Binding of NAP-22, a calmodulin-binding neuronal protein,to raft-like domains in model membranes

The cholesterol-binding protein NAP-22 is a major component of the detergent-insoluble low-density fraction of rat brain. In this study, we found, using fluorescence microscopy, that native NAP-22, but not a demyristoylated form, binds to cholesterol-rich raft-like domains in planar-supported monolayers and remains bound after nonionic detergent extraction. NAP-22 also protects the cholesterol-rich domains during extraction by methyl-beta-cyclodextrin. The lateral mobility of this protein is much lower than that of other raft components in model membranes, suggesting that both cholesterol binding and inter-NAP-22 interactions markedly reduce its lateral diffusion. This study suggests that NAP-22 binding may be employed to image cholesterol-rich regions, such as caveolae/rafts, on the plasma membrane of cells, and preliminary efforts in that direction are presented.     

Protein-induced formation of cholesterol-rich domains

A major protein of neuronal rafts, NAP-22, binds specifically to cholesterol. We demonstrate by circular dichroism that NAP-22 contains a significant amount of beta-structure that is not sensitive to binding of the protein to membranes, suggesting that a major portion of the protein does not insert deeply into the membrane. The free energy of binding of NAP-22 to liposomes of dioleoylphosphatidylcholine with 40% cholesterol is -7.3 +/- 0.5 kcal/mol. NAP-22 mixed with dipalmitoylphosphatidylcholine and 40% cholesterol partitions into the detergent insoluble fraction in the presence of 1% Triton X-100. NAP-22 also causes this insoluble fraction to become enriched in cholesterol relative to phospholipid, again demonstrating the ability of this protein to segregate cholesterol and phospholipids into domains. Differential scanning calorimetry results demonstrate that NAP-22 promotes domain formation in liposomes composed of cholesterol and phosphatidylcholine. This is shown by NAP-22-promoted changes in the shape and enthalpy of the phase transition of phosphatidylcholine as well as by the appearance of cholesterol crystallite transitions in membranes composed of phosphatidylcholine with either saturated or unsaturated acyl chains. In situ atomic force microscopy revealed a marked change in the surface morphology of a supported bilayer of dioleoylphosphatidylcholine with 0.4 mole fraction of cholesterol upon addition of NAP-22. Prior to the addition of the protein, the bilayer appears to be a molecularly smooth structure with uniform thickness. Addition of NAP-22 resulted in the rapid formation of localized raised bilayer domains. Remarkably, there was no gross disruption or erosion of the bilayer but rather simply an apparent rearrangement of the lipid bilayer structure due to the interaction of NAP-22 with the lipid. Our results demonstrate that NAP-22 can induce the formation of cholesterol-rich domains in membranes. This is likely to be relevant in neuronal membrane domains that are rich in NAP-22.     

Specificity of Membrane Binding of the Neuronal Protein NAP-22

NAP-22, a major protein of neuronal rafts is known to preferentially bind to membranes containing cholesterol. In this work we establish the requirements for membrane binding of NAP-22. We find that other sterols can replace cholesterol to promote binding. In addition, bilayers containing phosphatidylethanolamine bind NAP-22 in the absence of cholesterol. Thus, there is not a specific interaction of NAP-22 with cholesterol that determines its binding to membranes. Addition of a mol fraction of phosphatidylserine of 0.05 to membranes of phosphatidylcholine and cholesterol enhances the membrane binding of NAP-22. The dependence of binding on the mol fraction of phosphatidylserine indicates that NAP-22 binds to membranes with its amino-terminal segment closer to the membrane than the remainder of the protein. We have also determined which segments of NAP-22 are required for membrane binding. A non-myristoylated form binds only weakly to membranes. Truncating the protein from 226 amino acids to the myristoylated amino-terminal 60 amino acids does not prevent binding to membranes in a cholesterol-dependent manner, but this binding is of weaker affinity. However, myristoylation is not sufficient to promote binding to cholesterol-rich domains. An N-terminal 19-amino-acid, myristoylated peptide binds to membranes but without requiring specific lipids. Thus, the remainder of the protein contributes to the lipid specificity of the membrane binding of NAP-22.     

