Regulation of neurexin 1beta tertiary structure and ligand binding through alternative splicing

Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a "splice-insert signaling code." In particular, neurexin 1beta carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1beta+SS#4 reveals dramatic rearrangements to the "hypervariable surface," the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop beta10-beta11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca(2+)-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1beta isoforms acquire neuroligin splice isoform selectivity.     

Modulation of agrin function by alternative splicing and Ca2+ binding

The aggregation of acetylcholine receptors on postsynaptic membranes is a key step in neuromuscular junction development. This process depends on alternatively spliced forms of the proteoglycan agrin with "B-inserts" of 8, 11, or 19 residues in the protein's globular C-terminal domain, G3. Structures of the neural B8 and B11 forms of agrin-G3 were determined by X-ray crystallography. The structure of G3-B0, which lacks inserts, was determined by NMR. The agrin-G3 domain adopts a beta jellyroll fold. The B insert site is flanked by four loops on one edge of the beta sandwich. The loops form a surface that corresponds to a versatile interaction interface in the family of structurally related LNS proteins. NMR and X-ray data indicate that this interaction interface is flexible in agrin-G3 and that flexibility is reduced by Ca(2+) binding. The plasticity of the interaction interface could enable different splice forms of agrin to select between multiple binding partners.     

Crystal structure of Mg2+- and Ca2+-bound Gla domain of factor IX complexed with binding protein

Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a gamma-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla domain (IXGD1-46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1-46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1-46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1-46 and IX-bp, forced IXGD1-46 to rotate 4 degrees relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.     

Calmodulin binding to the C-terminus of the small-conductance Ca2+-activated K+ channel hSK1 is affected by alternative splicing

We identified three splice variants of hSK1 whose C-terminal structures are determined by the independent deletion of two contiguous nucleotide sequences. The upstream sequence extends 25 bases in length, is initiated by a donor splice site within exon 8, and terminates at the end of the exon. The downstream sequence consists of nine bases that compose exon 9. When the upstream sequence (hSK1(-)(25b)) or both sequences (hSK1(-)(34b)) are deleted, truncated proteins are encoded in which the terminal 118 amino acids are absent. The binding of calmodulin to these variants is diminished, particularly in the absence of Ca2+ ions. The first 20 amino acids of the segment deleted from hSK1(-)(25b) and hSK1(-)(34b) contain a 1-8-14 Ca2+ calmodulin binding motif, and synthetic oligopeptides based on this region bind calmodulin better in the presence than absence of Ca2+ ions. When the downstream sequence (hSK1(-)(9b)) alone is deleted, only the three amino acids A452, Q453, and K454 are removed, and calmodulin binding is not reduced. On the basis of the relative abundance of mRNA encoding each of the four isoforms, the full-length variant appears to account for most hSK1 in the human hippocampus, while hSK1(-)(34b) predominates in reticulocytes, and hSK1(-)(9b) is especially abundant in human erythroleukemia cells in culture. We conclude that the binding of calmodulin by hSK1 can be modulated through alternative splicing.     

The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1.

