(Received for publication, August 15, 1995; and in revised form, January 17, 1996)
From the
We used site-directed mutagenesis of rabbit muscle aldolase,
falling ball viscometry, co-sedimentation binding assays, and negative
stain electron microscopy, to identify specific residues involved in
the aldolase-actin interaction. Three mutants, R42A (Arg Ala),
K107A (Lys
Ala), and R148A (Arg
Ala), had minimal actin
binding activity relative to wild type (wt) aldolase, and one mutant,
K229A (Lys
Ala), had intermediate actin binding activity. A
mutant with
4,000-fold reduced catalytic activity, D33S (Asp
Ser), had normal actin binding activity. The aldolase substrates
and product, fructose 1,6-bisphosphate, fructose 1-phosphate, and
dihydroxyacetone phosphate, reversed the gelling of wt aldolase and
F-actin, consistent with at least partial overlap of catalytic and
actin-binding sites on aldolase. Molecular modeling reveals that the
actin-binding residues we have identified are clustered in or around
the catalytic pocket of the molecule. These data confirm that the
aldolase-actin interaction is due to specific binding, and they suggest
that electrostatic interactions between specific residues, rather than
net charge, mediate this interaction. Low concentration of wt and D33S
aldolase caused formation of high viscosity actin gel networks, while
high concentrations of wt and D33S aldolase resulted in solation of the
gel by bundling actin filaments, consistent with a potential role for
this enzyme in the regulation of cytoplasmic structure.
Most intermediary metabolism is catalyzed by enzymes that are not known to be associated with a discrete organelle or complex, such as the mitochondrion or fatty acid synthase complex. Because of this, metabolic pathways are generally treated as though they exist as a series of diffusion-limited reactions in the aqueous phase of cytoplasm (1) . While this assumption simplifies conceptualization and modeling of metabolism, there is substantial evidence that it is oversimplified, and perhaps incorrect, in at least some cases. Hypotheses involving elegant alternative organizational schemes for cellular biochemistry have been proposed over the years(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) , but they have been difficult to test experimentally due to the ephemeral nature of the interactions at this intermediate level of organization(13) . These are important hypotheses to test, due to their far-reaching implications for cytoplasmic structure and for metabolic regulation.
Glycolysis is a metabolic pathway that may be
organized around the cytoskeleton, rather than in a membrane-bound
compartment(14) . It has been known for many years that several
glycolytic enzymes can interact with cytoskeletal proteins (15) and it has been proposed that some glycolytic enzymes may
play structural and/or regulatory roles in cytoplasm, in addition to
their catalytic roles(16) . Aldolase has one of the highest
bound fractions to myofibrils, stress fibers, and F-actin among the
glycolytic
enzymes(15, 16, 17, 18, 19) .
In fact, aldolase was one of the first actin-binding proteins
identified(20, 21) . There are multiple binding sites
on one aldolase tetramer, demonstrated by its ability to cross-link
F-actin into a gel(16, 22, 23) . Aldolase
binding to F-actin is inhibited by the substrate fructose
1,6-bisphosphate
(FBP)()(16, 23, 24) , and its
catalytic parameters are also changed when bound to actin or actin
containing filaments(24, 25) . Both disassembly of the
actin cytoskeleton with cytochalasin D and inhibition of glycolytic
flux with 2-deoxyglucose result in rapid, reversible release of bound
aldolase in 3T3 cells, consistent with physiologically relevant
cytoskeletal binding of aldolase in vivo(26) .
Several methods have been used in studies aimed at localizing the F-actin binding and catalytic sites on aldolase. Proteolytic cleavage and chemical modification studies indicated spatial separation of substrate and actin-binding sites on aldolase(27, 28) . Kinetic studies have shown that actin filaments can modify the catalytic parameters of aldolase (24, 25) and that myofibrils can competitively inhibit FBP cleavage by aldolase(29) . More recently, a region of the aldolase molecule bearing sequence similarity to the actin-binding site on actin and actin-binding proteins was identified between residues 33 and 45 of aldolase(30) , and it was shown that a synthetic peptide corresponding to aldolase residues 32-52 binds to F-actin and specifically competes with native aldolase for F-actin binding(31) .