Quaternary structure of the neuronal protein NAP-22 in aqueous solution

NAP-22, a myristoylated, anionic protein, is a major protein component of the detergent-insoluble fraction of neurons. After extraction from the membrane, it is readily soluble in water. NAP-22 will partition only into membranes with specific lipid compositions. The lipid specificity is not expected for a monomeric myristoylated protein. We have studied the self-association of NAP-22 in solution. Sedimentation velocity experiments indicated that the protein is largely associated. The low concentration limiting s value is approximately 1.3 S, indicating a highly asymmetric monomer. In contrast, a nonmyristoylated form of the protein shows no evidence of oligomerization by velocity sedimentation and has an s value corresponding to the smallest component of NAP-22, but without the presence of higher oligomers. Sedimentation equilibrium runs indicate that there is a rapidly reversible equilibrium between monomeric and oligomeric forms of the protein followed by a slower, more irreversible association into larger aggregates. In situ atomic force microscopy of the protein deposited on mica from freshly prepared dilute solution revealed dimers on the mica surface. The values of the association constants obtained from the sedimentation equilibrium data suggest that the weight concentration of the monomer exceeds that of the dimer below a total protein concentration of 0.04 mg/ml. Since the concentration of NAP-22 in the neurons of the developing brain is approximately 0.6 mg/ml, if the protein were in solution, it would be in oligomeric form and bind specifically to cholesterol-rich domains. We demonstrate, using fluorescence resonance energy transfer, that at low concentrations, NAP-22 labeled with Texas Red binds equally well to liposomes of phosphatidylcholine either with or without the addition of 40 mol% cholesterol. Thus, oligomerization of NAP-22 contributes to its lipid selectivity during membrane binding.     

Morphological analysis on the distribution of membrane lipids and a membrane protein,NAP-22, during neuronal development in vitro

Specific localization of membrane proteins based on the interactions with membrane lipids at various microdomains (MDs) is under active investigation, since the elucidation of the molecular mechanism of the interactions could reveal a novel concept of cell organization. Due to the strong interactions not only between lipids but also between lipids and proteins, these MDs are considered to be recovered in a detergent-resistant low-density membrane fraction (DRM) after detergent extraction and density-gradient centrifugation. Neurons take well-developed membrane systems during maturation and specific localization of various membrane components, not only proteins but also lipids, is essential for the establishment of the nervous system. In previous studies, we showed that NAP-22 is a major protein of neuronal DRM and binds liposomes in a cholesterol-dependent manner. In this study, we analyzed the localization of membrane lipids during neuronal maturation in vitro and compared their distribution with that of NAP-22. In an attempt to detect DRM-associated lipids, we observed the staining patterns of neurons treated with Triton-X-100 at 4 degrees C before fixation. Our results showed the less staining patterns of cholesterol and sphingomyelin at the axonal tips and a different staining pattern of two gangliosides, GM(1) and GD(3). The enrichment of cholesterol at the NAP-22 localizing spots was observed after the treatment of the detergent. Since the application of maitotoxin, a calcium ion channel, caused the diminution of NAP-22 and cholesterol positive spots, the distribution of these molecules are considered under the calcium regulation.     

Molecular characterization of the detergent-insoluble cholesterol-rich membrane microdomain (raft) of the central nervous system

Many fundamental neurological issues such as neuronal polarity, the formation and remodeling of synapses, synaptic transmission, and the pathogenesis of the neuronal cell death are closely related to the membrane dynamics. The elucidation of functional roles of a detergent-insoluble cholesterol-rich domain (raft) could therefore provide good clues to the molecular understanding of these important phenomena, for the participation of the raft in the fundamental cell functions, such as signal transduction and selective transport of lipids and proteins, has been elucidated in nonneural cells. Interestingly, the brain is rich in raft and the brain-derived raft differs in its lipid and protein components from other tissue-derived rafts. Since many excellent reviews are written on the membrane lipid dynamics of this microdomain, signal transduction, and neuronal glycolipids, we review on the characterization of the raft proteins recovered in the detergent-insoluble low-density fraction from rat brain. Special focus is addressed on the biochemical characterization of a neuronal enriched protein, NAP-22, for the lipid organizing activity of this protein has become increasingly clear.     

