and active state populations.
This result is inconsistent with the L. mexicana
and T. cruzi pyruvate kinase crystal structures
The substitution of Thr405 to alanine results
in a 5-fold increase in the S0.5 for fructose-1,6-
bisphosphate, from 0.006±0.001 mM for the
wild-type enzyme to 0.029±0.009 mM for the
7

Conformational Dynamics and Allostery in Pyruvate Kinase
Thr405Ala variant. This is consistent with the
results in enzyme activation. Arg271Leu, which
proposed interaction of the Cα4’- and Cα3’-
helices, whereby fructose-1,6-bisphosphate
binding alters the hydrogen bonding pattern
between Gln379 and the Cα4’-helix.
The Gln408Ala variant, which bridges the A-
and C-domains, shows a significant right shift of
both ADP and phosphoenolpyruvate saturation
curves in the presence of fructose-1,6-
sits above the Cα3’-helix, affects both
phosphoenolpyruvate
and
fructose-1,6-
bisphosphate binding, consistent with a key role
of the Cα3’-helix in the allosteric mechanism.
Disruption of hydrogen bonds within the Cα4’-
helix by substitutions at positions Thr405, which
interacts with the end of the Cα3’-helix, and
Gln408, which interacts with residues at the
bottom of the A-domain, result in disruption of
fructose-1,6-bisphosphate binding and altered
catalysis. Substitution of Leu411 with glutamate
suggests that changes at this interface can be
destabilising.
The tetrameric A/A’ and C/C’ interfaces are
also altered upon fructose-1,6-bisphosphate
binding – The tetrameric C/C’ interface that is
formed by the allosteric domains shows
significantly increased conformational flexibility
bisphosphate.
In addition, the enzyme is
catalytically compromised in the absence of
fructose-1,6-bisphosphate (kcat
=
95±3 s-1
compared to wild-type kcat = 216±4 s-1). Thus,
the mechanism of allostery appears to be
changed: for this variant, fructose-1,6-
bisphosphate also affects the maximal rate (kcat
=
95±3 s-1 in the absence, but 283±30 s-1 in the
presence
Interestingly,
of
fructose-1,6-bisphosphate).
Gln408Ala has decreased
thermostability (47.5±0 °C), which is further
destabilised 10 °C by fructose-1,6-bisphosphate
(37.3±0.3 °C). This variant, which bridges the
Cα4’-helix with the A-domain via the main
chains of Ala187 and Asp214, and the side chain
of His216, suggests clear connections between
the A- and C-domains and is consistent with a
destabilising role for the Cα4’-helix, but
suggests that the level of destabilisation is
carefully tuned for activation.
The substitution of Lys404 with alanine
resulted in no change in function, suggesting that
this residue plays either a minor or no role in the
allosteric mechanism. The introduction of a
negative charge to the hydrophobic (β/α)8-barrel
in Leu411Glu, results in an inactive enzyme, and
an extremely low unfolding temperature (Tm ~23
°C), suggesting that mutations at the A/C
domain interface can destabilise the protein.
To summarise, hydrogen deuterium exchange
studies suggest that fructose-1,6-bisphosphate
binding results in large changes in
conformational flexibility of key elements
within the allosteric domain. Firstly, the effector
loop, which in the unbound pyruvate kinase type
upon
fructose-1,6-bisphosphate
binding,
suggesting that the β-strand (Cβ5’-strand;
residues 463−470) that forms this interface is
destabilised.
Figure 8A shows the C/C’
interface interactions in the unbound E. coli
pyruvate kinase type 1. These interactions
include hydrogen bonds between residues
Ala465 and Ala465’, Val467 and Asn463’ and
Asn463 and Val467’. Fructose-1,6-bisphosphate
binding causes a 38% increase in deuterium
exchange in the peptide that includes this strand.
Although peptide coverage across the A/A’
interface is poor (7 of 26 residues in the
fructose-1,6-bisphosphate unbound monomer are
within peptides that could be detected in both
samples), several peptides show altered
deuterium exchange (Fig. 8A). The peptide
containing residues 291−294 has an increase in
deuterium exchange (difference of +31%) and is
less structured.
In contrast, the peptide
containing residues 255−258, which is close to
the A/A’ interface, has decreased exchange
(difference of -38%) and is, therefore,
significantly more structured upon fructose-1,6-
bisphosphate binding.
1
structure lies across the fructose-1,6-
Our deuterium exchange data supports an
allosteric mechanism whereby, fructose-1,6-
bisphosphate binding destabilises and decouples
the subunits at the tetrameric interfaces to
bisphosphate binding site, has increased
conformational flexibility, consistent with its
displacement upon binding.
Cα3’-helix has decreased conformational
flexibility, consistent with fructose-1,6-
bisphosphate binding to this helix (Fig. 7B), as
seen in the S. cerevisiae structure. Finally, the
Cα4’-helix has increased conformational
flexibility (Fig. 7D), which we propose is due to
weakening interactions between the Cα3’ and
Cα4’-helices, as the Cα3’-helix binds fructose-
1,6-bisphosphate (Fig. 7D). This is corroborated
by mutagenic data. Substitution of Lys413 with
alanine at the end of the Cα4’-helix breaks a salt
bridge between the A- and C-domains and
Secondly, the
remove
phosphoenolpyruvate
cooperation
between active sites (homotropic activation).
This finding is corroborated by mutagenic
studies from two groups, Lovell et al. (1998) and
Fenton and Blair (2002), who both created
pyruvate kinase variants with amino acid
substitutions at the tetrameric interfaces (36,49).
Firstly, Fenton and Blair (2002) generated the
Glu392Ala pyruvate kinase variant at the C/C
interface, resulting in complete loss of
homotropic activation by phosphoenolpyruvate,
suggesting that in yeast, the active sites
8

