Crystal structure of the Ig1 domain of the neural cell adhesion molecule NCAM2 displays domain swapping

The crystal structure of the first immunoglobulin (Ig1) domain of neural cell adhesion molecule 2 (NCAM2/OCAM/RNCAM) is presented at a resolution of 2.7 Å. NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). In the structure, two Ig domains interact by domain swapping, as the two N-terminal β-strands are interchanged. β-Strand swapping at the terminal domain is the accepted mechanism of homophilic interactions amongst the cadherins, another class of CAMs, but it has not been observed within the IgCAM superfamily. Gel-filtration chromatography demonstrated the ability of NCAM2 Ig1 to form dimers in solution. Taken together, these observations suggest that β-strand swapping could have a role in the molecular mechanism of homophilic binding for NCAM2.     

Homophilic adhesion between Ig superfamily carcinoembryonic antigen molecules involves double reciprocal bonds

Both carcinoembryonic antigen (CEA) and neural cell adhesion molecule (NCAM) belong to the immunoglobulin supergene family and have been demonstrated to function as homotypic Ca(++)-independent intercellular adhesion molecules. CEA and NCAM cannot associate heterotypically indicating that they have different binding specificities. To define the domains of CEA involved in homotypic interaction, hybrid cDNAs consisting of various domains from CEA and NCAM were constructed and were transfected into a CHO-derived cell line; stable transfectant clones showing cell surface expression of CEA/NCAM chimeric-proteins were assessed for their adhesive properties by homotypic and heterotypic aggregation assays. The results indicate that all five of the Ig(C)-like domains of NCAM are required for intercellular adhesion while the COOH-terminal domain containing the fibronectin-like repeats is dispensable. The results also show that adhesion mediated by CEA involves binding between the Ig(V)-like amino-terminal domain and one of the Ig(C)-like internal repeat domains: thus while transfectants expressing constructs containing either the N domain or the internal domains alone were incapable of homotypic adhesion, they formed heterotypic aggregates when mixed. Furthermore, peptides consisting of both the N domain and the third internal repeat domain of CEA blocked CEA-mediated cell aggregation, thus providing direct evidence for the involvement of the two domains in adhesion. We therefore propose a novel model for interactions between immunoglobulin supergene family members in which especially strong binding is effected by double reciprocal interactions between the V-like domains and C-like domains of antiparallel CEA molecules on apposing cell surfaces.     

Single-molecule force spectroscopy of the aplysia cell adhesion molecule reveals two homophilic bonds

Aplysia californica neurons comprise a powerful model system for quantitative analysis of cellular and biophysical properties that are essential for neuronal development and function. The Aplysia cell adhesion molecule (apCAM), a member of the immunoglobulin superfamily of cell adhesion molecules, is present in the growth cone plasma membrane and involved in neurite growth, synapse formation, and synaptic plasticity. apCAM has been considered to be the Aplysia homolog of the vertebrate neural cell adhesion molecule (NCAM); however, whether apCAM exhibits similar binding properties and neuronal functions has not been fully established because of the lack of detailed binding data for the extracellular portion of apCAM. In this work, we used the atomic force microscope to perform single-molecule force spectroscopy of the extracellular region of apCAM and show for the first time (to our knowledge) that apCAM, like NCAM, is indeed a homophilic cell adhesion molecule. Furthermore, like NCAM, apCAM exhibits two distinct bonds in the trans configuration, although the kinetic and structural parameters of the apCAM bonds are quite different from those of NCAM. In summary, these single-molecule analyses further indicate that apCAM and NCAM are species homologs likely performing similar functions.     