Molecular characterization of the detergent-insoluble cholesterol-rich membrane microdomain (raft) of the central nervous system

Many fundamental neurological issues such as neuronal polarity, the formation and remodeling of synapses, synaptic transmission, and the pathogenesis of the neuronal cell death are closely related to the membrane dynamics. The elucidation of functional roles of a detergent-insoluble cholesterol-rich domain (raft) could therefore provide good clues to the molecular understanding of these important phenomena, for the participation of the raft in the fundamental cell functions, such as signal transduction and selective transport of lipids and proteins, has been elucidated in nonneural cells. Interestingly, the brain is rich in raft and the brain-derived raft differs in its lipid and protein components from other tissue-derived rafts. Since many excellent reviews are written on the membrane lipid dynamics of this microdomain, signal transduction, and neuronal glycolipids, we review on the characterization of the raft proteins recovered in the detergent-insoluble low-density fraction from rat brain. Special focus is addressed on the biochemical characterization of a neuronal enriched protein, NAP-22, for the lipid organizing activity of this protein has become increasingly clear.     

The arrangement of cholesterol in membranes and binding of NAP-22

Cholesterol forms crystals when the mol fraction of sterol in a membrane bilayer exceeds a certain value. The solubility limit of cholesterol is very dependent on the nature of the phospholipid with which it is mixed. NMR methods have proven useful in quantifying the amount of cholesterol monohydrate crystals present in mixtures with phospholipids. A protein, NAP-22, present in high abundance in the synaptic cell membrane and synaptic vesicle, promotes the formation of cholesterol crystallites in lipid mixtures in which cholesterol would be completely dissolved in the membrane in the absence of protein. This finding, along with effects of the protein on the phase transitions of mixtures of phosphatidylcholine (PC) and cholesterol indicate that NAP-22 facilitates the formation of cholesterol-rich domains. This protein will bind only to membranes of PC that contain either cholesterol or phosphatidylethanolamine (PE). The process requires the presence of a myristoyl group on the N-terminus of NAP-22. The phenomenon also does not occur with a 19 amino acid myristoylated peptide corresponding to the amino terminal segment of NAP-22. The basis of the selectivity of NAP-22 for interacting with membranes of specific composition is suggested to be due to the accessibility of the myristoyl group.     

Tracking peptide-membrane interactions: insights from in situ coupled confocal-atomic force microscopy imaging of NAP-22 peptide insertion and assembly

Elucidating the role that charged membrane proteins play in determining cell membrane structure and dynamics is an area of active study. We have applied in situ correlated atomic force and confocal microscopies to characterize the interaction of the NAP-22 peptide with model membranes prepared as supported planar bilayers containing both liquid-ordered and liquid-disordered domains. Our results demonstrated that the NAP-22 peptide interacts with membranes in a concentration-dependent manner, preferentially inserting into DOPC (ld) domains. While at low peptide concentrations, the NAP-22 peptide formed aggregate-like structures within the ld domains, at high peptide concentrations, it appeared to sequester cholesterol into the ld domains and recruited phosphatidyl-myo-inositol 4,5-bisphosphate by inducing a blending effect that homogenizes the phase-segregated domains into one liquid-ordered domain. This study describes a possible mechanism by which the NAP-22 peptide can affect neuronal morphology.     

