Enhancement by Mg2+ of domain specificity in Ca2+-dependent interactions of calmodulin with target sequences

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+-dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo-calmodulin. Apo-calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd approximately 70-90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C-domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+-saturated calmodulin-target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+-dependent target interactions.     

The complex structure of calmodulin bound to a calcineurin peptide

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25-residue peptide taken from the CaMBD, joined by a 5-glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25-residue CaMBD peptide without the glycine linker at a resolution of 2.1 A. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N-lobe from one molecule and C-lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully-designed CaM-sepharose pull-down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg-408, Val-409, and Phe-410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N- and C-lobes, ultimately keeping these lobes apart and forcing "head-to-tail" dimerization to attain the requisite N- and C-lobe pairing for CaMBD binding.     

Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding

Gap junctions are formed by a family of transmembrane proteins, connexins. Connexin43 is a widely studied member of the family, being ubiquitously expressed in a variety of tissues and a target of a large number of disease mutations. The intracellular loop of connexin43 has been shown to include a calmodulin binding domain, but detailed 3-dimensional data on the structure of the complex are not available. In this study, we used a synthetic peptide from this domain to reveal the conformation of the calmodulin-peptide complex by small angle X-ray scattering. Upon peptide binding, calmodulin lost its dumbbell shape, adopting a more globular conformation. We also studied the energetics of the interaction using calorimetry and computational methods. All our data indicate that calmodulin binds to the peptide from cx43 in the classical ‘collapsed’ conformation.     

Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences.

The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.     

Molecular Dynamics Simulations of a Calmodulin-peptide Complex in Solution

Abstract

We have performed an 4-ns MD simulation of calmodulin complexed with a target peptide in explicit water, under realistic conditions of constant temperature and pressure, in the presence of a physiological concentration of counterions and using Ewald summation to avoid truncation of long-range electrostatic forces. During the simulation the system tended to perform small fluctuations around a structure similar to, but somewhat looser than the starting crystal structure. The calmodulin-peptide complex was quite rigid and did not exhibit any large amplitude domain motions such as previously seen in apo- and calcium-bound calmodulin. We analyzed the calmodulin-peptide interactions by calculating buried surface areas, CHARMM interaction energies and continuum model interaction free energies. In the trajectory, the protein surface area buried by contact with the peptide is 1373 Ų, approximately evenly divided between the calmodulin N-terminal, C-terminal and central linker regions. A majority of this buried surface, 803 ·A², comes from nonpolar residues, in contrast to the protein as a whole, for which the surface is made up of mostly polar and charged groups. Our continuum calculations indicate that the largest favorable contribution to pep- tide binding comes from burial of molecular surface upon complex formation. Electrostatic contributions are favorable but smaller in the trajectory structures, and actually unfavorable for binding in the crystal structure. Since nonpolar groups make up most of buried surface of the protein, our calculations suggest that the hydrophobic effect is the main driving force for binding the helical peptide to calmodulin, consistent with thermodynamic analysis of experimental data. Besides the burial of nonpolar surface area, secondary contributions to peptide binding come from burial of polar surface and electrostatic interactions. In the nonpolar interactions a crucial role is played by the nine methionines of calmodulin. In the electrostatic interactions the negatively charged protein residues and positively charged peptide residues play a dominant role.     

Structure of the complex of calmodulin with the target sequence of calmodulin-dependent protein kinase I: studies of the kinase activation mechanism

Calcium-saturated calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to a region in the C-terminal regulatory sequence of the enzyme to relieve autoinhibition. The structure of CaM in a high-affinity complex with a 25-residue peptide of CaMKI (residues 294-318) has been determined by X-ray crystallography at 1.7 A resolution. Upon complex formation, the CaMKI peptide adopts an alpha-helical conformation, while changes in the CaM domain linker enable both its N- and C-domains to wrap around the peptide helix. Target peptide residues Trp-303 (interacting with the CaM C-domain) and Met-316 (with the CaM N-domain) define the mode of binding as 1-14. In addition, two basic patches on the peptide form complementary charge interactions with CaM. The CaM-peptide affinity is approximately 1 pM, compared with 30 nM for the CaM-kinase complex, indicating that activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactions between the CaM-binding sequence and the rest of the enzyme. We present biochemical and structural evidence indicating the involvement of both CaM domains in the activation process: while the C-domain exhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystal structure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 from the target peptide interact directly with CaM, demonstrating overlap between the autoinhibitory and CaM-binding sequences. Thus, the kinase activation mechanism involves the binding of CaM to residues associated with the inhibitory pseudosubstrate sequence.     