Conformational Dynamics and Allostery in Pyruvate Kinase
communicate through this interface (36).
Moreover, Lovell et al. (1998) propose that the
equivalent A/A interface residue of E. coli
loops at the N-terminus of the (β/α)8-barrel.
Residue Gln408 of the key Cα4’-helix forms
interactions with Asp187 of loop α3β3 and
Asn214 of loop α4β4, while Lys404 of the Cα4’-
helix interacts with Gly185 of loop α3β3 (Fig.
7E). Given the large change in conformational
flexibility of the Cα4’-helix, unsurprisingly, the
β-strands following the αβ-loops (β-strands 4
and 5, respectively) show an increase in
deuterium exchange of 23% and 31%,
respectively. Furthermore, β-strands 1, 2 and 3
also showed some destabilisation (increase in
deuterium exchange of 36%, 22% and 27%,
respectively), resulting in a largely unstable
(β/α)8-barrel core. The destabilisation of these
β-strands suggests the allosteric signal is
communicated through the core of the (β/α)8-
barrel to the active site.
Changes in deuterium exchange were also
identified in the active site loops at the top of the
(β/α)8-barrel (Fig. 9). Exchange in the peptide
containing loop β8α8 increased by 20% and the
peptide with the neighbouring loop β1α1
increased by 36%. Furthermore, β-strands 1, 2,
3, 4 and 5 in the core of the (β/α)8-barrel, near
the substrate binding sites, were also
destabilised. Specific substrate binding regions
are modelled from L. mexicana pyruvate kinase
crystal structures (PDB ID: 3HQP). This reveals
that the adenosine ring of the ATP (or ADP) is
located within interaction distance of loops β1α1,
β2α2, β3α3 and β8α8, while the oxalate molecule
(an analogue of phosphoenolpyruvate/pyruvate)
and catalytic ions are situated near β-strands 4
and 5 (Fig. 9). Thus, many of the catalytically
pyruvate
kinase
Gln279
in
B.
stearothermophilus pyruvate kinase is important
in coupling phosphoenolpyruvate binding with
cooperative and allosteric functions (49).
Overall, the peptides mapped to the
tetrameric interfaces demonstrate increased
conformational flexibility, indicating that the
interfaces are destabilised upon fructose-1,6-
bisphosphate binding. Interestingly, this result,
together with the increased conformational
sampling evidenced from the global hydrogen-
deuterium
exchange
experiment
above,
contradict the general hypothesis put forward by
Morgan and co-workers (10), that “while the
regulatory molecules and molecular mechanisms
may vary between species, the underlying
principle of tetrameric stabilisation in response
to effector binding is conserved.” That study
used thermal unfolding to infer conformation
stability, whereas the data from the hydrogen-
deuterium exchange directly assesses the degree
of conformational flexibility, both globally and
within specific regions within the protein. In
any event, together with the dramatic
destabilisation at the A/C inter-domain interface
Cα4’-helix (peptide 398−408), our results
connect the domain and tetrameric interfaces,
and in particular their destabilisation, to the
allosteric mechanism of E. coli pyruvate kinase
type 1 enzyme (30).
Lastly, our data are consistent with the affect
that
fructose-1,6-bisphosphate
has
on
phosphoenolpyruvate affinity.
Fructose-1,6-
important
residues
show
increased
bisphosphate binding blocks the homotropic
allosteric mechanism by phosphoenolpyruvate:
in the absence of fructose-1,6-bisphosphate,
titration of phosphoenolpyruvate results in a
sigmoidal response, a signature of cooperativity,
whereas in the presence of fructose-1,6-
bisphosphate a rectangular hyperbolic curve is
observed, signalling a loss of cooperativity.
Destabilisation of the domain and tetrameric
interfaces, such that the active site containing A-
domains are decoupled from one another, is
consistent with the loss of phosphoenolpyruvate
cooperation between active sites.
Signalling the catalytic site for activation –
Next, we traced changes in conformational
flexibility associated with the active site (β/α)8-
barrel domain to understand the mechanism by
which fructose-1,6-bisphosphate binding affects
catalysis.
conformational flexibility. A caveat to this
analysis is that the peptides, in addition to
residues belonging to β-strands 1, 3 and 4, these
peptides also include residues that are with the
βα-loops, which are likely to be dynamic.
Unfortunately, the resolution limits of this
experiment do not allow us to confirm if the
loop regions are experiencing high exchange,
thus causing an averaged apparent high
exchange of the peptide, or if the entire peptide
is experiencing high exchange.
Increased exchange in these specific active
site loops in the E. coli pyruvate kinase type 1
enzyme implies that substrate binding is likely
conserved across pyruvate kinase enzymes and
that the binding of fructose-1,6-bisphosphate
increases the flexibility of these active site loops
(Fig. 9).
Structural analysis comparing
temperature factors of the L. mexicana structures
in the presence and absence of fructose-1,6-
bisphosphate identifies two active site loops
(β7α7, and β8α8,) and their preceding β-strands (7
and 8), in the core of the (β/α)8-barrel that have
Increased conformational flexibility of the β-
strands within the (β/α)8-barrel accompanies
destabilisation of the Cα4’-helix, which sits at
the inter-domain interface between the allosteric
and catalytic domains and interacts with the αβ-
increased
B-factors
upon
fructose-1,6-
9