The structure of Ca-prothrombin fragment 1 (residues 1-156 prothrombin) has been solved and refined at 2.2-A resolution by X-ray crystallographic methods. The first two-thirds of the Gla domain (residues 1-48) and two carbohydrate chains (approximately 5 kDa) are disordered in crystals of apo-fragment 1. When crystals are grown in the presence of Ca2+ ions, the Gla domain exhibits a well-defined structure binding seven Ca2+ ions, but the carbohydrate is still disordered. Even so, the crystallographic R factor reduced to 0.171. The folding of the Gla domain is dominated by 9-10 turns of three different alpha-helices. These turns produce two internal carboxylate surfaces composed of Gla side chains. A polymeric array of five Ca2+ ions separated by about 4.0 A intercalates between the carboxylate surfaces. The coordination of the Ca2+ ions with Gla carboxylate oxygen atoms and water molecules leads to distorted polyhedral arrangements with mu-oxo bridges in a highly complex array that most likely orchestrates the folding of the domain. The overall mode of interaction of the Ca2+ ions is new and different from any Ca2+ ion-protein interactions heretofore observed or described. The fluorescence quenching event observed upon Ca2+ ion binding is due to a disulfide-pi-electron interaction that causes a 100 degrees reorientation of Trp42 of the Gla domain. The Ca2+ ion interaction also affords the N-terminus protection from acetylation because the latter is buried in the folded structure and makes hydrogen-bonding salt bridges with Gla17, Gla21, and Gla27. The Gla domain and its trailing disulfide unit associate intimately and together give rise to a domain-like structure. Electrostatic potential calculations indicate that the Gla domain is very electronegative. Since most of the carboxylate oxygen atoms of Gla residues are involved in Ca2+ ion binding, leaving only a few for bridging Ca2+ ion-phospholipid interactions, the role of bridging Ca2+ ions might be generally unspecific, with Ca2+ ions simply intervening between the negative Gla domain and negative head groups of the membrane surface. The folding of the kringle structure in apo- and Ca-fragment 1 is essentially the same. However, the Ser36-Ala47 helix of the Gla domain pivots around Cys48, shifting by approximately 30 degrees, and the helix encroaches on the kringle producing some concomitant changes. These might be related to the protection of carbohydrate carrying Asn101 from acetylation in the Ca-fragment 1 structure.     

Structure and evolution of neurexin genes: insight into the mechanism of alternative splicing

Neurexins are neuron-specific vertebrate proteins with hundreds of differentially spliced isoforms that may function in synapse organization. We now show that Drosophila melanogaster and Caenorhabditis elegans express a single gene encoding only an alpha-neurexin, whereas humans and mice express three genes, each of which encodes alpha- and beta-neurexins transcribed from separate promoters. The neurexin genes are very large (up to 1.62 Mb), with the neurexin-3 gene occupying almost 2% of human chromosome 14. Although invertebrate and vertebrate neurexins exhibit a high degree of evolutionary conservation, only vertebrate neurexins are subject to extensive alternative splicing that uses mechanisms ranging from strings of mini-exons to multiple alternative splice donor and acceptor sites. Consistent with their proposed role in synapse specification, neurexins thus have evolved from relatively simple genes in invertebrates to diversified genes in vertebrates with multiple promoters and extensive alternative splicing.     

Crystal structure of the N-terminal NC4 domain of collagen IX, a zinc binding member of the laminin-neurexin-sex hormone binding globulin (LNS) domain family

Collagen IX, located on the surface of collagen fibrils, is crucial for cartilage integrity and stability. The N-terminal NC4 domain of the alpha1(IX) chain is probably important in this because it interacts with various macromolecules such as proteoglycans and cartilage oligomeric matrix protein. At least 17 distinct collagen polypeptides carry an NC4-like unit near their N terminus, but this report, describing the crystal structure of NC4 at 1.8-A resolution, represents the first atomic level structure for these domains. The structure is similar to previously characterized laminin-neurexin-sex hormone binding globulin (LNS) structures, dominated by an antiparallel beta-sheet sandwich. In addition, a zinc ion was found in a position similar to that of the metal binding site of other LNS domains. A partial backbone NMR assignment of NC4 was obtained and utilized in NMR titration studies to investigate the zinc binding in solution state and to quantitate the affinity of metal binding. The K(d) of 11.5 mM suggests a regulatory rather than a structural role for zinc in solution. NMR titration with a heparin tetrasaccharide revealed the presence of a secondary binding site for heparin on NC4, showing structural and functional conservation with thrombospondin-1, but a markedly reduced affinity for the ligand. Also the overall arrangement of the N and C termini of NC4 resembles most closely the N-terminal domain of thrombospondin-1, distinguishing the two from the majority of the published LNS structures.     