Free-energy simulations of the oxidation of c-terminal methionines in calmodulin

In the course of aging or under conditions of oxidative stress, methionine residues of calmodulin undergo oxidation, leading to loss of biological activity of the protein. We have performed free-energy simulations of the effects of C-terminal methionine side-chain oxidation on the properties of calmodulin. The simulation results indicate that oxidation should have a destabilizing effect on all three protein functional states: calcium free, calcium loaded, and with both calcium and target peptide bound. Because the different states are destabilized by different amounts, this leads to a more complex pattern in the observable effects on protein thermal stability, calcium affinity, and binding of a target peptide. The influence of oxidation on the free energy of CaM unfolding is estimated by comparing the free-energy cost of oxidizing a Met residue in a Gly-Met-Gly peptide and in the protein. The protein thermal stability of the oxidized forms is lowered by a moderate amount 1-3 kcal/mol, in qualitative agreement with experimental results of 0.3 kcal/mol. The calculated changes in affinity for calcium and for the target peptide show opposing trends. Oxidation at position 144 is predicted to enhance peptide binding and weaken calcium binding, whereas oxidation at 145 weakens peptide binding and enhances affinity for calcium. The lower affinity of Met 145-oxidized calmodulin toward the target peptide correlates with experimentally observed lowering of calmodulin-activated Ca-ATPase activity when oxidized calmodulin from aged rat brains is used. Thus, our simulations suggest that Met 145 is the oxidation site in the C-terminal fragment of calmodulin. The microscopic mechanism behind the calculated free energy changes appears to be a greater affinity for water of the oxidized Met side-chain relative to normal Met. Structures with Met exposed to solvent had consistently lower free energies than those with buried Met sidechains.     

Formation of cyclic imide-like structures upon the treatment of calmodulin and a calmodulin peptide with heat

Protein cyclic imide is the putative intermediate in the formation of sites of carboxyl-methylation in eukaryotic proteins. Conditions known to induce the formation of a cyclic imide in model peptides have been applied to a protein, calmodulin. Heating of calmodulin in the dry state at 100 degrees C for 24 h after lyophilization from a pH 2.0 or pH 6.0 solution produces derivatives with altered chromatographic properties in anion-exchange HPLC. At pH 6.0, complete activity of calmodulin was retained. Analysis with Fourier transform infrared (FTIR)-photoacoustic spectroscopy demonstrated the presence of a new structure in the calmodulin molecule consistent with modification of carboxylic acid groups. The conversion of calmodulin is dependent upon the absence of Ca2+ (the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid). A peptide analogous to the calcium binding regions of calmodulin, Asp-Lys-Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys-Glu, is also converted, upon heating, to chromatographically different forms in reversed-phase chromatography. This process is also dependent upon the absence of calcium. Sequence analysis of the peptide derivatives reveals a second amino terminus, implicating peptide bond hydrolysis in the product. A dipeptide, Asp-Gly, known to form a cyclic imide structure under similar conditions is also hydrolyzed during sequence analysis consistent with cleavage occurring at the position of the cyclic imide structure. Asp3 is suggested to be the site of cyclic imide formation in the calmodulin peptide. The presence of a cyclic imide structure is also confirmed by the application of FTIR-photoacoustic spectroscopy. These data suggest that cyclic imide formation in calmodulin has been induced, possibly at one, or more, of the calcium binding loops of the protein. These modification reactions may provide a basis for future investigations of cyclic imide formation in proteins.     

Structure and dynamics of calmodulin in solution.

