The solution structures of calmodulin and its complexes with synthetic peptides based on target enzyme binding domains

doi:10.1016/0143-4160(92)90051-S

Cell Calcium

Volume 13, Issues 6–7, June–July 1992, Pages 377-390

https://doi.org/10.1016/0143-4160(92)90051-S Get rights and content

Abstract

Small-angle X-ray and neutron scattering experiments have given important information on the solution structures of calmodulin and its complexes with synthetic peptides used to model target enzyme interactions. In combination with crystallographic data, site directed mutagenesis and various spectroscopic studies, these experiments have contributed to our understanding of the solution structure of calmodulin in different functional states. We have gained important insights into the conformational flexibility in calmodulin that appears to be crucial to its regulatory functions. Specifically, flexibility in the interconnecting helix region of calmodulin has been shown to play a critical role in facilitating calmodulin's binding to a wide variety of target enzymes whose activities are thus regulated. This review will focus mainly on the contributions small-angle scattering has made to our understanding of the solution structure of calmodulin in the context of other studies, with particular regard to circular dichroism and Fourier transform infrared studies that complement the small-angle scattering data.

References (78)

T Tanaka et al.
Hydrophobic regions function in calmodulin-enzyme(s) interactions
J. Biol. Chem.
(1980)
K Ibel et al.
Comparison of neutron and X-ray scattering of dilute myoglobin solutions
J. Mol. Biol.
(1975)
SR Hubbard et al.
Small-angle scattering investigation of the solution structure of troponin C
J. Biol. Chem.
(1988)
C-LA Wang et al.
pH-dependent structural transition in rabbit skeletal troponin C
J. Biol. Chem.
(1987)
A Persechini et al.
The central helix of calmodulin functions as a flexible tether
J. Biol. Chem.
(1988)
A Persechini et al.
The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding
J. Biol. Chem.
(1989)
MFA VanBerkum et al.
Calmodulin activation of target enzymes: consequences of deletions in the central helix
J. Biol. Chem.
(1990)
Z Sheng et al.
Effects of mutations in the central helix of troponin C on its biological activity
J. Biol. Chem.
(1991)
PM Bayley et al.
The conformation of calmodulin: a substantial environmentally sensitive helical transition in Ca₄-calmodulin with potential mechanistic function
FEBS Lett.
(1988)
CB Klee et al.
Calmodulin. Adv. Prot. Chem.
(1982)

O Herzberg et al.

A model for the Ca²⁺-induced conformational transition of troponin C

J. Biol. Chem.

(1986)

H Susi et al.

Protein structure by Fourier transform infrared spectroscopy: second derivative spectra

Biochem. Biophys. Res. Commun.

(1983)

YS Babu et al.

Structure of calmodulin refined at 2.2 Å resolution

J. Mol. Biol.

(1988)

KA Satyshur et al.

Refined structure of chicken skeletal muscle troponin C in the two-calcium state at 2-Å resolution

J. Biol. Chem.

(1988)

PJ Kennelly et al.

Rabbit skeletal muscle myosin light chain kinase: the calmodulin binding domain as a potential active site-directed inhibitory domain

J. Biol. Chem.

(1987)

KT O'Neil et al.

How calmodulin binds its targets: sequence independent recognition of amphiphilic α-helices

Trends Biochem. Sci.

(1990)

RE Klevit et al.

¹H NMR studies of calmodulin-peptide interactions

L Garone et al.

The interaction of calmodulin with the C-terminal M5 peptide of myosin light chain kinase

Arch. Biochem. Biophys.

(1990)

JA Putkey et al.

Genetically engineered calmodulins differentially activate target enzymes

J. Biol. Chem.

(1986)

H Yoshino et al.

Calcium-induced shape change of calmodulin with mastoparan studied by solution X-ray scattering

J. Biol. Chem.

(1989)

P Cohen

The role of cyclic-AMP-dependent protein kinase in the regulation of glycogen metabolism in mammalian skeletal muscle

Curr. Top. Cell. Regul.

(1978)

MR Trempe et al.

Analyses of phosphorylase kinase by transmission and scanning transmission electron microscopy

J. Biol. Chem.

(1986)

RD Edstrom et al.

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunnelling and atomic force microscopy

Biophys. J.

(1990)

JA Talbot et al.

Synthetic studies on the inhibitory region of rabbit skeletal troponin I

J. Biol. Chem.

(1981)

DC Dalgarno et al.

Interaction between troponin I and troponin C

FEBS Lett.

(1982)

RH Kretsinger

Hypothesis: calcium modulated proteins contain EF hands

WY Cheung

Calmodulin plays a pivotal role in cellular regulation

Science

(1980)

DC La Porte et al.

Calcium-induced exposure of a hydrophobic surface on calmodulin

Biochemistry

(1980)

I Pilz

Proteins

LA Feigen et al.