Conformational Dynamics and Allostery in Pyruvate Kinase
bisphosphate binding. Increased B-factors in
favoured states can be shifted by the allosteric
event (24,25,27,43,44).
these active site regions suggests allosteric
activation by fructose-1,6-bisphosphate binding
increases the flexibility of active site loops for
substrate and catalytic ion binding. The binding
of the allosteric activator can redistribute protein
conformational ensembles and alter the rates of
interchange between these populations; this can
manifest as a change in flexibility at the active
site (27). The increased exchange can be
explained by these loops having greater
flexibility and therefore an increased range of
conformational sampling, which would increase
the chances of substrate binding for catalysis
(50,51). Increased exchange in the active site at
early labelling time-points is consistent with the
'intensification' model for catalysis-linked
dynamics whereby the energy barrier between
conformational sub-states is lowered upon
activator binding (46). This supports the K-type
binding mechanism, which proposes that
allosteric activation increases the binding
affinity of substrate in the active site (5).
Dynamic allosteric activation mechanism –
We present a step-by-step interpretation of the
observed dynamic changes during the allosteric
transition to the active bound state. We propose
that the following scheme results in allosteric
activation of E. coli pyruvate kinase type 1 upon
fructose-1,6-bisphosphate binding:
1. The presence of fructose-1,6-bisphosphate
destabilises the effector loop and causes a
structural rearrangement, displacing it to allow
binding.
2. The fructose-1,6-bisphosphate then forms
interactions with the Cα3’-helix, increasing the
stability of the helix and weakening an
interaction between the loop preceding Cα3’
and the Cα4’-helix.
3. Destabilisation of the Cα4’-helix results in
destabilisation of the inter-domain interface
that links the allosteric domain (C-domain) and
the (β/α)8-barrel (A-domain) within the
monomer.
The flexible lid domain (B-domain) is not
allosterically controlled by fructose-1,6-
bisphosphate – The exchange profiles of
peptides derived from the lid domain (B-
domain) of pyruvate kinase type 1 are largely
unchanged when the enzyme is pre-treated with
fructose-1,6-bisphosphate, suggesting that ligand
binding in the allosteric site has only a minor
effect upon the secondary structure of the lid
domain. This result is consistent with previous
studies have shown that the movement of the
flexible lid domain is not allosterically
controlled (17,21,52); however, it is controlled
by the presence, type and position of substrates
bound within the active site.
4. Altered interactions of the destabilised
Cα4’-helix at the interface between the A and
C-domains and loops α3β3 and α4β4 result in
destabilised β-strands following the αβ-loops,
consequently
producing
a
more
conformationally flexible (β/α)8-barrel core.
This destabilisation is consistent with the β-
strands being involved in allosteric signal
transfer through the core of the (β/α)8-barrel,
to the active site.
5. The signal transfer through the core of the
(β/α)8-barrel destabilises the substrate binding
loops at the active site. We propose that the
active site loops then sample a greater number
of conformations, some of which facilitate
binding of the substrate and catalytic ions.
6. The destabilisation of the tetrameric
interfaces (A/A’ and C/C’) decouples the
CONCLUSION
The results presented here provide an
experimental characterisation of site-specific
dynamics for the allosteric activation of E. coli
pyruvate kinase type 1, a central metabolic
monomers
attenuating
the
phosphoenolpyruvate homotropic allosteric
signal between active sites, explaining why
fructose-1,6-bisphosphate binding blocks the
phosphoenolpyruvate allosteric mechanism.
enzyme.
The deuterium exchange data
demonstrates that fructose-1,6-bisphosphate
binding alters the conformational flexibility of
specific regions, largely increasing the
conformational sampling in the bound state. In
addition, the data reveal altered dynamics in the
allosteric binding region and also at the substrate
binding site approximately 40 Å away. The
results are consistent with the view of allosteric
activation that describes proteins as dynamic
molecules existing as an ensemble of
conformations (40,41,44), fluctuating between
conformational states, towards which the
In conclusion, we propose
a
new
mechanism by which fructose-1,6-
bisphosphate allosterically regulates pyruvate
kinase. This work extends previous knowledge
of the allosteric activation mechanism by
explaining how fructose-1,6-bisphosphate
promotes substrate binding and attenuating the
phosphoenolpyruvate homotropic allosteric
signal, despite the allosteric binding site being
40 Å away from the active site.
Acknowledgements: We thank Grant Pearce and Jeremy Keown for their graphic assistance. RCJD
acknowledges the following for funding support, in part: 1) the Ministry of Business, Innovation and
10