The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II

A limited number of mammalian proteins are modified by polysialic acid, with the neural cell adhesion molecule (NCAM) being the most abundant of these. We hypothesize that polysialylation is a protein-specific glycosylation event and that an initial protein-protein interaction between polysialyltransferases and glycoprotein substrates mediates this specificity. To evaluate the regions of NCAM required for recognition and polysialylation by PST/ST8Sia IV and STX/ST8Sia II, a series of domain deletion proteins were generated, co-expressed with each enzyme, and their polysialylation analyzed. A protein consisting of the fifth immunoglobulin-like domain (Ig5), which contains the reported sites of polysialylation, and the first fibronectin type III repeat (FN1) was polysialylated by both enzymes, whereas a protein consisting of Ig5 alone was not polysialylated by either enzyme. This demonstrates that the Ig5 domain of NCAM and FN1 are sufficient for polysialylation, and suggests that the FN1 may constitute an enzyme recognition and docking site. Two other NCAM mutants, NCAM-6 (Ig1-5) and NCAM-7 (FN1-FN2), were weakly polysialylated by PST/ST8Sia IV, suggesting that a weaker enzyme recognition site may exist within the Ig domains, and that glycans in the FN region are polysialylated. Further analysis indicated that O-linked oligosaccharides in NCAM-7, and O-linked and N-linked glycans in full-length NCAM, are polysialylated when these proteins are co-expressed with the polysialyltransferases in COS-1 cells. Our data support a model in which the polysialyltransferases bind to the FN1 of NCAM to polymerize polysialic acid chains on appropriately presented glycans in adjacent regions.     

Insights into GFRalpha1 regulation of neural cell adhesion molecule (NCAM) function from structure-function analysis of the NCAM/GFRalpha1 receptor complex

The neural cell adhesion molecule NCAM binds glial cell line-derived neurotrophic factor (GDNF) through specific determinants located in its third immunoglobulin (Ig) domain. However, high affinity GDNF binding and downstream signaling depend upon NCAM co-expression with the GDNF co-receptor GFRalpha1. GFRalpha1 promotes high affinity GDNF binding to NCAM and down-regulates NCAM-mediated homophilic cell adhesion, but the mechanisms underlying these effects are unknown. NCAM and GFRalpha1 interact at the plasma membrane, but the molecular determinants involved have not been characterized nor is it clear whether their interaction is required for GFRalpha1 regulation of NCAM function. We have investigated the structure-function relationships underlying GFRalpha1 binding to NCAM in intact cells. The fourth Ig domain of NCAM was both necessary and sufficient for the interaction of NCAM with GFRalpha1. Moreover, although the N-terminal domain of GFRalpha1 had previously been shown to be dispensable for GDNF binding, we found that it was both necessary and sufficient for the efficient interaction of this receptor with NCAM. GFRalpha1 lacking its N-terminal domain was still able to potentiate GDNF binding to NCAM and assemble into a tripartite receptor complex but showed a reduced capacity to attenuate NCAM-mediated cell adhesion. On its own, the GFRalpha1 N-terminal domain was sufficient to decrease NCAM-mediated cell adhesion. These results indicate that direct receptor-receptor interactions are not required for high affinity GDNF binding to NCAM but play an important role in the regulation of NCAM-mediated cell adhesion by GFRalpha1.     

Magnetic Tweezers-Based Force Clamp Reveals Mechanically Distinct apCAM Domain Interactions

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) play a crucial role in cell-cell interactions during nervous system development and function. The Aplysia CAM (apCAM), an invertebrate IgCAM, shares structural and functional similarities with vertebrate NCAM and therefore has been considered as the Aplysia homolog of NCAM. Despite these similarities, the binding properties of apCAM have not been investigated thus far. Using magnetic tweezers, we applied physiologically relevant, constant forces to apCAM-coated magnetic particles interacting with apCAM-coated model surfaces and characterized the kinetics of bond rupture. The average bond lifetime decreased with increasing external force, as predicted by theoretical considerations. Mathematical simulations suggest that the apCAM homophilic interaction is mediated by two distinct bonds, one involving all five immunoglobulin (Ig)-like domains in an antiparallel alignment and the other involving only two Ig domains. In summary, this study provides biophysical evidence that apCAM undergoes homophilic interactions, and that magnetic tweezers-based, force-clamp measurements provide a rapid and reliable method for characterizing relatively weak CAM interactions.     

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.     

Structural requirements for neural cell adhesion molecule-heparin interaction.