DB Heidorn et al.

Comparison of the crystal and solution structures of calmodulin and troponin C

Biochemistry

(1988)

DB Heidorn et al.

Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase

Biochemistry

(1989)

J Trewhella et al.

Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase

Biochemistry

(1990)

DB Heidorn et al.

Low-resolution structural studies of proteins in solution: calmodulin

Comm. Mol. Cell. Biophys.

(1990)

O Glatter

Interpretation

BA Seaton et al.

Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle scattering

Biochemistry

(1985)

T Fujisawa et al.

X-ray scattering from a troponin C solution and its interpretation with a dumbbell-shaped molecule model

J. Appl. Crystallogr.

(1987)

T Fujisawa et al.

Structural change of the troponin C molecule upon Ca²⁺ binding measured in solution by the X-ray scattering technique

J. Biochem.

(1989)

C-LA Wang

pH-dependent conformational changes of wheat germ calmodulin

Biochemistry

(1989)

Cited by (37)

Interplay between conformational selection and induced fit in multidomain protein-ligand binding probed by paramagnetic relaxation enhancement
2014, Biophysical Chemistry
Citation Excerpt :
With one exception [33], all crystal structures of free CaM–4Ca2 + appear as an extended dumbbell structure with a contiguous helix connecting the two domains separated by a center-to-center distance of ~ 40 Å [34–36] (Fig. 5, top). However, fluorescence [37], and small-angle X-ray and neutron scattering in solution [38] are indicative of an average structure that is somewhat more compact than the extended dumbbell structure. NMR relaxation studies have shown that the central residues (77–81) of the linker are highly mobile (with S2 order parameters of 0.5–0.6) [30] and that the two domains reorient almost independently of one another on a time scale of ~ 3 ns undergoing restricted diffusion within a cone of semi-angle ~ 30° [39].
The binding of ligands and substrates to proteins has been extensively studied for many years and can be described, in its simplest form, by two limiting mechanisms: conformational selection and induced fit. Conformational selection involves the binding of ligand to a pre-existing sparsely-populated conformation of the free protein that is the same as that in the final protein–ligand complex. In the case of induced fit, the ligand binds to the major conformation of the free protein and only subsequent to binding undergoes a conformational change to the final protein–ligand complex. While these two mechanisms can be dissected and distinguished by transient kinetic measurements, direct direction, characterization and visualization of transient, sparsely-populated states of proteins are experimentally challenging. Unless trapped, sparsely-populated states are generally invisible to conventional structural and biophysical techniques, including crystallography and most NMR measurements. In this review we summarize some recent developments in the use of paramagnetic relaxation enhancement to directly study sparsely-populated states of proteins and illustrate the application of this approach to two proteins, maltose binding protein and calmodulin, both of which undergo large rigid body conformational rearrangements upon ligand binding from an open apo state to a closed ligand-bound holo state. We show that the apo state ensemble comprises a small population of partially-closed configurations that are similar but not identical to that of the holo state. These results highlight the complementarity and interplay of induced fit and conformational selection and suggest that the existence of partially-closed states in the absence of ligand facilitates the transition to the closed ligand-bound state.
Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker
2011, Protein Expression and Purification
Citation Excerpt :
In the presence of calcium, the small (148 residue), monomeric alpha-helical protein CaM undergoes large conformational changes throughout the protein to recognize and bind to its numerous cellular target proteins [12–20]. Most of these structural changes occur in the protein’s unique central linker region, which serves as a molecular hinge between the lobed N- and C-terminal dual EF-hand calcium binding domains (Fig. 1A) [13,21–25]. For example, the crystal structure of CaM complexed with a peptide of the CaM binding site of smooth muscle myosin light chain kinase (smMLCK, Fig. 1B) shows that the two lobed domains are brought into close proximity to simultaneously interact with the target by bending the central hinge region to form a canonical binding motif [26].
Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.
Protein-protein docking and analysis reveal that two homologous bacterial adenylyl cyclase toxins interact with calmodulin differently
2008, Journal of Biological Chemistry
Calmodulin (CaM), a eukaryotic calcium sensor that regulates diverse biological activities, consists of N- and C-terminal globular domains (N-CaM and C-CaM, respectively). CaM serves as the activator of CyaA, a 188-kDa adenylyl cyclase toxin secreted by Bordetella pertussis, which is the etiologic agent for whooping cough. Upon insertion of the N-terminal adenylyl cyclase domain (ACD) of CyaA to its targeted eukaryotic cells, CaM binds to this domain tightly (∼200 pm affinity). This interaction activates the adenylyl cyclase activity of CyaA, leading to a rise in intracellular cAMP levels to disrupt normal cellular signaling. We recently solved the structure of CyaA-ACD in complex with C-CaM to elucidate the mechanism of catalytic activation. However, the structure of the interface between N-CaM and CyaA, the formation of which contributes a 400-fold increase of binding affinity between CyaA and CaM, remains elusive. Here, we used site-directed mutations and molecular dynamic simulations to generate several working models of CaM-bound CyaA-ACD. The validity of these models was evaluated by disulfide bond cross-linking, point mutations, and fluorescence resonance energy transfer experiments. Our study reveals that a β-hairpin region (amino acids 259–273) of CyaA-ACD likely makes contacts with the second calcium binding motif of the extended CaM. This mode of interaction differs from the interaction of N-CaM with anthrax edema factor, which binds N-CaM via its helical domain. Thus, two structurally conserved, bacterial adenylyl cyclase toxins have evolved to utilize distinct binding surfaces and modes of activation in their interaction with CaM, a highly conserved eukaryotic signaling protein.
Structural aspects of calcium-binding proteins and their interactions with targets
2007, New Comprehensive Biochemistry
Citation Excerpt :
When the structure of Ca2+-loaded CaM complexed with a peptide from myosin light chain kinase was solved, the capability of CaM to undergo a large conformational change for target recognition was appreciated [38,39]. This structure revealed that a portion of the central helix region of CaM is highly flexible (residues 78–81); furthermore, this region can be dramatically bent upon binding to a target protein (Fig. 1B) [23,27,40,41]. The flexibility of the central helix domain not only permits a more fitted interaction within domain linker region of CaM, but also allows the two domains of CaM to change independently in order to accommodate the three-dimensional characteristics of the peptide [6].
Calcium (Ca²⁺) sensor proteins are critical and ubiquitous mediators of Ca²⁺ messages in eukaryotic cells. These proteins “sense” local changes in intracellular Ca²⁺ concentrations by undergoing a conformational response to binding or release of Ca²⁺ ions. The structural changes intercede interactions with a specific target protein, ultimately regulating its function. Despite the sequence homology between Ca²⁺-binding motifs (EF-hands), Ca²⁺ sensors show a remarkable diversity in regulatory mechanism and biological function. This chapter reviews the structural aspects of Ca²⁺ sensor proteins and the various mechanisms of target-binding specificity and regulation. A structural survey of calmodulin, neuronal Ca²⁺ sensor proteins, S100 proteins, penta-EF-hand, and several “other” Ca²⁺ sensors [stromal interaction molecule (STIM), Eps15 homology (EH)-domain-containing, nucleobindin, BM40, and calcium-binding protein 40 (CBP40)] reveal some common themes and important disparities in the molecular mechanisms of target recognition. Diversity of target recognition for each Ca²⁺ sensor is considered in terms of two distinct evolutionary mechanisms.
Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments
2004, Analytical Biochemistry
We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP · CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP · CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP · CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP · CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP · CaM-DA molecules. The extension of this method to other biomolecules is also proposed.
A closed compact structure of native Ca<sup>2+</sup>-calmodulin
2003, Structure
Calmodulin has been a subject of intense scrutiny since its discovery because of its unusual properties in regulating the functions of about 100 diverse target enzymes and structural proteins. The original and to date only crystal conformation of native eukaryotic Ca²⁺-calmodulin (Ca²⁺-CaM) is a very extended molecule with two widely separated globular domains linked by an exposed long helix. Here we report the 1.7 Å X-ray structure of a new native Ca²⁺-CaM that is in a compact ellipsoidal conformation and shows a sharp bend in the linker helix and a more contracted N-terminal domain. This conformation may offer advantages for recognition of kinase-type calmodulin targets or small organic molecule drugs.

