Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7^KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis.     

Autonomous functions for the Sec14p/spectrin-repeat region of Kalirin

Kalirin is a GDP/GTP exchange factor (GEF) for Rho proteins that modulates the actin cytoskeleton in neurons. Alternative splicing generates Δ-isoforms, which encode the RhoGEF domain, but lack the N-terminal Sec14p domain and first 4 spectrin-like repeats of the full-length isoforms. Splicing has functional consequences, with Kal7 but not ΔKal7 causing formation of dendritic spines. Cells lacking endogenous Kalirin were used to explore differences between these splice variants. Expression of ΔKal7 in this system induces extensive lamellipodial sheets, while expression of Kal7 induces formation of adherent compact, round cells with abundant cortical actin. Based on in vitro and cell-based assays, Kal7 and ΔKal7 are equally active GEFs, suggesting that other domains are involved in controlling cell morphology. Catalytically inactive Kal7 and a Kalirin fragment which includes only Sec14p and spectrin-like domains retain the ability to produce compact, round cells and fractionate as high molecular weight complexes. Separating the Sec14p domain from the spectrin-like repeats eliminates the ability of Kal7 to cause this response. The isolated Sec14p domain binds PI(3,5)P2 and PI3P, but does not alter cell morphology. We conclude that the Sec14p and N-terminal spectrin-like domains of Kalirin play critical roles in distinguishing the actions of full-length and Δ-Kalirin proteins.     

Crystal structure of a rigid four-spectrin-repeat fragment of the human desmoplakin plakin domain

The plakin protein family serves to connect cell-cell and cell-matrix adhesion molecules to the intermediate filament cytoskeleton. Desmoplakin (DP) is an integral part of desmosomes, where it links desmosomal cadherins to the intermediate filaments. The 1056-amino-acid N-terminal region of DP contains a plakin domain common to members of the plakin family. Plakin domains contain multiple copies of spectrin repeats (SRs). We determined the crystal structure of a fragment of DP, residues 175-630, consisting of four SRs and an inserted SH3 domain. The four repeats form an elongated, rigid structure. The SH3 domain is present in a loop between two helices of an SR and interacts extensively with the preceding SR in a manner that appears to limit inter-repeat flexibility. The intimate intramolecular association of the SH3 domain with the preceding SR is also observed in plectin, another plakin protein, but not in α-spectrin, suggesting that the SH3 domain of plakins contributes to the stability and rigidity of this subfamily of SR-containing proteins.     

A comparative and phylogenetic analysis of the alpha-actinin rod domain

Alpha-actinin is a ubiquitous actin-binding protein, composed of 3 domains; an actin-binding domain and a calcium-binding domain at the termini, connected by a rod domain composed by 1, 2, or 4 spectrin repeats (SRs). To understand how the rod domain has evolved during evolution, we have analyzed and compared the amino acid residue heterogeneity and phylogeny of the SRs of alpha-actinins of vertebrates, invertebrates, fungi, and several protozoa. The repeats of vertebrate alpha-actinins show a high degree of similarity, whereas repeats of invertebrates, fungi, and, in particular, of protozoa are more divergent. In the phylogeny, SR1 of all species were clustered together, independent of the number of repeats in the protein. It was also obvious that the second and last repeat in fungi (SR2) grouped with the fourth and last repeat of vertebrates and invertebrates (SR4). Therefore, the phylogeny implied that the rod domain of the cenancestral alpha-actinin only contained one SR. It was also obvious that SR2 of fungi are related to SR4 of vertebrates and invertebrates, implying that in the second intragenic duplication 2 repeats (i.e., what become SR2 and SR3) were inserted between the initial 2 repeats that become SR1 and SR4.     

The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat

Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.     

