(Received for publication, October 4, 1995; and in revised form, January 18, 1996)
From the
Calcium-dependent regulation of intracellular processes is
mediated by proteins that on binding Ca assume a new
conformation, which enables them to bind to their specific target
proteins and to modulate their function. Calmodulin (CaM) and troponin
C, the two best characterized Ca
-regulatory proteins,
are members of the family of Ca
-binding proteins
utilizing the helix-loop-helix structural motif (EF-hand). Herzberg,
Moult, and James (Herzberg, O., Moult, J., and James, M. N. G.(1986) J. Biol. Chem. 261, 2638-2644) proposed that the
Ca
-induced conformational transition in troponin C
involves opening of the interface between the
-helical segments in
the N-terminal domain of this protein. Here we have tested the
hypothesis that a similar transition is the key
Ca
-induced regulatory event in calmodulin. Using
site-directed mutagenesis we have substituted cysteine residues for
Gln
and Lys
(CaM41/75) or Ile
and Leu
(CaM85/112) in the N-terminal and C-terminal
domains, respectively, of human liver calmodulin. Based on molecular
modeling, cysteines at these positions were expected to form
intramolecular disulfide bonds in the Ca
-free
conformation of the protein, thus blocking the putative
Ca
-induced transition. We found that intramolecular
disulfide bonds are readily formed in both mutants causing a decrease
in affinity for Ca
and the loss of ability to
activate target enzymes, phosphodiesterase and calcineurin. The
regulatory activity is fully recovered in CaM41/75 and partially
recovered in CaM85/112 upon reduction of the disulfide bonds with
dithiotreitol and blocking the Cys residues by carboxyamidomethylation
or cyanylation. These results indicate that the
Ca
-induced opening of the interfaces between helical
segments in both domains of CaM is critical for its regulatory
properties consistent with the Herzberg-Moult-James model.
Troponin C (TnC) ()and calmodulin (CaM) are the two
best characterized Ca
-binding regulatory proteins and
members of the EF-hand family of Ca
-binding proteins.
The tissue distribution and the function of these proteins are
different; TnC is the striated muscle-specific protein, whereas CaM is
an ubiquitous protein involved in regulation of a number of
intracellular enzymatic systems. In spite of different functions the
two proteins have remarkably similar
structures(1, 2, 3) . There are eight helical
segments (A-H) associated with the four helix-loop-helix
Ca
-binding sites equally distributed between two
globular domains. The most significant difference between
crystallographic structures of the two proteins is the lack of
Ca
in sites I and II in TnC that results in a more
compact packing of the N-terminal domain of this protein. The helices
flanking the Ca
-binding loops in this domain make
tight contacts through the side chains and run in an antiparallel
fashion. In contrast, in the two Ca
-binding sites in
the C-terminal domain of TnC and in all four sites in CaM the
interhelical dihedral angle of a helix-loop-helix unit is close to 90
°. This difference forms the basis for the mechanism of the
Ca
-induced conformational transition proposed by
Herzberg et al.(4) . They proposed that upon
Ca
binding to site I and II in TnC, the B/C pair of
helices moves away from the A/D pair thereby exposing a patch of
hydrophobic residues, the interaction site for TnI. This model,
referred to as the Herzberg-Moult-James (HMJ) model, has a strong
experimental support based on several lines of evidence (for review see
Grabarek et al.(5) ). The atomic resolution structure
of the Ca
-filled N-terminal domain fragment of TnC
recently solved by NMR (6) and x-ray crystallography (7) have proved the HMJ model to be correct.
Here we address
the question of whether the HMJ model is unique for the N-terminal
domain of TnC or whether it is also applicable to calmodulin. We have
designed two mutants of human liver CaM in which the putative
Ca-induced opening of the interhelical interfaces in
each of the two domains could be blocked by a disulfide bond. Cysteine
residues have been introduced at position 41 and 75 in the N-terminal
domain (CaM41/75) and at position 85 and 112 in the C-terminal domain
(CaM85/112). We found that the cysteine residues in both mutants form
stable disulfide bonds. The oxidized forms of these mutants are unable
to activate phosphodiesterase and calcineurin. Upon reduction of the
disulfide and blocking SH groups, the regulatory properties are fully
restored in CaM41/75 and to a large extent in CaM85/112. These data
strongly support the view that a TnC-like opening of the interhelical
interface in each of the two domains of CaM represents the key
Ca
-induced conformational transition in CaM.
