Review
|
|
|
file on Synergy OPEN |
Acta Biochim Biophys
Sin 2008, 40: 663-669
doi:10.1111/j.1745-7270.2008.00443.x
Modulation of
allostery of pyruvate kinase by shifting of an ensemble of microstates
J. Ching Lee*
Department of
Biochemistry and Molecular Biology, The University of Texas Medical Branch at
Galveston, Galveston, Texas 77555-1055, USA
Received: June 10,
2008
Accepted: June 11,
2008
This work was
supported by grants from the National Institutes of Health (GM77551) and the
Robert A. Welch Foundation
*Corresponding
author: Tel, 409-772-2281; Fax, 409-772-4298; E-mail, [email protected]
Since the introduction of the concepts of
allostery about four decades ago, much advancement has been made in elucidating
the structure-function correlation in allostery. However, there are still a
number of issues that remain unresolved. In this review we used mammalian
pyruvate kinase (PK) as a model system to understand the role of protein
dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)
(a/b)8 barrel structural motif. PK is an ideal system to
address basic questions regarding regulatory mechanisms about this common (a/b)8 structural motif. The simplest model accounting for
all of the solution thermodynamic and kinetic data on ligand-enzyme
interactions involves two conformational states, inactive ET and active ER. These conformational states are
represented by domain movements. Further studies provide the first evidence for
a differential effect of ligand binding on the dynamics of the structural
elements, not major secondary structural changes. These data are consistent
with our model that allosteric regulation of PK is the consequence of
perturbation of the distribution of an ensemble of states in which the inactive
ET and active ER represent the two extreme end states.
Sequence differences and ligands can modulate the distribution of states
leading to alterations of functions. The future work includes: defining the
network of functionally connected residues; elucidating the chemical principles
governing the sequence differences which affect functions; and probing the
nature of mutations on the stability of the secondary structural elements,
which in turn modulate allostery.
Keywords allostery; thermodynamics; protein dynamics; protein fold;
human genetics
One of the major aims of the post-genomic era is to establish the
correlation between protein structural folds and functions. The premise is
based on the assumption that protein folds are designed for specific functions,
analogous to the early concept of one gene for one function. However, it is
soon recognized that diverse functions are carried out by proteins with
essentially identical folds but different sequences. Many protein structural
folds have been identified, for example, the various folds involved in signal
transduction [1–3]. The chemical principles behind these observations are still
unclear. Thus, we focus on the investigation of the chemical principles that
govern the relationship linking sequence-fold-function.We have chosen the biological phenomenon of allosteric regulation as
the focus to tackle the issue of fold-function correlation. The rationale for
the choice is that allostery is a predominant regulatory mechanism and an
allosteric system consists of a variety of functions that can be modulated by a
change in sequence.An ideal system that has an outstanding chance for revealing the
chemical principles of allostery should consist of the following properties: (1)
Structure: defined at the atomic level; (2) Function: identified linked
reactions that lead to allostery; (3) Identities of natural mutants that affect
allostery. Rabbit muscle pyruvate kinase (RMPK) consists of properties that
satisfy these criteria.The outstanding features that make RMPK an ideal system are: (1) The
allosteric behavior can be modulated from a non-allosteric to an allosteric
form; (2) Only 22 amino acid changes out of 530 residues per subunit are
required to accomplish this significant functional change; (3) No significant
structural changes in PK are associated with these changes in residues [4,5].Pyruvate kinase has a triosephosphate isomerase (TIM) (a/b)8 barrel structural motif [6–9]. Although, there is a paucity of knowledge
on the chemical ground rules on how its functions are regulated, PK is an ideal
system to address basic questions regarding regulatory mechanisms about this
common (a/b)8 structural motif [10]. Muscle PK is a homo-tetramer [11–14], as shown in
Fig. 1. Each subunit consists of 530 amino acids and multiple domains.