An isoform of kalirin, a brain-specific GDP/GTP exchange factor, is enriched in the postsynaptic density fraction

Communication between membranes and the actin cytoskeleton is an important aspect of neuronal function. Regulators of actin cytoskeletal dynamics include the Rho-like small GTP-binding proteins and their exchange factors. Kalirin is a brain-specific protein, first identified through its interaction with peptidylglycine-alpha-amidating monooxygenase. In this study, we cloned rat Kalirin-7, a 7-kilobase mRNA form of Kalirin. Kalirin-7 contains nine spectrin-like repeats, a Dbl homology domain, and a pleckstrin homology domain. We found that the majority of Kalirin-7 protein is associated with synaptosomal membranes, but a fraction is cytosolic. We also detected higher molecular weight Kalirin proteins. In rat cerebral cortex, Kalirin-7 is highly enriched in the postsynaptic density fraction. In primary cultures of neurons, Kalirin-7 is detected in spine-like structures, while other forms of Kalirin are visualized in the cell soma and throughout the neurites. Kalirin-7 and its Dbl homology-pleckstrin homology domain induce formation of lamellipodia and membrane ruffling, when transiently expressed in fibroblasts, indicative of Rac1 activation. Using Rac1, the Dbl homology-pleckstrin homology domain catalyzed the in vitro exchange of bound GDP with GTP. Kalirin-7 is the first guanine-nucleotide exchange factor identified in the postsynaptic density, where it is positioned optimally to regulate signal transduction pathways connecting membrane proteins and the actin cytoskeleton.     

The nonlinear structure of the desmoplakin plakin domain and the effects of cardiomyopathy-linked mutations

Desmoplakin is a cytoplasmic desmosomal protein that plays a vital role in normal intercellular adhesion. Mutations in desmoplakin can result in devastating skin blistering diseases and arrhythmogenic right ventricular cardiomyopathy, a heart muscle disorder associated with ventricular arrhythmias, heart failure, and sudden death. The desmoplakin N-terminal region is a 1056-amino-acid sequence of unknown structure. It mediates interactions with other desmosomal proteins, is found in a variety of plakin proteins, and spans what has been termed the “plakin domain,” which includes residues 180-1022 and consists of six spectrin repeats (SRs) and an Src homology 3 domain. Herein we elucidate the architecture of desmoplakin's plakin domain, as well as its constituent tandem SRs. Small-angle X-ray scattering analysis shows that the entire plakin domain has an “L” shape, with a long arm and a short arm held at a perpendicular angle. The long arm is 24.0 nm long and accommodates four stably folded SRs arranged in tandem. In contrast, the short arm is 17.9 nm in length and accommodates two independently folded repeats and an extended C-terminus. We show that mutations linked to arrhythmogenic right ventricular cardiomyopathy (K470E and R808C) cause local conformational alterations, while the overall folded structure is maintained. This provides the first structural and mechanistic insights into an entire plakin domain and provides a basis for understanding the critical role of desmoplakin in desmosome function.     

Cooperativity in forced unfolding of tandem spectrin repeats

Force-driven conformational changes provide a broad basis for protein extensibility, and multidomain proteins broaden the possibilities further by allowing for a multiplicity of forcibly extended states. Red cell spectrin is prototypical in being an extensible, multidomain protein widely recognized for its contribution to erythrocyte flexibility. Atomic force microscopy has already shown that single repeats of various spectrin family proteins can be forced to unfold reversibly under extension. Recent structural data indicates, however, that the linker between triple-helical spectrin repeats is often a contiguous helix, thus raising questions as to what the linker contributes and what defines a domain mechanically. We have examined the extensible unfolding of red cell spectrins as monomeric constructs of just two, three, or four repeats from the actin-binding ends of both alpha- and beta-chains, i.e., alpha(18-21) and beta(1-4) or their subfragments. In addition to single repeat unfolding evident in sawtooth patterns peaked at relatively low forces (<50 pN at 1 nm/ms extension rates), tandem repeat unfolding is also demonstrated in ensemble-scale analyses of thousands of atomic force microscopy contacts. Evidence for extending two chains and loops is provided by force versus length scatterplots which also indicate that tandem repeat unfolding occurs at a significant frequency relative to single repeat unfolding. Cooperativity in forced unfolding of spectrin is also clearly demonstrated by a common force scale for the unfolding of both single and tandem repeats.     