Two biological domains have been identified in the amino terminal region of the neural cell adhesion molecule (NCAM): a homophilic-binding domain, responsible for NCAM-NCAM interactions, and a heparin-binding domain (HBD). It is not known whether these two domains exist as distinct structural entities in the NCAM molecule. To approach this question, we have further defined the relationship between NCAM-heparin binding and cell adhesion. A putative HBD consisting of two clusters of basic amino acid residues located close to each other in the linear amino acid sequence of NCAM has previously been identified. Synthetic peptides corresponding to this domain were shown to bind both heparin and retinal cells. Here we report the construction of NCAM cDNAs with targeted mutations in the HBD. Mouse fibroblast cells transfected with the mutant cDNAs express NCAM polypeptides with altered HBD (NCAM-102 and NCAM-104) or deleted HBD (HBD-) at levels similar to those of wild-type NCAM. Mutant NCAM polypeptides purified from transfected cell lines have substantially reduced binding to heparin and fail to promote chick retinal cell attachment. Furthermore, whereas a synthetic peptide that contains both basic amino acid clusters inhibits retinal-cell adhesion to NCAM-coated dishes, synthetic peptides in which either one of the two basic regions is altered to contain only neutral amino acids do not inhibit this adhesion. These results confirm that this region of the NCAM polypeptide does indeed mediate not only the large majority of NCAM's affinity for heparin but also a significant portion of the cell-adhesion-mediating capability of NCAM.     

Regulation of cell adhesion by polysialic acid. Effects on cadherin, immunoglobulin cell adhesion molecule, and integrin function and independence from neural cell adhesion molecule binding or signaling activity.

The polysialylation of neural cell adhesion molecule (NCAM) evolved in vertebrates to carry out biological functions related to changes in cell position and morphology. Many of these effects involve the attenuation of cell interactions that are not mediated through NCAM's own adhesion properties. A proposed mechanism for this global effect on cell interaction is the steric inhibition of membrane-membrane apposition based solely on polysialic acid (PSA) biophysical properties. However, it remains possible that the intrinsic binding or signaling properties of the NCAM polypeptide are also involved. To help resolve this issue, this study uses a quantitative cell detachment assay together with cells engineered to display different adhesion receptors together with a variety of polysialylated NCAM polypeptide isoforms and functional domain deletion mutations. The results obtained indicate that regulation by PSA occurs with adhesion receptors as diverse as an IgCAM, a cadherin and an integrin, and does not require NCAM functional domains other than those minimally required for polysialylation. These findings are most consistent with the cell apposition mechanism for PSA action, as this model predicts that the inhibitory effects of PSA-NCAM on cell adhesion should be independent of the nature of the adhesion system and of any intrinsic binding or signaling properties of the NCAM polypeptide itself.     

The neural cell adhesion molecule is associated with major components of the cytoskeleton

The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily. Two of the three major isoforms (NCAM 140 and NCAM 180) are transmembrane glycoproteins, which differ in their intracellular domains. The present study is concerned with the identification of novel intracellular binding partners of NCAM. We expressed and purified both cytoplasmic domains of NCAM. Using ligand affinity chromatography followed by peptide mass fingerprinting, we could identify several novel binding partners of the cytoplasmic domains of NCAM 140 and 180. We present data that alpha- and beta-tubulin as well as alpha-actinin 1 are associated with both NCAM 140 and 180. In contrast, beta-actin, tropomyosin, microtubuli-associated protein MAP 1A, and rhoA-binding kinase-alpha preferentially bind to NCAM 180. Furthermore, we demonstrate that inhibition of rhoA-binding kinase-alpha stimulates neurite outgrowth independently from NCAM.     

Sequences at the interface of the fifth immunoglobulin domain and first fibronectin type III repeat of the neural cell adhesion molecule are critical for its polysialylation