Conformational Dynamics and Allostery in Pyruvate Kinase
Employment (contract UOCX1208); 2) the New Zealand Royal Society Marsden Fund (contract
UOC1013) and 3) the US Army Research Laboratory and US Army Research Office under
contract/grant number W911NF-11-1-0481. KAD acknowledges the following for funding support:
1) Maurice Wilkins Centre flexible research funding; 2) Journal of Cell Science travel fellowship.
DJW acknowledges support from the Natural Sciences and Engineering Research Council (NSERC)
of Canada Discovery Grant. We acknowledge Jackie Healy for effervescent technical support.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of
this article.
Author contributions: KAD, RCJD and DJW designed and coordinated the study and wrote the
article. KAD, SZ and PL designed, performed and analysed the experiments. All authors were
involved in the interpretation of the data. All authors critically revised the article, reviewed the results
and approved the final version of the manuscript.
11

Conformational Dynamics and Allostery in Pyruvate Kinase
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14

Conformational Dynamics and Allostery in Pyruvate Kinase
FIGURE LEGENDS
Figure 1. (A) The reaction catalysed by pyruvate kinase. (B) Structure of Escherichia coli pyruvate
kinase type 1 tetramer with the tetrameric A/A’ and C/C’ interfaces labelled. (C) Pyruvate kinase
type 1 monomer showing the active site and allosteric binding site. The monomer is coloured by
domain and the helix that connects the allosteric domain with the active site domain is circled.
Figure 2. Schematic representation of the two proposed allosteric activation models. (A) The ‘domain
and subunit rotation model’ proposes that fructose-1,6-bisphosphate binding causes a 16° subunit
rotation and rotations of the B- (17°) and C-domains (15°). (B) The ‘rigid-body rotation model’
suggests a ~6−8° symmetrical rigid domain rocking motion of the AC-domain cores around a pivot
point.
Figure 3. Global deuterium exchange kinetics for E. coli pyruvate kinase type 1. The global
hydrogen deuterium exchange experiment was measured in the absence (!) and presence (") of
fructose-1,6-bisphosphate, providing initial exchange rates of 1.9 ± 0.5 s-1 and 2.9 ± 0.8 s-1,
respectively. Kinetic data was fitted using single exponential non-linear regression analysis (y=y0 +
a(1-e-kt)). The first time point is at 14 ms. The binding of fructose-1,6-bisphosphate increased the
total amount of deuterium exchanged from 1884 Da (without ligand) to 1966 Da (with ligand). This
DMass is the amount of deuterium exchanged into the protein, exclusive of the mass of fructose-1,6-
bisphosphate.
Figure 4. Analysis of site-specific hydrogen-deuterium exchange analysis. (A) Representative
spectra for peptides derived from pyruvate kinase for non-deuterated pyruvate kinase (top row),
deuterated pyruvate kinase from 1 s labelling time (second row), deuterated pyruvate kinase with