The structure of a tandem pair of spectrin repeats of plectin reveals a modular organization of the plakin domain

Plectin is a large and versatile cytoskeletal linker and member of the plakin protein family. Plakins share a conserved region called the plakin domain located near their N terminus. We have determined the crystal structure of an N-terminal fragment of the plakin domain of plectin to 2.05 A resolution. This region is adjacent to the actin-binding domain and is required for efficient binding to the integrin alpha6beta4 in hemidesmosomes. The structure is formed by two spectrin repeats connected by an alpha-helix that spans these two repeats. While the first repeat is very similar to other known structures, the second repeat is structurally different with a hydrophobic core, narrower than that in canonical spectrin repeats. Sequence analysis of the plakin domain revealed the presence of up to nine consecutive spectrin repeats organized in an array of tandem modules, and a Src-homology 3 domain inserted in the central spectrin repeat. The structure of the plakin domain is reminiscent of the modular organization of members of the spectrin family. The architecture of the plakin domain suggests that it forms an elongated and flexible structure, and provides a novel molecular explanation for the contribution of plectin and other plakins to the elasticity and stability of tissues subjected to mechanical stress, such as the skin and striated muscle.     

The crystal structures of dystrophin and utrophin spectrin repeats: implications for domain boundaries

Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.     

Identification and characterization of beta V spectrin, a mammalian ortholog of Drosophila beta H spectrin

Four mammalian beta-spectrin genes are currently recognized, all encode proteins of approximately 240-280,000 M(r) and display 17 triple helical homologous approximately 106-residue repeat units. In Drosophila and Caenorhabditis elegans, a variant beta spectrin with unusual properties has been recognized. Termed beta heavy (beta(H)), this spectrin contains 30 spectrin repeats, has a molecular weight in excess of 400,000, and associates with the apical domain of polarized epithelia. We have cloned and characterized from a human retina cDNA library a mammalian ortholog of Drosophila beta(H) spectrin, and in accord with standard spectrin naming conventions we term this new mammalian spectrin beta 5 (betaV). The gene for human betaV spectrin (HUBSPECV) is on chromosome 15q21. The 11, 722-nucleotide cDNA of betaV spectrin is generated from 68 exons and is predicted to encode a protein with a molecular weight of 416,960. Like its fly counterpart, the derived amino acid sequence of this unusual mammalian spectrin displays 30 spectrin repeats, a modestly conserved actin-binding domain, a conserved membrane association domain 1, a conserved self-association domain, and a pleckstrin homology domain near its COOH terminus. Its putative ankyrin-binding domain is poorly conserved and may be inactive. These structural features suggest that betaV spectrin is likely to form heterodimers and oligomers with alpha spectrin and to interact directly with cellular membranes. Unlike its Drosophila ortholog, betaV spectrin does not contain an SH3 domain but displays in repeat 5 a 45-residue insertion that displays 42% identity to amino acids 85-115 of the E4 protein of type 75 human papilloma virus. Human betaV spectrin is expressed at low levels in many tissues. By indirect immunofluorescence, it is detected prominently in the outer segments of photoreceptor rods and cones and in the basolateral membrane and cytosol of gastric epithelial cells. Unlike its Drosophila ortholog, a distinct apical distribution of betaV spectrin is inapparent in the epithelial cell populations examined, although it is confined to the outer segments of photoreceptor cells. The complete cDNA sequence of human betaV spectrin is available from GenBank(TM) as accession number.     

Folding of spectrin's SH3 domain in the presence of spectrin repeats

The multifunctional protein spectrin contains several different structural motifs, such as spectrin repeats and a SH3 domain. Both triple-helix spectrin repeats and the SH3 domain are believed to form independent structural entities. In alpha-spectrins the SH3 domain is localized to repeat 9, where it is positioned between helix B and helix C in the repeat unit. The presence of SH3 in repeat 9 decreases the thermal stability considerably of this repeat unit while another insert in helix C does not seem to affect the stability. Addition of one or two adjacent repeat units increases the thermal stability from ca 25 degrees C to 41 and 48 degrees C, respectively. Despite the differences in thermal stability, the folding properties of peptides comprising the SH3 domain only or together with one or more repeats are more or less the same.     

The structure of interfaces between subunits of dimeric and tetrameric proteins

The structures of the interfaces of nine dimeric and nine tetrameric proteins have been analyzed and have been seen to follow general principles. These interfaces are combinations of four structural motifs, which resemble features of monomeric proteins. These are: (i) extended beta sheet; (ii) helix-helix packing; (iii) sheet-sheet packing; and (iv) loop interactions. Other common structural features in the interfaces studied are two-fold symmetry, charged hydrogen bonds and channel formation (found only in tetramers). Monomer-monomer interfaces are intermediate in hydrophobicity and charge between the interfaces between secondary structures of monomeric proteins and the exteriors of monomeric proteins. A typical interface has one of the first three of the structural motifs at its centre and loop interactions around the outside, where most of the charge resides.     