To characterize the dynamic behavior of calmodulin in solution, we have carried out molecular dynamics (MD) simulations of the Ca2+-loaded structure. The crystal structure of calmodulin was placed in a solvent sphere of radius 44 A, and 6 Cl- and 22 Na+ ions were included to neutralize the system and to model a 150 mM salt concentration. The total number of atoms was 32,867. During the 3-ns simulation, the structure exhibits large conformational changes on the nanosecond time scale. The central alpha-helix, which has been shown to unwind locally upon binding of calmodulin to target proteins, bends and unwinds near residue Arg74. We interpret this result as a preparative step in the more extensive structural transition observed in the "flexible linker" region 74-82 of the central helix upon complex formation. The major structural change is a reorientation of the two Ca2+-binding domains with respect to each other and a rearrangement of alpha-helices in the N-terminus domain that makes the hydrophobic target peptide binding site more accessible. This structural rearrangement brings the domains to a more favorable position for target binding, poised to achieve the orientation observed in the complex of calmodulin with myosin light-chain kinase. Analysis of solvent structure reveals an inhomogeneity in the mobility of water in the vicinity of the protein, which is attributable to the hydrophobic effect exerted by calmodulin's binding sites for target peptides.     

Recombinant preparation and characterization of interactions for a calmodulin-binding chromogranin A peptide and calmodulin.

Chromogranin-derived peptides have important and varied biological activities. They affect a wide spectrum of targets such as fungal membranes, blood vessels, myocardial cells, and pancreatic cells. Despite the biological significance and the diverse activities, the molecular mechanisms of the interactions between the peptides and the target proteins have not been well understood. Here, we studied the interaction between a chromogranin A-derived peptide (CGA40-65) and its target protein, calmodulin, with NMR spectroscopy. Calmodulin was easily prepared with standard recombinant technology, but CGA40-65 posed challenges requiring multistep procedures. The recombinantly produced peptide retained the calmodulin-binding property of the full-length CGA, as shown by the HSQC binding experiment. By applying resonance assignments, we identified the residues in calmodulin involved in the CGA40-65 binding. We also found that the peak changes are close to those exhibited by the peptides having the wrap-around binding mechanism. Further analysis revealed that the CGA40-65-induced changes are more similar to those by CaMKIp peptide than those by smMLCKp peptide among the wrap-around binding peptides, suggesting that CGA40-65 can be categorized as a CaMKIp-like peptide. Our report is the first residue-resolution mechanistic study involving chromogranin peptides and their target proteins. Our approaches should be applicable to interaction studies involving other chromogranin-derived peptides and their cellular target proteins.     

Peptide and metal ion-dependent association of isolated helix-loop-helix calcium binding domains: studies of thrombic fragments of calmodulin

Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium-binding regulatory protein, has been cleaved by thrombin to create two fragments. TM1 (1-106) and TM2 (107-148). NMR and CD results indicate that TMI and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix-loop-helix domains, but rather within an alpha-helix. Cadmium-113 NMR results show that this complex has enhanced metal-ion binding properties when compared to either TM1 or TM2 alone. This complex can bind several CaM-binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl-13C-Met-labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex. Cadmium-113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine. However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM-dependent enzymes. This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage. The results presented here reflect the importance of the existence of helix-loop-helix Ca2+-binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.     

Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution

A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb^{3 +})₄ is effectively transmitted to the target protein (DHFR). ¹H-¹⁵N HSQC IPAP experiments on the tagged complex containing ¹⁵N-labelled DHFR-M13 protein and unlabelled CaM(Tb^{3 +})₄ allow one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to –7.4 Hz were measured at 600 MHz. Comparisons of ¹H-¹⁵N HSQC spectra of ¹⁵N-DHFR-M13 alone and its complexes with CaM(Ca^{2 +})₄ and CaM(Tb³⁺)₄ indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb³⁺) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).     