In each subunit, there is a complete set of binding sites to bind the
substrates [ADP and phosphoenolpyruvate (PEP)], inhibitor (Phe) and activator
(fructose-1,6-bisphosphate, FBP), as shown in Fig. 2. The active site
resides between the A and B domains and at a long distance from the effectors
binding sites that are distributed throughout the subunit structure. Thus, the
regulatory mechanism of RMPK involves communication through distant sites. The
tetrameric RMPK communicates through two intersubunit interfaces mainly between
adjacent A domains and C domains. The only amino acid sequence differences
between the mammalian allosteric kidney PK and
the non-allosteric muscle PK isozymes are located in the C-domain
and are involved in intersubunit interactions (Figs. 1,2). Thus,
embedded in these two isoforms of PK are the rules involved in engineering the
popular TIM (a/b)8 motif to modulate its allosteric properties.
Functional Linkage Scheme of
Allostery for RMPK
As a result of a series of extensive steady-state kinetic,
equilibrium and solution structural studies, a concerted, allosteric model was
developed for quantitative interpretation of the kinetic and equilibrium
binding data of RMPK [15–18]. The simplest model accounting for all of the experimental data
involves two conformational states, inactive ET and
active ER, shown in Fig. 3. E, S, P and I are enzyme, substrate (PEP
or ADP), product (pyruvate) and inhibitor (Phe), respectively.The upper and lower faces of the cube contain all of those species
assuming ER and ET state, respectively. The species are
interconnected with equilibrium constant, L=[ET]/[ER]; KR and KT are the ligand binding
equilibrium constants associated to the ER and ET state, respectively, while KI and KS are the equilibrium constants associated with the binding of
inhibitor Phe and substrate, respectively. The conceptual significance of this
model is that the two states are in a pre-existing equilibrium, L. This crucial
aspect of the model was independently substantiated by published reports
[19,20]. The distribution of ligands bound to these two states is defined by
their respective equilibrium constants to these states. The ligands shift the
state changes in PK by mass action. This model differs from models that assume
a mechanism consisting of a ligand induced state change not in a pre-existing
equilibrium. The simple model shown in Fig. 3 is not adequate to account
for the experimentally defined energy landscape of the linked equilibria that
govern the enzymatic reactions. Added novel features are derived from global
analysis of calorimetric and fluorescence data. These features are: (1)
Coupling between the bindings of ADP and Phe; (2) Temperature dependence of the
preferential binding of ADP to the ER or ET state of PK; (3) At high temperature, ADP actually prefers binding
to the inactive ET state. The consequence is a more sigmoidal
response of PK activity to fluctuation of substrate concentration (Herman and
Lee, unpublished data).In summary, our thermodynamic studies have established
quantitatively the intricate network of interactions among ligands and PK.
Structural Properties of the ER and ET States
The transition between the ER and ET state of RMPK can be monitored by sedimentation velocity or gel
filtration techniques [15,21]. These results imply significant changes in the
quaternary structure of the enzyme. To characterize this change in global
structure of RMPK, we studied the effects of ligands on the structure of RMPK
by small angle neutron scattering [22]. The radius of gyration, RG, decreases by about 1 in
the presence of substrate PEP but increases by the same magnitude in the
presence of inhibitor Phe. When the scattering data were analyzed as a function
of P(r) versus r, where P(r) is the frequency
distribution of all the point-to-point pair distances, and r is distance
between the scattering centers of the particle, the results indicate that the
increase in RG is associated with a pronounced increase in
the probability for interatomic distance between 80 and 110 , shown in Fig.
4 (upper panel). With the aid of computer modeling, these changes in
interatomic distance are consistent with the rotation of the B domain relative
to the A domain, leading to the closure or opening of the cleft between these
domains as a consequence of binding to PEP and Phe, respectively, as shown in
the lower panel of Fig. 4. These results show that one of the dynamic
motions in RMPK includes changes in domain-domain interaction between A and B
domains.We further studied the structural perturbations by Fourier transform
infrared (FT-IR) spectroscopy [23], shown in Fig. 5. The experiments
were designed to monitor the secondary structure of RMPK in the presence of
saturating amounts of various ligands. Mg2+ is a
divalent cation essential for activity. PEP and ADP are the two substrates,
whereas Phe is the inhibitor. Fig. 5 shows that in all experimental
conditions there is no significant change in the areas encompassed by the peaks
assigned to either a or b structures. This implies that there is no detectable conversion of
secondary structure of PK in the presence of all of these ligands. A closer
examination of Fig. 5 shows that the maximum wavenumber associated with a-helix remains
the same, whereas that of the b-strand shifts as a function of ligand. In the presence of Mg2+, PEP and ADP, the maximum wavenumber is less than that in buffer
alone or Phe. These results imply that the local environments of the b-strands are
perturbed by these ligands, although the amount of b strands has not changed.