bound fructose-1,6-bisphosphate from 1 s labelling time (third row). The raw spectra illustrate the
observed shifts in isotopic distribution following hydrogen-deuterium exchange and black circles
denote the theoretical isotopic distribution generated using FORTRAN (developed in the Wilson
Laboratory, York University). The raw percentage of deuterium exchange for the illustrated replicate
is indicated on each spectra. (B) Hydrogen-deuterium exchange kinetic plots of representative
peptides for pyruvate kinase type 1 in the absence (¾) and presence (l) of fructose-1,6-bisphosphate.
Data represent an average of triplicate runs (error bars denote SEM). Kinetic data was fitted using
single exponential non-linear regression analysis [y=y0+a(1-e-kt)].
Figure 5. Average deuterium exchange mapped and colour-coded onto the E. coli pyruvate kinase
structure (PDB ID: 4YNG (53)). (A) Unbound pyruvate kinase viewing both sides of the subunit and
looking down the (β/α)8-barrel. (B) Pyruvate kinase with the allosteric activator, fructose-1,6-
bisphosphate (magenta) modelled into the structure (based upon 1A3W) viewing both sides of the
subunit and looking down the (β/α)8-barrel. The grey regions were not identified in the hydrogen
deuterium exchange experiment. The Aα4’-helix, residues 398-408, is circled in black. Only
peptides that are identified in both experiments (with and without fructose-1,6-bisphosphate) are
displayed.
Figure 6. Change in average deuterium exchange between the unbound and bound pyruvate kinase
peptides. (A) Amount of deuterium exchange in peptides derived from unbound (blue) and fructose-
1,6-bisphosphate bound (red) pyruvate kinase. Only peptides significant difference (>15% absolute
difference in normalised deuterium exchange) are plotted. (B) Absolute difference (>15%) in
deuterium exchange are plotted as an increase (red) or a decrease in exchange (blue) when in the
presence of fructose-1,6-bisphosphate. (C) Absolute differences (>15%) are mapped onto the
pyruvate kinase subunit as an increase (red) or a decrease in exchange (blue). The allosteric activator
fructose-1,6-bisphosphate (green stick) has been modelled into the structure to show the binding
position and is based upon the structure of S. cerevisiae pyruvate kinase PDB ID: 1A3W). Data
represent an average of triplicate runs and error bars represent the standard error of the mean.
Figure 7. Dynamic changes to the pyruvate kinase allosteric domain. (A) S. cerevisiae allosteric
domain with fructose-1,6-bisphosphate bound (green) (PDB ID: 1A3W). (B) E. coli type 1 pyruvate
kinase allosteric domain with fructose-1,6-bisphosphate (green) modelled from 1A3W into the
binding site showing proposed interactions. (C) (β/α)8-barrel and allosteric domains with the Cα4’-
helix circled. (D) Interactions between Cα3’ and Cα4’-helices. (E) Interactions between the Cα4’-
helix and loops α3β3 and α4β4. Significant changes (>15% absolute difference) in deuterium
15