The role of cholesterol and glycosylphosphatidylinositol-anchored proteins of erythrocyte rafts in regulating raft protein content and malarial infection

Human erythrocytes are terminally differentiated, nonendocytic cells that lack all intracellular organelles. Here we show that their plasma membranes contain detergent-resistant membrane rafts that constitute a small fraction (4%) of the total membrane protein, with a complex mixture of proteins that differentially associate with rafts. Depletion of raft-cholesterol abrogates association of all proteins with no significant effect on cholesterol:protein ratios in the rest of the membrane, lipid asymmetry, deformability, or transport properties of the bilayer, indicating that cholesterol is critical for protein assembly into rafts and suggesting that rafts have little influence on several erythrocyte functions. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which lack glycosylphosphatidylinositol-anchored proteins, show significant elevation in raft-cholesterol but no increase in raft protein association, suggesting that raft assembly does not require glycosylphosphatidylinositol-anchored proteins, raft proteins do not bind directly to cholesterol, and only threshold levels of raft-cholesterol are critical for protein recruitment. Loss of glycosylphosphatidylinositol-anchored proteins had no effect on erythrocytic infection by malarial parasite or movement of raft markers into the parasite's vacuole. However, infection is blocked following raft-cholesterol disruption, suggesting that erythrocyte rafts can be functionally exploited and providing the first evidence for the involvement of host rafts in an apicomplexan infection.     

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca(2+) concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.     

Cholesterol effects on BAX pore activation

The importance of BCL-2 family proteins in the control of cell death has been clearly established. One of the key members of this family, BAX, has soluble, membrane-bound, and membrane-integrated forms that are central to the regulation of apoptosis. Using purified monomeric human BAX, defined liposomes, and isolated human mitochondria, we have characterized the soluble to membrane transition and pore formation by this protein. For the purified protein, activation, but not oligomerization, is required for membrane binding. The transition to the membrane environment includes a binding step that is reversible and distinct from the membrane integration step. Oligomerization and pore activation occur after the membrane integration. In cells, BAX targets several intracellular membranes but notably does not target the plasma membrane while initiating apoptosis. When cholesterol was added to either the liposome bilayer or mitochondrial membranes, we observed increased binding but markedly reduced integration of BAX into both membranes. This cholesterol inhibition of membrane integration accounts for the reduction of BAX pore activation in liposomes and mitochondrial membranes. Our results indicate that the presence of cholesterol in membranes inhibits the pore-forming activity of BAX by reducing the ability of BAX to transition from a membrane-associated protein to a membrane-integral protein.     

Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100

The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55(gag). We have analyzed whether Pr55(gag) has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55(gag) has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55(gag) particles (VLPs) yield buoyant Pr55(gag) complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55(gag) complexes cannot be taken as a proof for raft association of Pr55(gag), since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55(gag). However, Pr55(gag) might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55(gag). Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55(gag) complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55(gag) localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55(gag) VLPs.     

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca2+ concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca²⁺-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-β-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-β-cyclodextrin complexes restored the Ca²⁺-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca²⁺, annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca²⁺ concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca²⁺ concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.     

HIV‐1 Assembly Differentially Alters Dynamics and Partitioning of Tetraspanins and Raft Components

Partitioning of membrane proteins into various types of microdomains is crucial for many cellular functions. Tetraspanin‐enriched microdomains (TEMs) are a unique type of protein‐based microdomain, clearly distinct from membrane rafts, and important for several cellular processes such as fusion, migration and signaling. Paradoxically, HIV‐1 assembly/egress occurs at TEMs, yet the viral particles also incorporate raft lipids. Using different quantitative microscopy approaches, we investigated the dynamic relationship between TEMs, membrane rafts and HIV‐1 exit sites, focusing mainly on the tetraspanin CD9. Our results show that clustering of CD9 correlates with multimerization of the major viral structural component, Gag, at the plasma membrane. CD9 exhibited confined behavior and reduced lateral mobility at viral assembly sites, suggesting that Gag locally traps tetraspanins. In contrast, the raft lipid GM1 and the raft‐associated protein CD55, while also recruited to assembly/budding sites, were only transiently trapped in these membrane areas. CD9 recruitment and confinement were found to be partially dependent on cholesterol, while those of CD55 were completely dependent on cholesterol. Importantly, our findings support the emerging concept that cellular and viral components, instead of clustering at preexisting microdomain platforms, direct the formation of distinct domains for the execution of specific functions.     