Increase in the molecular weight and radius of gyration of apocalmodulin induced by binding of target peptide: evidence for complex formation

Small-angle X-ray scattering was used to investigate a complex state of apocalmodulin induced by the binding of a Ca(2+)/calmodulin-dependent protein kinase IV calmodulin target site. Upon binding of the peptide, the molecular weight for apocalmodulin increased by 8.4%, which provides direct evidence for the formation of a calmodulin/target peptide complex. Comparison of the radius of gyration and Kratky plots of the apocalmodulin/peptide complex with those of apocalmodulin indicates that the overall conformation remains unchanged but the flexibility of the central linker decreases. An analysis of residue pairs between calmodulin and the target peptides suggests that the complex formation is induced by electrostatic interactions and subsequent van der Waals interactions.     

Modulating calmodulin binding specificity through computational protein design

We report the computational redesign of the protein-binding interface of calmodulin (CaM), a small, ubiquitous Ca(2+)-binding protein that is known to bind to and regulate a variety of functionally and structurally diverse proteins. The CaM binding interface was optimized to improve binding specificity towards one of its natural targets, smooth muscle myosin light chain kinase (smMLCK). The optimization was performed using optimization of rotamers by iterative techniques (ORBIT), a protein design program that utilizes a physically based force-field and the Dead-End Elimination theorem to compute sequences that are optimal for a given protein scaffold. Starting from the structure of the CaM-smMLCK complex, the program considered 10(22) amino acid residue sequences to obtain the lowest-energy CaM sequence. The resulting eightfold mutant, CaM_8, was constructed and tested for binding to a set of seven CaM target peptides. CaM_8 displayed high binding affinity to the smMLCK peptide (1.3nM), similar to that of the wild-type protein (1.8nM). The affinity of CaM_8 to six other target peptides was reduced, as intended, by 1.5-fold to 86-fold. Hence, CaM_8 exhibited increased binding specificity, preferring the smMLCK peptide to the other targets. Studies of this type may increase our understanding of the origins of binding specificity in protein-ligand complexes and may provide valuable information that can be used in the design of novel protein receptors and/or ligands.     

Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

Complex of calmodulin with a ryanodine receptor target reveals a novel, flexible binding mode

Calmodulin regulates ryanodine receptor-mediated Ca(2+) release through a conserved binding site. The crystal structure of Ca(2+)-calmodulin bound to this conserved site reveals that calmodulin recognizes two hydrophobic anchor residues at a novel "1-17" spacing that brings the calmodulin lobes close together but prevents them from contacting one another. NMR residual dipolar couplings demonstrate that the detailed structure of each lobe is preserved in solution but also show that the lobes experience domain motions within the complex. FRET measurements confirm the close approach of the lobes in binding the 1-17 target and show that calmodulin binds with one lobe to a peptide lacking the second anchor. We suggest that calmodulin regulates the Ca(2+) channel by switching between the contiguous binding mode seen in our crystal structure and a state where one lobe of calmodulin contacts the conserved binding site while the other interacts with a noncontiguous site on the channel.     

Natively unfolded C-terminal domain of caldesmon remains substantially unstructured after the effective binding to calmodulin

The structure of C-terminal domain (CaD136, C-terminal residues 636-771) of chicken gizzard caldesmon has been analyzed by a variety of physico-chemical methods. We are showing here that CaD136 does not have globular structure, has low secondary structure content, is essentially noncompact, as it follows from high R(g) and R(S) values, and is characterized by the absence of distinct heat absorption peaks, i.e. it belongs to the family of natively unfolded (or intrinsically unstructured) proteins. Surprisingly, effective binding of single calmodulin molecule (K(d) = 1.4 +/- 0.2 microM) leads only to a very moderate folding of this protein and CaD136 remains substantially unfolded within its tight complex with calmodulin. The biological significance of these observations is discussed.     

Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin.

The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk-MLCK) target sequence has been studied using 1H-NMR. The 18-residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C-domain and the Phe 17 side chain with the N-domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H-NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C-domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C-domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H-NMR spectra of CaM bound to WFF, a Trp 4-->Phe analogue (FFF), or a Trp 4-->Phe/Phe 17-->Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C-domain and the side chain in position 17 interacting with the N-domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34:2087-2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N-domain of CaM.