The origin of the differential perturbations by ligands in the environments of
secondary structures might be the modulation of structural dynamics of RMPK.
Thus, the structural dynamics of RMPK in the presence of various ligands was
probed by H/D exchange monitored by FT-IR. The second derivative spectra as a
function of time of H/D exchange were monitored and compared. The spectra in
the presence of K+ and Mg2+ were
compared with those of RMPK in buffer. They show that the basic pattern of
exchange was retained. However, a larger change in intensity was observed even
at the 1-min time point. These results imply that the activating metal ions
induce an increase in the number of rapidly exchangeable amide protons. The
presence of either PEP or ADP shows a pattern of exchange that is quite similar
to each other, namely, a very rapid exchange was observed in both the helices
and sheets. There is a clear indication of the presence of two different
populations of helices. The change in the second derivative spectra reflecting
the amide proton exchange in the presence of Phe showed that within the time
frame of the experiment no exchangeable amide proton was detected in the b-sheets, an
observation that differs from that of the helices. Thus, these H/D exchange
experiments show that substrates (ADP or PEP) and activating metal ions (Mg2+) lock PK in a more dynamic ER
structure, whereas Phe exerts an opposite effect.These results provide the first evidence for a differential effect
of ligand binding on the dynamics of the structural elements, not major
conformational changes, in RMPK. These data are consistent with our model that
allosteric regulation of RMPK is the consequence of perturbation of the
distribution of an ensemble of states in which the observed change in RG represents the two extreme end states. Sequence differences
and ligands can modulate the distribution of states leading to alterations of
functions.
Composite Effects in the Model
of Allostery Induced by the 22 Residues that Differ between the Isozymes of PK
Composite Effects in the Model
of Allostery Induced by the 22 Residues that Differ between the Isozymes of PK
The differences in the various parameters of the allostery model
induced by the 22 amino acids that are different between the RMPK and rabbit
kidney PK (RKPK) isozymes are summarized in Table 1. It is apparent that
a change of <5% of amino acids in strategically important structural elements
leads to changes in allosteric parameters. Six out of nine parameters are
affected and the magnitude of changes is significant, up to 1000-fold. Thus, a
change in a small number of residues has a major impact on the behavior of the
RMPK.These changes are not elicited either by changes in the sequences
involving the active sites or by changes in the other ligand binding sites.
These changes in the binding constants of ligands are strictly elicited by
changes of 22 amino acids in a localized region, as shown in Figs. 1 and
2. These amino acids are located in the C-domain of each subunit and are
obviously involved in intersubunit interactions. They are far removed from the
active sites and yet changes in these residues lead to very significant changes
in the allosteric properties of the enzyme including favoring the ET state and higher affinities of substrate and inhibitor to both
states.
Effects of Single Residue
Mutation Derived from the 22 Amino Acids that Differ between the Isozymes
S402P
In our study, to elucidate the role of each of these 22 residues in conferring
allosteric properties to the RMPK, we mutated residue 402 of RMPK from S to P,
in accordance with the difference in sequence between the two isozymes.
Converting S402 to P changes neither the secondary, nor the tetrameric
structure [24]. The S402P RMPK mutant exhibits steady-state kinetic behavior
that indicates that it is more responsive to regulation by effectors, as shown
in Fig. 6. The data are shown as initial enzyme velocity versus substrate
PEP concentration. The sigmoidicity of the curves is a reflection of
cooperativity of substrate binding. The RMPK data show almost no sigmoidicity,
as expected for an enzyme exhibiting little allosteric behavior, whereas the
RKPK shows pronounced sigmoidicity. The data for the S402P RMPK are
intermediary to the muscle and kidney isozymes. The presence of 12 mM inhibitor
Phe shifts the curve to the right, as expected, since Phe would shift the
conformational state equilibrium towards ET, which
has a weaker affinity for PEP. The presence of 10 nM activator FBP in addition
to 12 mM Phe shifts the curve to the left. In RMPK, it would require millimolar
concentrations of FBP to achieve the same effect. Thus, a S402P mutation
confers partial restoration of allosteric behavior to the RMPK. We have elucidated the atomic structure of the S402P RMPK variant by
X-ray crystallography [25]. Although the overall S402P PK structure is nearly
identical to the wild-type (WT) structure within experimental error, significant
differences in the conformation of the backbone are found at the site of
mutation. We found that the ratio of B-factors of mutant/WT provides a good
representation of the pattern of long range communications between distant
sites [25]. The most obvious increase in B-factor in the S402P PK is around the
residue 402 indicating a significant increase in dynamics in that region. There
are also significant changes in the ratio of B-factor for residues 50 to 200.