In this study we have used site-directed mutagenesis, falling ball viscometry (FBV), co-sedimentation binding assays, and negative stain electron microscopy (EM) to identify residues that are involved in the actin binding activity of aldolase. We present evidence that a specific molecular interaction involving several residues in and near the catalytic site of aldolase mediate its actin binding activity, and we show that modifications of single residues on this enzyme can result in significant alterations of its ability to form gel networks with F-actin.
Fig. 1shows the locations of these residues on a MOLSCRIPT model of the 3.0-Å coordinates of human muscle aldolase. Human and rabbit muscle aldolase (aldolase A) share 99% sequence identity, with most of the differing residues in the amino-terminal end of the molecule, and none of them at or near the residues of interest in this report(35, 40, 41) . Asp-33, Lys-107, Arg-148, and Lys-229 are located in the central catalytic site, and Arg-42 is just outside the catalytic site pocket. The CD spectra of the wild type and mutant proteins were similar (Fig. 2), indicating that these mutations did not cause major perturbations in the secondary structure. The substantial activity of all three mutant enzymes shown in Fig. 2further indicated that these mutations did not cause major perturbations in the tertiary structure of the active site.
Figure 1: A ribbon model of aldolase, highlighting residues Asp-33, Arg-42, Lys-107, Arg-148, Lys-229.
Figure 2:
Circular dichroism spectra of wild type
and mutant aldolases at 25 °C. One spectrum is shown for each
enzyme; recombinant wild type rabbit muscle aldolase from pPB14
(), R42A (
), K107A (
), R148A (
). CD spectra of
D33S and K229A have been published
previously(33
Figure 3:
F-actin gelling activity of wt and mutant
aldolases. Different concentrations of wt and mutant aldolases were
mixed with 0.5 mg/ml F-actin and incubated at 37 167 C for 30 min.
before apparent viscosity of the samples were measured with a falling
ball viscometer. wt (), D33S (
), R42A (
), K107A
(
), R148A (
), K229A (
).
We also investigated the effects of FBP, fructose 2,6-bisphosphate, fructose 1-phosphate, fructose 6-phosphate, D-ribose-5-phosphate, glyceraldehyde 3-phosphate, and dihydroxyacetone phosphate on the F-actin gelling activity of wt aldolase. Fig. 4shows that FBP, DHAP and F1P all inhibited aldolase-F-actin gel formation (2 µM aldolase, 1 mg/ml actin) at less than 40 µM concentrations. Structurally similar compounds F6P, R5P, F-2,6-P, and the aldolase product G3P did not have any detectable effect on aldolase-F-actin gel in the same experimental concentration range, although they eventually did reverse the aldolase-F-actin gel at 500-1,000 µM concentrations (data not shown).
Figure 4:
Effects of phosphosugars on
aldolase-F-actin gels. Different concentrations of reagents were mixed
with 2 µM wt aldolase and 1 mg/ml F-actin before viscosity
measurements were made. FBP (), DHAP (
), and F1P (
)
solated the gel. F6P (
), F-2,6-P (
), G3P (
), and
R5P (
) did not solate the gel and are indistinguishable in this
figure.
Figure 5: Co-sedimentation of wt and mutant aldolases with F-actin. Wild type and mutant aldolases (3 µM) were mixed with 1 mg/ml F-actin before sedimentation. Samples of resuspended pellets were run on a 10% SDS acrylamide gel and then Coomassie-stained (inset).
Figure 6: Rescue of aldolase-F-actin gelation by FBP at high aldolase concentrations. FBP was mixed with 10 µM wt aldolase and 0.5 mg/ml F-actin and incubated at 37 °C for 30 min before apparent viscosity of the samples was measured.