View all citing articles on Scopus

View full text

The solution structures of calmodulin and its complexes with synthetic peptides based on target enzyme binding domains

Abstract

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Calmodulin. Adv. Prot. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Trends Biochem. Sci.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Curr. Top. Cell. Regul.

J. Biol. Chem.

Biophys. J.

J. Biol. Chem.

FEBS Lett.

Hypothesis: calcium modulated proteins contain EF hands

Calmodulin plays a pivotal role in cellular regulation

Science

Calcium-induced exposure of a hydrophobic surface on calmodulin

Biochemistry

Proteins

Comparison of the crystal and solution structures of calmodulin and troponin C

Biochemistry

Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase

Biochemistry

Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase

Biochemistry

Low-resolution structural studies of proteins in solution: calmodulin

Comm. Mol. Cell. Biophys.

Interpretation

Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle scattering

Biochemistry

X-ray scattering from a troponin C solution and its interpretation with a dumbbell-shaped molecule model

J. Appl. Crystallogr.

Structural change of the troponin C molecule upon Ca2+ binding measured in solution by the X-ray scattering technique

J. Biochem.

pH-dependent conformational changes of wheat germ calmodulin

Biochemistry

Structural change of the troponin C molecule upon Ca²⁺ binding measured in solution by the X-ray scattering technique