Polysialic acid is an anti-adhesive glycan that modifies a select group of mammalian proteins. The primary substrate of the polysialyltransferases (polySTs) is the neural cell adhesion molecule (NCAM). Polysialic acid negatively regulates cell adhesion, is required for proper brain development, and is expressed in specific areas of the adult brain where it promotes on-going cell migration and synaptic plasticity. The first fibronectin type III repeat (FN1) of NCAM is required for polysialylation of the N-glycans on the adjacent immunoglobulin-like domain (Ig5), and acidic residues on the surface of FN1 play a role in polyST recognition. Recent work demonstrated that the FN1 domain from the unpolysialylated olfactory cell adhesion molecule (OCAM) was able to partially replace NCAM FN1 (Foley, D. A., Swartzentruber, K. G., Thompson, M. G., Mendiratta, S. S., and Colley, K. J. (2010) J. Biol. Chem. 285, 35056-35067). Here we demonstrate that individually replacing three identical regions shared by NCAM and OCAM FN1, (500)PSSP(503) (PSSP), (526)GGVPI(530) (GGVPI), and (580)NGKG(583) (NGKG), dramatically reduces NCAM polysialylation. In addition, we show that the polyST, ST8SiaIV/PST, specifically binds NCAM and that this binding requires the FN1 domain. Replacing the FN1 PSSP sequences and the acidic patch residues decreases NCAM-polyST binding, whereas replacing the GGVPI and NGKG sequences has no effect. The location of GGVPI and NGKG in loops that flank the Ig5-FN1 linker and the proximity of PSSP to this linker suggest that GGVPI and NGKG sequences may be critical for stabilizing the Ig5-FN1 linker, whereas PSSP may play a dual role maintaining the Ig5-FN1 interface and a polyST recognition site.     

Multiple-bond kinetics from single-molecule pulling experiments: evidence for multiple NCAM bonds

The kinetic parameters of single bonds between neural cell adhesion molecules were determined from atomic force microscope measurements of the forced dissociation of the homophilic protein-protein bonds. The analytical approach described provides a systematic procedure for obtaining rupture kinetics for single protein bonds from bond breakage frequency distributions obtained from single-molecule pulling experiments. For these studies, we used the neural cell adhesion molecule (NCAM), which was recently shown to form two independent protein bonds. The analysis of the bond rupture data at different loading rates, using the single-bond full microscopic model, indicates that the breakage frequency distribution is most sensitive to the distance to the transition state and least sensitive to the molecular spring constant. The analysis of bond failure data, however, motivates the use of a double-bond microscopic model that requires an additional kinetic parameter. This double-bond microscopic model assumes two independent NCAM-NCAM bonds, and more accurately describes the breakage frequency distribution, particularly at high loading rates. This finding agrees with recent surface-force measurements, which showed that NCAM forms two spatially distinct bonds between opposed proteins.     

Elasticity and Rupture of a Multi-Domain Neural Cell Adhesion Molecule Complex

The neural cell adhesion molecule (NCAM) plays an important role in nervous system development. NCAM forms a complex between its terminal domains Ig1 and Ig2. When NCAM of cell A and of cell B connect to each other through complexes Ig12(A)/Ig12(B), the relative mobility of cells A and B and membrane tension exerts a force on the Ig12(A)/Ig12(B) complex. In this study, we investigated the response of the complex to force, using steered molecular dynamics. Starting from the structure of the complex from the Ig1-Ig2-Ig3 fragment, we first demonstrated that the complex, which differs in dimensions from a previous structure from the Ig1-Ig2 fragment in the crystal environment, assumes the same extension when equilibrated in solvent. We then showed that, when the Ig12(A)/Ig12(B) complex is pulled apart with forces 30-70 pN, it exhibits elastic behavior (with a spring constant of ∼0.03 N/m) because of the relative reorientation of domains Ig1 and Ig2. At higher forces, the complex ruptures; i.e., Ig12(A) and Ig12(B) separate. The interfacial interactions between Ig12(A) and Ig12(B), monitored throughout elastic extension and rupture, identify E16, F19, K98, and L175 as key side chains stabilizing the complex.     

Structure and interactions of NCAM Ig1-2-3 suggest a novel zipper mechanism for homophilic adhesion

The neural cell adhesion molecule, NCAM, mediates Ca(2+)-independent cell-cell and cell-substratum adhesion via homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. NCAM plays a key role in neural development, regeneration, and synaptic plasticity, including learning and memory consolidation. The crystal structure of a fragment comprising the three N-terminal Ig modules of rat NCAM has been determined to 2.0 A resolution. Based on crystallographic data and biological experiments we present a novel model for NCAM homophilic binding. The Ig1 and Ig2 modules mediate dimerization of NCAM molecules situated on the same cell surface (cis interactions), whereas the Ig3 module mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans interactions) through simultaneous binding to the Ig1 and Ig2 modules. This arrangement results in two perpendicular zippers forming a double zipper-like NCAM adhesion complex.     