At high aldolase concentrations the F-actin-aldolase gel is solated (Fig. 3), suggesting three possibilities. First, aldolase might sever F-actin (30) and bind to actin monomers or short oligomers, thereby reducing the mass of polymer, and solating the gel(17) . Second, aldolase might bundle F-actin, thus reducing the highly cross-linked network of filaments, and induce solation. Third, at high concentration, aldolase might saturate all binding sites on the actin filaments, thereby allowing the gel to solate. The viscosity decrease below that of F-actin alone at high aldolase concentrations (Fig. 3) is inconsistent with the third possibility. We used negative stain EM to distinguish between the first two possibilities. At 1 µM wt aldolase concentration, 0.5 mg/ml F-actin was extensively bundled and cross-linked, as revealed by the formation of dense network of F-actin filaments (Fig. 7A). Actin filaments were also longer and straighter compared to F-actin alone (Fig. 7E). At 3 µM wt aldolase, F-actin was bundled to a higher degree, but these bundles were no longer cross-linked (Fig. 7C). Samples with 1 µM R42A and 0.5 mg/ml F-actin (Fig. 7B) were not distinguishable from those with F-actin alone; when R42A was increased to 3 µM, we observed limited F-actin bundle formation (Fig. 7D).
Figure 7: Negative stain electron microscopy of mixtures of aldolase with 0.5 mg/ml F-actin. 1 µM wt aldolase bundled F-actin and formed an extensive network (A); 3 µM wt aldolase bundled F-actin and there was no longer an F-actin network (C). Samples with 1 µM R42A (B) were indistinguishable from those with F-actin alone (E); there was limited network bundle formation in the presence of 3 µM R42A (D). Scale bar is 1 µm; magnification is identical in all images.
The acidic N-terminal region of actin is an attractive candidate for interaction with the positively charged residues we have identified on aldolase. However, there is evidence from a study with affinity-purified polyclonal antibodies that aldolase does not bind to sequence regions 1-7, 18-28, or 40-113 on actin(43) . Instead, the same study indicated that aldolase binds to the region around residue 299 on actin(43) . In our experiments, aldolase gels F-actin at a stoichiometry of about 1 aldolase/25 actin monomers, so it must occupy a relatively small percentage of the potential aldolase binding sites on actin at this concentration.
The effect of F-actin on the
catalytic activity of aldolase has been studied previously. Two early
studies showed that F-actin (or actin-containing filaments) caused an
increase in aldolase V and K
for FBP(24, 25) . A more recent study, however,
found that myofibrils acted as competitive inhibitors of aldolase and
caused an increase in K
but not V
(29) . We have demonstrated that the
active site residues Lys-107 and Arg-148 are necessary for normal
aldolase-F-actin binding activity. However, Lys-107 and Arg-148 are
also involved in FBP binding to aldolase(33) , which is
reinforced by our findings with site-directed mutagenesis of these
residues. Consequently, FBP must compete with actin for the substrate
binding site, giving rise to an increase in K
.
The simplest model consistent with our data is one in which actin
and substrate compete for a single domain which includes both
actin-binding and catalytic activities, with actin competitively
inhibiting aldolase activity. An alternate model consistent with the
reported increase in V in the presence of actin (24, 25) would require two actin-binding sites on each
aldolase monomer, one of which is within the catalytic site, and the
other of which is distinct from the catalytic site. This second site
might involve Arg-42, or a residue in that vicinity. The increase in V
could be due to effects of binding at this
site on the conformation or electrostatic configuration of the active
site. Muscle aldolase exists as a tetramer(48) , and kinetic
evidence indicates that each subunit acts independently(49) .
It is thus unlikely that, by binding to F-actin with one subunit, the
catalytic activity of the other three subunits can be altered
significantly to account for the observed increase in V
. Another possibility is that the active site
mutants caused conformational changes in the Arg-42 vicinity which
alter their apparent actin binding activity, while in fact the
catalytic site is not directly involved in actin binding. This is also
unlikely, however, since site-directed mutagenesis of many other
residues in or near the active site does not cause major changes in
aldolase structure(33, 45, 50) .