Conformational Dynamics and Allostery in Pyruvate Kinase
exchange are mapped onto the pyruvate kinase subunit as an increase (red) or a decrease in exchange
(blue). Relevant residues are shown as sticks and key regions of the protein have been labelled.
Interactions are displayed as dashed lines.
Figure 8. Changes in conformational flexibility within the C/C’ and A/A’ tetrameric interfaces.
Change in deuterium exchange mapped onto the E. coli pyruvate kinase type 1 structure highlighting
exchange changes at the (A) C/C’ interface (interactions are shown as dashed lines) and (B) A/A’
interface. Relevant residues are displayed as sticks and labelled.
Figure 9. Dynamic changes to the catalytic site of the (β/α)8-barrel. (A) L. mexicana (β/α)8-barrel,
showing how the substrates bound in the active site (PDB ID: 3HQP). Hydrogen bonds between the
substrates and residues are shown in red with interacting residues shown as sticks and labelled. The
substrates bound in the active site are coloured as follows: ATP (green), pyruvate (magenta),
magnesium ions (orange) and potassium ion (cyan). (B) E. coli type 1 pyruvate kinase (β/α)8-barrel
(looking down the barrel). The active site substrates and equivalent interacting residues are modelled
from the L. mexicana crystal structure (PDB ID: 3HQP) are presented as sticks and labelled. (C) E.
coli type 1 pyruvate kinase (β/α)8-barrel (looking up the barrel).
16

Conformational Dynamics and Allostery in Pyruvate Kinase
TABLES
Table 1. Kinetic parameters for variant enzymes with substitutions in the Cα4’-helix. The variants
were designed to probe the role of the hydrogen bonding network in this helix with the Cα3’-helix
that binds to fructose-1,6-bisphosphate (Thr405Ala) and the interface with the A-domain (Lys404Ala
and Gln408Ala). Pyruvate kinase initial rate data were measured using the lactate dehydrogenase-
coupled spectrophotometric assay, adapted from Valentini and colleagues (30). All measurements
were performed in duplicate and the data were fit to the Hill cooperative model (31) to derive Vmax
S0.5 and nH values.
,
Phosphoenolpyruvate titration (ADP fixed at 4 mM)
With fructose-1,6-bisphosphate (4 mM)
w/o fructose-1,6-bisphosphate
kcat (s-1)
271 ±4
292 ±6
299 ±8
217 ±7
S0.5 (mM)
0.083 ±0.005
0.071 ±0.005
0.061 ±0.006
0.4 ±0.2
nH
kcat (s-1)
216 ±4
259 ±16
135 ±10
95 ±3
S0.5 (mM)
1.34 ±0.03
1.4 ±0.1
nH
Wild-type
Lys404Ala
Thr405Ala
Gln408Ala
1.12 ±0.07
1.15 ±0.08
1.2 ±0.1
3.5 ±0.2
2.9 ±0.4
3.2 ±0.4
4.5 ±0.5
1.9 ±0.1
0.59 ±0.09
1.69 ±0.05
ADP titration (phosphoenolpyruvate fixed at 4 mM)
With fructose-1,6-bisphosphate (4 mM) w/o fructose-1,6-bisphosphate
kcat (s-1)
297 ±8
303 ±7
329 ±6
283 ±30
S0.5 (mM)
0.55 ±0.04
0.53 ±0.03
0.41 ±0.02
2.2 ±0.5
nH
kcat (s-1)
226 ±4
276 ±22
261 ±10
96 ±2
S0.5 (mM)
0.40 ±0.02
0.7 ±0.1
nH
Wild-type
Lys404Ala
Thr405Ala
Gln408Ala
1.27 ±0.07
1.30 ±0.07
1.29 ±0.06
1.00 (fixed)
1.30 ±0.06
1.0 ±0.1
1.2 ±0.1
1.28 ±0.08
0.39 ±0.04
0.41 ±0.03
Fructose-1,6-bisphosphate titration
(ADP fixed at 4 mM, phosphoenolpyruvate fixed at 1 mM)
Vmax (U/mg)
177 ±6
S0.5 (mM)
nH
Wild-type
Lys404Ala
Thr405Ala
Gln408Ala
0.006 ±0.001
0.003 ±0.001
0.029 ±0.009
0.010 ±0.003
0.8 ±0.1
0.4 ±0.1
0.6 ±0.3
0.6 ±0.1
225 ±16
321 ±37
101 ±7
17

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 1
18

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 2
19

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 3
x
20

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 4
21

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 5
22

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 6
23

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 7
24

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 8
25

Conformational Dynamics and Allostery in Pyruvate Kinase
Figure 9
26

Conformational Dynamics and Allostery in Pyruvate Kinase
Katherine A. Donovan, Shaolong Zhu, Peter Liuni, Fen Peng, Sarah A. Kessans, Derek
J. Wilson and Renwick C. J. Dobson
J. Biol. Chem. published online February 15, 2016
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