It was
less clear what residues should be mutated in the C-terminal domain
because the Ca-free conformation of neither of the
two proteins was known. We assumed that the Ca
-free
conformation of the C-terminal domain of CaM is similar to that of the
N-terminal domain of TnC, in which case the F/G linker would undergo
the largest displacement upon Ca
-binding, and
disulfide cross-linking of this segment to either helix H or helix E
should be effective in blocking the transition. We have chosen the
latter alternative (Fig. 1) and substituted Cys for Ile
in helix E and for Leu
, which is located at the
beginning of the F/G linker (CaM85/112), the selection being based on
the observation that the side chains of the residues at the homologous
positions in the Ca
-free N-terminal domain of TnC are
in contact.
Figure 1: Schematic representation of the mutant calmodulins showing positions of the disulfide cross-links. Note that although each of the disulfide bonds appear to span a single EF-hand, most likely an entire two-site domain is blocked owing to strong interhelical interactions in the three-dimensional structure (helices A/D, B/C, E/H, and F/G).
Figure 2: Effect of DTT on the electrophoretic mobility of CaM mutants. Electrophoresis on 10% polyacrylamide gel in the presence of 80 mM Tris-Gly buffer, pH 8.6, and 1 mM EDTA. The gel for CaM41/75 contains also 4.5 M urea. Prior to electrophoresis the protein samples (2-3 mg/ml) were incubated at 25 °C with 10 mM DTT in a solution containing 0.1 M NaCl and 50 mM Hepes, pH 7.5. The incubation was terminated by addition of excess of iodoacetamide. SH and S-S indicate the reduced and disulfide cross-linked forms of the CaM mutants, respectively.
Figure 3:
Electrophoretic mobility of CaM mutants.
Electrophoresis on 10% polyacrylamide gel in the presence of 80 mM Tris-Gly buffer, pH 8.6, with or without 4.5 M urea and 1
mM CaCl or 1 mM EDTA as indicated. Lanes a, wild type human liver CaM; lanes b,
ox-CaM41/75; lanes c, carboxyamidomethylated CaM41/75; lanes d, ox-CaM85/112; lanes e,
carboxyamidomethylated CaM85/112; lanes f, cyanylated
CaM85/112. Each lane contains 4 µg of
protein
Figure 4:
Calcium binding properties of CaM85/112.
Ca binding was monitored by tyrosine fluorescence
(
= 280 nm,
= 308
nm). Owing to the location of Tyr residues only sites III and IV are
``visible.''
, wild type human liver calmodulin;
, disulfide cross-linked CaM85/112;
,
carboxyamidomethylated CaM85/112. Each data point is an average of two
titrations. The solid lines represent the best fit with the
Hill equation. Parameters of the fit are given below. For CaM, the K
value is 5.0
10
and
the Hill coefficient is 2.0. For disulfide cross-linked CaM85/112, the K
value is 8.9
10
and
the Hill coefficient is 1.05. For carboxyamidomethylated CaM85/112, the K
value is 1.4
10
and
the Hill coefficient is 1.36.
The effect of the disulfide bond on the
Ca affinity of CaM85/112 is consistent with the HMJ
model and can be explained on the basis of the coupling between
Ca
binding and the opening of the interhelical
interface in a two-EF-hand domain. Such coupling requires that the most
favorable energetically interaction between Ca
and
its ligands can be achieved only in the open conformation in which the
two
-helices flanking the loop are perpendicular to each other. In
CaM85/112 the disulfide bond precludes such a conformation, which
results in less then optimal interaction with Ca
and
a lower apparent binding constant. In the carboxyamidomethylated
CaM85/112 there is no restriction of the helical movement. In addition
there is a decrease in hydrophobic interactions between the helices in
the Ca
-free (closed) conformation due to the absence
of Ile
and Leu
. Thus the transition to the
open conformation requires less energy, resulting in an increased
affinity for Ca
.