Triple effect of mimetic peptides interfering with neural cell adhesion molecule homophilic cis interactions

The neural cell adhesion molecule (NCAM) is pivotal in neural development, regeneration, and learning. Here we characterize two peptides, termed P1-B and P2, derived from the homophilic binding sites in the first two N-terminal immunoglobulin (Ig) modules of NCAM, with regard to their effects on neurite extension and adhesion. To evaluate how interference of these mimetic peptides with NCAM homophilic interactions in cis influences NCAM binding in trans, we employed a coculture system in which PC12-E2 cells were grown on monolayers of fibroblasts with or without NCAM expression and the rate of neurite outgrowth subsequently was analyzed. P2, but not P1-B, induced neurite outgrowth in the absence of NCAM binding in trans. When PC12-E2 cells were grown on monolayers of NCAM-expressing fibroblasts, the effect of both P1-B and P2 on neurite outgrowth was dependent on peptide concentrations. P1-B and P2 acted as conventional antagonists, agonists, and reverse agonists of NCAM at low, intermediate, and high peptide concentrations, respectively. The demonstrated in vitro triple pharmacological effect of mimetic peptides interfering with the NCAM homophilic cis binding will be valuable for the understanding of the actions of these mimetics in vivo.     

DICAM, a novel dual immunoglobulin domain containing cell adhesion molecule interacts with alphavbeta3 integrin

Immunoglobulin (Ig) superfamily members are abundant with diverse functions including cell adhesion in various tissues. Here, we identified and characterized a novel adhesion molecule that belongs to the CTX protein family and named as DICAM (Dual Ig domain containing cell adhesion molecule). DICAM is a type I transmembrane protein with two V-type Ig domains in the extracellular region and a short cytoplasmic tail of 442 amino acids. DICAM is found to be expressed ubiquitously in various organs and cell lines. Subcellular localization of DICAM was observed in the cell-cell contact region and nucleus of cultured epithelial cells. Cell-cell contact region was colocalized with tight junction protein, ZO-1. The DICAM increased MDCK cell adhesion to 60% levels of fibronectin. DICAM mediated cell adhesion was specific for the alphavbeta3 integrin; other integrins, alpha2, alpha5, beta1, alpha2beta1, alpha5beta1, were not involved in cell adhesion. In identifying the interacting domain of DICAM with alphavbeta3, the Ig domain 2 showed higher cell adhesion activity than that of Ig domain 1. Although RGD motif in Ig domain 2 was engaged in cell adhesion, it was not participated in DICAM-alphavbeta3 mediated cell adhesion. Furthermore, differentially expressing DICAM stable cells showed well correlated cell to cell adhesion capability with integrin beta3-overexpressing cells. Collectively, these results indicate that DICAM, a novel dual Ig domain containing adhesion molecule, mediates cell adhesion via alphavbeta3 integrin.     

Chemistry on a single protein, vascular cell adhesion molecule-1, during forced unfolding

Proteins of many types experience tensile forces in their normal function, and vascular cell adhesion molecule-1 (VCAM-1) is typical in this. VCAM has seven Ig domains, and each has a disulfide bond (-S-S-) buried in its core that covalently stabilizes about half of each domain against unfolding. VCAM is extended here by single molecule atomic force microscopy in the presence or absence of reducing agents. In the absence of reducing agent, a sawtooth pattern of forced unfolding reveals an average period and total length consistent with disulfide locations in VCAM. With increasing reducing agent, accessible disulfides are specifically reduced (to SH); the average period for unfolding increases up to saturation together with additional metrics of unfolding. Steered molecular dynamics simulations of unfolding indeed show that the core disulfide bond is solvent-exposed in the very earliest stages of protein extension. Michaelis-Menten kinetics emerge with reduction catalyzed by force (tau(reduction) approximately 10(-4) s). The results establish single molecule reduction, one bond at a time, and show that mechanical forces can play a key role in modulating the redox state of cell adhesion proteins that are invariably stressed in cell adhesion.     

Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM

The neural cell adhesion molecule NCAM is capable of mediating cell-cell adhesion via homophilic interactions. In this study, three strategies have been combined to identify regions of NCAM that participate directly in NCAM-NCAM binding: analysis of domain deletion mutations, mapping of epitopes of monoclonal antibodies, and use of synthetic peptides to inhibit NCAM activity. Studies on L cells transfected with NCAM mutant cDNAs using cell aggregation and NCAM-covasphere binding assays indicate that the third immunoglobulin-like domain is involved in homophilic binding. The epitopes of four monoclonal antibodies that have been previously shown to affect cell-cell adhesion mediated by NCAM were also mapped to domain 3. Overlapping hexapeptides were synthesized on plastic pins and assayed for binding with these monoclonal antibodies. One of them (PP) reacted specifically with the sequence KYSFNY. Synthetic oligopeptides containing the PP epitope were potent and specific inhibitors of NCAM binding activity. A substratum containing immobilized peptide conjugates also exhibited adhesiveness for neural retinal cells. Cell attachment was specifically inhibited by peptides that contained the PP-epitope and by anti-NCAM univalent antibodies. The shortest active peptide has the sequence KYSFNYDGSE, suggesting that this site is directly involved in NCAM homophilic interaction.     

Specific amino acids in the first fibronectin type III repeat of the neural cell adhesion molecule play a role in its recognition and polysialylation by the polysialyltransferase ST8Sia IV/PST

Polysialic acid is an anti-adhesive protein modification that promotes cell migration and the plasticity of cell interactions. Because so few proteins carry polysialic acid, we hypothesized that polysialylation is a protein-specific event and that a specific polysialyltransferase-substrate interaction is the basis of this specificity. The major substrate for the polysialyltransferases is the neural cell adhesion molecule, NCAM. Previous work demonstrates that the first fibronectin type III repeat of NCAM (FN1) was necessary for the polysialylation of the N-glycans on the adjacent immunoglobulin domain (Ig5) (Close, B. E., Mendiratta, S. S., Geiger, K. M., Broom, L. J., Ho, L. L., and Colley, K. J. (2003) J. Biol. Chem. 278, 30796-30805). This suggested that FN1 may be a recognition site for the polysialyltransferases. In this study, we showed that the second fibronectin type III repeat (FN2) of NCAM cannot replace FN1. Arg substitution of three unique acidic amino acids on the surface of FN1 eliminated polysialylation not only of a minimal Ig5-FN1 substrate but also of full-length NCAM. Ala substitution of these residues eliminated Ig5-FN1 polysialylation but not that of full-length NCAM, suggesting that the two proteins are interacting differently with the enzymes and that multiple residues are involved in the enzyme-NCAM interaction. By using another truncated protein, Ig5-FN1-FN2, we confirmed the importance of enzyme-substrate positioning for optimal recognition and polysialylation. In sum, we have found that acidic residues on the surface of FN1 are part of a larger protein interaction region that is critical for NCAM recognition and polysialylation by the polysialyltransferases.     

Backbone dynamics of the first, second, and third immunoglobulin modules of the neural cell adhesion molecule (NCAM)

The neural cell adhesion molecule (NCAM) is a cell surface multimodular protein, which plays an important role in cell-cell adhesion by homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. In the present study, the backbone dynamics of the first three immunoglobulin-like (Ig) modules of NCAM have been investigated by NMR spectroscopy. Ig1, Ig2, and Ig3 share low sequence identity but possess the same fold and have very similar three-dimensional structures. (15)N longitudinal and transverse relaxation rates and heteronuclear NOEs have been measured and subsequently analyzed by the axial symmetric Lipari-Szabo modelfree formalism to characterize fast (pico- to nanosecond) and slow (micro- to millisecond) motions in the three protein modules. We found that backbone motions of residues located in the beta-strand regions are generally restricted, while increased flexibility is observed in turns and loops. In all three modules, residues located in the segments connecting the C- and D-strand plus residues located in the segment connecting the E- and F-strand show significant chemical exchange on the micro- to millisecond time scale. In addition, a number of residues with small chemical exchange contribution seem to form contiguous regions in the beta sheets, suggesting that these motions might be correlated. Only few residues in the homophilic binding sites in the NCAM Ig1 and Ig2 modules show increased flexibility, indicating that the Ig1-Ig2-mediated NCAM homophilic binding does not depend on the local backbone mobility of the interacting modules.