Comprehensive analysis of all triple helical repeats in beta-spectrins reveals patterns of selective evolutionary conservation

The spectrin superfamily (spectrin, alpha-actinin, utrophin and dystrophin) has in common a triple helical repeating unit of ~106 amino acid residues. In spectrin, alpha and beta chains contain multiple copies of this repeat. beta-spectrin chains contain the majority of binding activities in spectrin and are essential for animal life. Canonical beta-spectrins have 17 repeats; beta-heavy spectrins have 30. Here, the repeats of five human beta-spectrins, plus beta-spectrins from several other vertebrates and invertebrates, have been analysed. Repeats 1, 2, 14 and 17 in canonical beta are highly conserved between invertebrates and vertebrates, and repeat 8 in some isoforms. This is consistent with conservation of critical functions, since repeats 1, 2 and 17 bind alpha-spectrin. Repeats 1 of beta-spectrins are not always detected by SMART or Pfam tools. A profile hidden Markov model of beta-spectrin repeat 1 detects alpha-actinins, but not utrophin or dystrophin. Novel examples of repeat 1 were detected in the spectraplakins MACF1, BPAG1 and plectin close to the actin-binding domain. Ankyrin binds to the C-terminal portion of repeat 14; the high conservation of this entire repeat may point to additional, undiscovered ligand-binding activities. This analysis indicates that the basic triple helical repeat pattern was adapted early in the evolution of the spectrin superfamily to encompass essential binding activities, which characterise individual repeats in proteins extant today.     

Molecular epitopes of the ankyrin-spectrin interaction

Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for beta-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin-spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30-60% unstructured. In contrast, human erythroid beta-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and beta-spectrin repeats 14-15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with beta-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of beta-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th beta-spectrin repeats comprise the minimal, phased region of beta-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.     

Interaction between the N-terminal domain of human DNA topoisomerase I and the arginine-serine domain of its substrate determines phosphorylation of SF2/ASF splicing factor.

Human DNA topoisomerase I, known for its DNA-relaxing activity, is possibly one of the kinases phosphorylating members of the SR protein family of splicing factors, in vivo. Little is known about the mechanism of action of this novel kinase. Using the prototypical SR protein SF2/ASF (SRp30a) as model substrate, we demonstrate that serine residues phosphorylated by topo I/kinase exclusively located within the most extended arginine-serine repeats of the SF2/ASF RS domain. Unlike other kinases such as cdc2 and SRPK1, which also phosphorylated serines at the RS domain, topo I/kinase required several SR dipeptide repeats. These repeats possibly contribute to a versatile structure in the RS domain thereby facilitating phosphorylation. Furthermore, far-western, fluorescence spectroscopy and kinase assays using the SF2/ASF mutants, demonstrated that kinase activity and binding were tightly coupled. Since the deletion of N-terminal 174 amino acids of Topo I destroys SF2/ASF binding and kinase activity but not ATP binding, we conclude that at least two distinct domains of Topo I are necessary for kinase activity: one in the C-terminal region contributing to the ATP binding site and the other one in the N-terminal region that allows binding of SF2/ASF.     

Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.     

The role of Kalirin9 in p75/nogo receptor-mediated RhoA activation in cerebellar granule neurons

p75 and the Nogo receptor form a signaling unit for myelin inhibitory molecules, with p75 being responsible for RhoA activation. Because p75 lacks the GDP/GTP exchange factor domain, it has remained unclear how p75 activates RhoA. Here, we report that Kalirin9, a dual RhoGEF, binds p75 directly and regulates p75-Nogo receptor-dependent RhoA activation and neurite inhibition in response to myelin-associated glycoprotein. The region of p75 that Kalirin9 binds includes its mastoparan-like fifth helix, which was shown to recruit RhoGDI-RhoA. As predicted from the presence of a shared binding site, we found that Kalirin9 competes with RhoGDI for p75 binding in a dose-dependent manner in vitro. In line with these data, myelin-associated glycoprotein addition to cerebellar granule neurons resulted in a reduction in the association of Kalirin9 with p75, and a simultaneous increase in the binding of RhoGDI to p75. These results reveal a mechanism by which the fifth helix of p75 regulates RhoA activation.     

A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.