In addition, there is an increase in the number of heterogeneity in the angle
assumed by the B-domain with respect to the A-domain (i.e., increase dynamics
in domain movements). Closer examination of the X-ray data shows a disruption
of a salt bridge between residues 341 and 177 of an adjacent subunit. This salt
bridge and residue 402 reside in different subunit interfaces. Thus, these
structural data show a communication between these two different subunit
interfaces. A similar conclusion was derived from the results of our study of
subunit assembly [26,27]. It is evident that a mutation at residue 402 leads to
increased dynamics in distant sites through long range communication without
significant changes in secondary structures.
Establishment of Functional
Coupling among Residues
T340M RMPK mutant
In an effort to establish functional coupling among residues in
RMPK, we incorporated into our studies the human genetic data [28–33]. Our choice
of residue 340 is based on the combined results of our structural studies and
human genetic data. The crystallographic structures of yeast and mammalian PK
have identified two intersubunit interfaces [4,5,11–14,34], one involves the
C-domain while the other comprises mainly the A domain, as shown in Fig. 1.
In our modeling study we have further identified the residues whose inter-a carbon
distances are within 15 [35] along the
axis between the adjacent A-domains. These include residues 330–350. Human
genetic data identified T340M as a mutant that is observed in patients
suffering from pyruvate kinase deficiency. The T340M RMPK and RKPK mutants are only half as active as the WT PKs. The T340M
RMPK enzyme is more susceptible than RKPK to
inhibition by Phe or to the activator FBP. Evidently the 22 residues in the
C-domain modulate the effect of residue 340, which reside in different subunit
interfaces. The result is a differential effect on the functional energetics of
PK. This study demonstrates the linkage between distant residues in different
interfacial interactions (i.e. establishing the functional coupling among
residues and the identities of these residues) [24,26,27].
Prospectus
Although we were successful in discovering that protein dynamics are
intimately coupled to allostery and the effect of differences in protein
sequence on the functional energy landscape of PK, there are serious
limitations: (1) Identification of connectivity among residues to modulate
functions is a very time consuming effort and is limited to a pair at a time.
It is very inefficient trying to establish the goal of defining the network of
functionally connected residues; (2) Even if the complete network has been
identified, we might not have gained deeper insight into the chemical
principles involved in these results that are basically simply phenomenological.
It is difficult to assign significance in sequence differences to the chemical
principles that govern the changes in functions (i.e. failure to establish the
ground rules in linking sequence-fold-function). Hence, our future direction of
research on RMPK focuses on: (1) Cast both structure and function in terms of
energetics, allowing us to correlate the energy landscapes; (2) Elucidate the
chemical principles governing the sequence differences that affect functions.
Probe the nature of mutations on the stability of the secondary structural
elements, for example, N- and C-caps of helices, helices and loops; (3) Use the
algorithm such as COREX/BEST [36–41] to probe the connectivity among distant
residues. Probe the changes in this connectivity pattern due to mutations; (4)
Integration of results from 1 to 3 will enhance our chances to identify the
chemical principles that govern the correlation of sequence-fold-function.