Membrane localization of Junín virus glycoproteins requires cholesterol and cholesterol rich membranes

Arenavirus morphogenesis and budding occurs at cellular plasma membrane; however, the nature of membrane assembly sites remains poorly understood. In this study we examined the effect of different cholesterol-lowering agents on Junín virus (JUNV) multiplication. We found that cholesterol cell depletion reduced JUNV glycoproteins (GPs) membrane expression and virus budding. Analysis of membrane protein insolubility in Triton X-100 suggested that JUNV GPs associate with cholesterol enriched membranes. Rafts dissociation conditions as warm detergent extraction and cholesterol removal by methyl-β-cyclodextrin compound showed to impair GPs cholesterol enriched membrane association. Analysis of GPs transfected cells showed similar results suggesting that membrane raft association is independent of other viral proteins.     

Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.     

Microvillar membrane microdomains exist at physiological temperature. Role of galectin-4 as lipid raft stabilizer revealed by "superrafts"

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by "conventional" extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant "superrafts." These were enriched in galectin-4, a beta-galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.     

Compartmentalization of beta-secretase (Asp2) into low-buoyant density, noncaveolar lipid rafts

Recent epidemiological studies show a reduced prevalence of Alzheimer's disease (AD) in patients treated with inhibitors of cholesterol biosynthesis. Moreover, the cholesterol-transport protein, apolipoprotein E4, and elevated cholesterol are important risk factors for AD. Additionally, in vitro and in vivo studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein (APP) to beta-amyloid, the major constituent of senile plaques. Cholesterol plays a crucial role in maintaining lipid rafts in a functional state. Lipid rafts are cholesterol-enriched membrane microdomains implicated in signal transduction, protein trafficking, and proteolytic processing. Since APP, beta-amyloid, and the putative gamma-secretase, presenilin-1 (PS-1), have all been found in lipid rafts, we hypothesized that the recently identified beta-secretase, Asp2 (BACE1), might also be present in rafts. Here, we report that recombinant Asp2 expressed in three distinct cell lines is raft associated. Using both detergent and nondetergent methods, Asp2 protein and activity were found in a light membrane raft fraction that also contained other components of the amyloidogenic pathway. Immunoisolation of caveolin-containing vesicles indicated that Asp2 was present in a unique raft population distinct from caveolae. Finally, depletion of raft cholesterol abrogated association of Asp2 with the light membrane fraction. These observations are consistent with the raft localization of APP processing and suggest that the partitioning of Asp2 into lipid rafts may underlie the cholesterol sensitivity of beta-amyloid production.     

Crystal structure of a myristoylated CAP-23/NAP-22 N-terminal domain complexed with Ca2+/calmodulin

A variety of viral and signal transduction proteins are known to be myristoylated. Although the role of myristoylation in protein-lipid interaction is well established, the involvement of myristoylation in protein-protein interactions is less well understood. CAP-23/NAP-22 is a brain-specific protein kinase C substrate protein that is involved in axon regeneration. Although the protein lacks any canonical calmodulin (CaM)-binding domain, it binds CaM with high affinity. The binding of CAP-23/NAP-22 to CaM is myristoylation dependent and the N-terminal myristoyl group is directly involved in the protein-protein interaction. Here we show the crystal structure of Ca2+-CaM bound to a myristoylated peptide corresponding to the N-terminal domain of CAP-23/NAP-22. The myristoyl moiety of the peptide goes through a hydrophobic tunnel created by the hydrophobic pockets in the N- and C-terminal domains of CaM. In addition to the myristoyl group, several amino-acid residues in the peptide are important for CaM binding. This is a novel mode of binding and is very different from the mechanism of binding in other CaM-target complexes.