Figure 5:
Effect of disulfide cross-linking on the
ability of mutant CaMs to activate phosphodiesterase (A) and
calcineurin (B). , wild type human liver calmodulin;
, disulfide cross-linked CaM41/75;
, disulfide
cross-linked CaM85/112;
, carboxyamidomethylated CaM41/75;
, carboxyamidomethylated CaM85/112;
, cyanylated
CaM85/112.
The loss of
activity upon intradomain disulfide cross-linking of mutant CaM
results, apparently, from a dramatic decrease in affinity for the
target proteins because these mutants do not compete with the wild type
CaM (data not shown). Although each of the two domains in CaM
contributes one binding site for the target enzyme, no interaction
occurs under experimental conditions when one of the domains is
inaccessible. Such interpretation is consistent with the observation
that proteolytic fragments of CaM comprising either half of the
molecule are incapable of activating target enzymes(16) . Our
results indicate that the Ca-induced opening of the
structure in both domains of CaM is essential for the activation of
targets by CaM.
Recently the three-dimensional structures of the complexes of calmodulin with synthetic peptides corresponding to the CaM-binding sites of myosin light chain kinase from skeletal (19) and smooth muscle (20) as well as CaM-dependent kinase II (21) have been reported. Unlike the free CaM, the complex has a compact globular structure. The two domains of CaM (residues 6-73 and 83-146) remain essentially unchanged; however, the long central helix is disrupted into two helices connected by a long flexible loop, thereby enabling the two domains to clamp the bound peptide that adopts a helical conformation. In this respect the structure is similar to the model proposed earlier by Persechini and Kretsinger(22) . The peptide is located in a hydrophobic channel formed by the hydrophobic faces of the two globular domains of CaM (for review see (23) ). It is clear from these structures that the disulfide bonds in CaM41/75 and CaM85/112 would preclude the formation of such complexes consistent with our data.
After this
work was completed two independent reports were published on the
three-dimensional structure of Ca-free calmodulin
determined by multidimensional heteronuclear
NMR(24, 25) . Also the Ca
-bound and
Ca
-free structures of the C-terminal domain fragment
of calmodulin (26) and the N-terminal domain of troponin C (27) were determined by NMR. These studies show that both
domains of CaM and the N-terminal domain of TnC undergo a
Ca
-induced transition consistent with the HMJ model.
The distances between C
atoms of Gln
and
Lys
and between C
of Ile
and
Leu
calculated from the averaged atomic coordinates of
the Ca
-free CaM (25) are 7.8 and 6.9 Å,
respectively. This is slightly longer than the 3.4-4.2 Å
range predicted to be optimal for a disulfide bond
formation(28) . However, the segments of the polypeptide chain
at all four mutation sites show significant
flexibility(24, 25, 26) ; thus it is unlikely
that any significant long range structural alterations would be
necessary for the disulfide bonds to form in CaM41/75 and CaM85/112.
The corresponding distances in the Ca
-bound
conformation are 17.1 and 16.4 Å in the N- and C-terminal
domains, respectively, which clearly would be impossible in the
presence of the disulfide bonds.
Our present data and the recent NMR
studies indicate that the transition between the closed and open
conformation in each of the two domains of CaM consistent with the HMJ
model (4) represents the key Ca-induced
regulatory transition in this protein. Most likely it is a general
mechanism of Ca
-regulation in proteins of the EF-hand
family. This applies only to the so called ``regulatory''
proteins such as CaM and TnC, but presumably not to those referred to
as Ca
buffers. In the latter case exemplified by
calbindin D
there is no Ca
-induced
increase in hydrophobicity and no change in interhelical
angles(29) . At this point it is not clear what structural
features are responsible for the tight coupling between Ca
binding and the conformational transitions in Ca
regulators and the lack of it in Ca
buffers.
A preliminary account of this work has been published (Grabarek, Z., Tan, R.-Y. and Gergely, J.(1991) Biophys. J.59, 23a).