References
1 Pawson T, Nash P. Assembly of cell regulatory
systems through protein interaction domains. Science 2003, 300: 445–452
2 Copley RR, Bork P. Homology among (ba)8
barrels: implications for the evolution of metabolic pathways. J Mol Biol 2000,
303: 627–640
3 Heggi H, Gerstein M. The relationship between
protein structure and function: a comprehensive survey with application to the
yeast genome. J Mol Biol 1999, 288: 147–164
4 Williams R, Holyoak T, McDonald G, Gui C,
Fenton AW. Differentiating a ligand? chemical requirements
for allosteric interactions from those for protein binding. Phenylalanine
inhibition of pyruvate kinase. Biochemistry 2006, 45: 5421–5429
5 Dombrauckas JD, Santarsiero BD, Mesecar AD.
Structural basis for tumor pyruvate kinase M2 allosteric regulation and
catalysis. Biochemistry 2005, 44: 9417–9429
6 Gerlt JA, Babbitt PC. Divergent evolution of
enzymatic function: mechanistically diverse superfamilies and functionally
distinct superfamilies. Annu Rev Biochem 2001, 70: 209–246
7 Wierenga RK. The TIM-barrel fold: a versatile
framework for efficient enzymes. FEBS Lett 2001, 492: 193–198
8 Farber GK, Petsko GA. The evolution of a/b barrel enzymes. Trends
Biochem Sci 1990, 15: 228–234
9 Branden C, Tooze J. Introduction to Protein Structure,
2nd edn. New York, Garland Publishing, Inc. 1999
10 Mattevi A, Bolognesi M, Valentini G. The
allosteric regulation of pyruvate kinase. FEBS Lett 1996, 289: 15–19
11 Muirhead H, Clayden DA, Barford D, Lorimer CG,
Fothergill-Gilmore LA, Schiltz E, Schmitt W. The structure of cat muscle
pyruvate kinase. EMBO J 1986, 5: 475–481
12 Larsen TM, Laughlin T, Holden HM, Rayment I,
Reed GH. Structure of rabbit muscle pyruvate kinase complexed with Mn2+,
K+, and pyruvate. Biochemistry 1994, 33: 6301–6309
13 Larsen TM, Benning MM, Wesenberg GE, Rayment
I, Reed GH. Ligand-induced domain movement in pyruvate kinase: structure of the
enzyme from rabbit muscle with Mg2+, K+,
and L-phospholactate at 2.7 resolution.
Arch Biochem Biophys 1997, 345: 199–206
14 Larsen TM, Benning MM, Rayment I, Reed GH.
Structure of the bis (Mg2+)-ATP-oxalate complex of
the rabbit muscle pyruvate kinase at 2.1 resolution: ATP binding over a barrel.
Biochemistry 1998, 37: 6247–6255
15 Oberfelder RW, Lee LL, Lee, JC. Thermodynamic
linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural
studies. Biochemistry 1984, 23: 3813–3821
16 Oberfelder RW, Barisas BG, Lee JC.
Thermodynamic linkages in rabbit muscle pyruvate kinase: analysis of
experimental data by a two-state model. Biochemistry 1984, 23: 3822–3826
17 Consler TG, Jennewein MJ, Cai GZ, Lee JC.
Synergistic effects of proton and phenylalanine on the regulation of muscle
pyruvate kinase. Biochemistry 1990, 29: 10765–10771
18 Consler TG, Jennewein MJ, Cai GZ, Lee JC.
Energetics of allosteric regulation in muscle pyruvate kinase. Biochemistry
1992, 31: 7870–7878
19 Lonhienne TG, Reilly PE, Winzor DJ. Further
evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon
isomerization of the ternary complex between enzyme and products. Biophys Chem
2003, 104: 189–198
20 Lonhienne TG, Winzor DJ. Calorimetric
demonstration of the potential of molecular crowding to emulate the effect of
an allosteric activator on pyruvate kinase kinetics. Biochemistry 2002, 41:
6897–6901
21 Heyduk E, Heyduk T, Lee JC. Global
conformational changes in allosteric proteins. A study of Escherichia coli
cAMP receptor protein and muscle pyruvate kinase. J Biol Chem 1992, 267:
3200–3204
22 Consler TG, Uberbacher EC, Bunick GJ, Liebman
MN, Lee JC. Domain-interaction in rabbit muscle pyruvate kinase, II:
Small-angle neutron scattering and computer simulation. J Biol Chem 1988, 263:
2794–2801
23 Yu S, Lee L, Lee JC. Effects of metabolites on
the structural dynamics of rabbit muscle pyruvate kinase. Biophys Chem 2003,
103: 1–11
24 Friesen RH, Castellani RJ, Lee JC, Braun W.
Allostery in rabbit pyruvate kinase: development of a strategy to elucidate the
mechanism. Biochemistry 1998, 37: 15266–15276
25 Wooll JO, Friesen RH, White MA, Watowich SJ,
Fox RO, Lee JC, Czerwinski EW. Structural and functional linkages between
subunit interfaces in mammalian pyruvate kinase. J Mol Biol 2001, 312: 525–540
26 Friesen RH, Chin AJ, Ledman DW, Lee JC.
Interfacial communications in recombinant rabbit kidney pyruvate kinase.
Biochemistry 1998, 37: 2949–2960
27 Friesen RH, Lee JC. The negative dominant
effects of T340M mutation on mammalian pyruvate kinase. J Biol Chem 1998, 273:
14772–14779
28 Zanella A, Fermo E, Bianchi P, Chiarelli LR,
Valentini G. Pyruvate kinase deficiency: the genotype-phenotype association.
Blood Rev 2007, 21: 217–231
29 Miwa S, Kanno H, Fujii H. Concise review:
pyruvate kinase deficiency: historical perspective and recent progress of
molecular genetics. Am J Hematol 1993,
42: 31–35
30 Neubauer B, Lakomek M, Winkler H, Parke M,
Hofferbert S, Schr?er W. Point mutations in the L-type pyruvate
kinase gene of two children with hemolytic anemia caused by pyruvate kinase
deficiency. Blood 1991, 77: 1871–1875
31 Valentine WN, Tanaka KR, Paglia DE. Pyruvate
kinase and other enzyme deficiency disorders of the erythrocyte. In: Seriver
CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease,
Vol. 6, New York, McGraw-Hill 1989
32 Kanno H, Fujii H, Hirono A, Miwa S. cDNA cloning
of human R-type pyruvate kinase and identification of a single amino acid
substitution (Thr384?et) affecting enzymatic
stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary
hemolytic anemia. Proc Natl Acad Sci USA 1991, 88: 8218–8221
33 Baronciani L, Beutler E. Molecular study of
pyruvate kinase deficient patients with hereditary nonspherocytic hemolytic
anemia. J Clin Invest 1995, 95: 1702–1709
34 Jurica MS, Mesecar A, Heath PJ, Shi W, Nowak
T, Stoddard BL. The allosteric regulation of pyruvate kinase by
fructose-1,6-bisphosphate. Structure 1998, 6: 195–210
35 Consler TG, Liebman, MN, Lee JC. Structural
elements involved in the allosteric switch in mammalian pyruvate kinase. In:
Liebman MN, Kumosinski T, eds. Molecular Modeling. ACS Symposium Series 576.
1994
36 Pan H, Lee JC, Hilser VJ. Binding sites in Escherichia
coli dihydrofolate reductase communicate by modulating the conformational
ensemble. Proc Natl Acad Sci USA 2000,
97: 12020–12025
37 Hilser VJ, Freire E. Structure-based
calculation of the equilibrium folding pathway of proteins. Correlation with
hydrogen exchange protection factors. J Mol Biol 1996, 262: 756–772
38 Hilser VJ, Freire E. Predicting the
equilibrium protein folding pathway: structure-based analysis of staphylococcal
nuclease. Proteins 1997, 27: 171–183
39 Hilser VJ, Townsend BD, Freire E.
Structure-based statistical thermodynamic analysis of T4 lysozyme mutants:
structural mapping of cooperative interactions. Biophys Chem 1997, 64: 69–79
40 Hilser VJ, Dowdy D, Oas TG, Freire E. The
structural distribution of cooperative interactions in proteins: analysis of
the native state ensemble. Proc Natl Acad Sci USA 1998, 95: 9903–9908
41 Friere E. The propagation of binding interactions
to remote sites in proteins: analysis of the binding of the monoclonal antibody
D1.3 to lysozyme. Proc Natl Acad Sci USA 1999, 96: 10118–10122
42 Consler TG, Woodard SH, Lee JC. Effects of
primary sequence differences on the global structure and function of an enzyme:
a study of pyruvate kinase isozymes. Biochemistry 1989, 28: 8756–8764