Ca2+ binding effects on the C2 domain conformation of human cytosolic phospholipase A2

It has been reported that the cooperative binding of calcium ions indicated a local conformational change of the human cytosolic phospholipase A2 (cPLA2) C2 domain (Nalefski et al., (1997) Biochemistry 36, 12011-12018). However its structural evidence is less known (Malmberg et al., (2003) Biochemistry 42, 13227-13240). In this letter, life-time decay and fluorescence quenching techniques were employed to compare the calcium-induced conformational changes. The life-time decay parameters and fluorescence quenching constant changes were small between the apo- and holo-C2 domains when tryptophan residue was excited at 295 nm. In contrast, the quenching constant change was large, from 0.52 M(-1) for the apo-C2 to 8.8 M(-1) for the holo-C2 domain, when tyrosine residues were excited at 284 nm. Our results provide new information on amino acid side chain orientation change at calcium binding loop 3, which is necessary for Ca2+ binding regulated membrane targeting of human cytosolic phospholipase A2.     

Crystal structure and cell surface anchorage sites of laminin alpha1LG4-5

The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin alpha1LG4-5 domains and provide a mutational analysis of heparin, alpha-dystroglycan, and galactosylsulfatide binding. The two domains of alpha1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin alpha2 LG4-5 but with a substantially different interdomain angle. Recombinant alpha1LG4-5 binding to heparin, alpha-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to alpha-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands.     

Analysis of the human neurexin genes: alternative splicing and the generation of protein diversity

The neurexins are neuronal proteins that function as cell adhesion molecules during synaptogenesis and in intercellular signaling. Although mammalian genomes contain only three neurexin genes, thousands of neurexin isoforms may be expressed through the use of two alternative promoters and alternative splicing at up to five different positions in the pre-mRNA. To begin understanding how the expression of the neurexin genes is regulated, we have determined the complete nucleotide sequence of all three human neurexin genes: NRXN1, NRXN2, and NRXN3. Unexpectedly, two of these, NRXN1 ( approximately 1.1 Mb) and NRXN3 ( approximately 1.7 Mb), are among the largest known human genes. In addition, we have identified several conserved intronic sequence elements that may participate in the regulation of alternative splicing. The sequences of these genes provide insight into the mechanisms used to generate the diversity of neurexin protein isoforms and raise several interesting questions regarding the expression mechanism of large genes.     

Crystal structure of the RIM1alpha C2B domain at 1.7 A resolution

RIM proteins play critical roles in synaptic vesicle priming and diverse forms of presynaptic plasticity. The C-terminal C2B domain is the only module that is common to all RIMs but is only distantly related to well-studied C2 domains, and its three-dimensional structure and interactions have not been characterized in detail. Using NMR spectroscopy, we now show that N- and C-terminal extensions beyond the predicted C2B domain core sequence are necessary to form a folded, stable RIM1alpha C2B domain. We also find that the isolated RIM1alpha C2B domain is not sufficient for previously described protein-protein interactions involving the RIM1alpha C-terminus, suggesting that additional sequences adjacent to the C2B domain might be required for these interactions. However, analytical ultracentrifugation shows that the RIM1alpha C2B domain forms weak dimers in solution. The crystal structure of the RIM1alpha C2B domain dimer at 1.7 A resolution reveals that it forms a beta-sandwich characteristic of C2 domains and that the unique N- and C-terminal extensions form a small subdomain that packs against the beta-sandwich and mediates dimerization. Our results provide a structural basis to understand the function of RIM C2B domains and suggest that dimerization may be a crucial aspect of RIM function.     

Crystal structure of the ligand binding domain of netrin G2

Netrin G proteins represent a small family of synaptic cell adhesion molecules related to netrins and to the polymerization domains of laminins. Two netrin G proteins are encoded in vertebrate genomes, netrins G1 and G2, which are known to bind the leucine-rich repeat proteins netrin G ligand (NGL)-1 and NGL-2, respectively. Netrin G proteins share a common multi-domain architecture comprising a laminin N-terminal (LN) domain followed by three laminin epidermal growth factor-like (LE) domains and a C′ region containing a glycosylphosphatidylinositol anchor. Here, we use deletion analysis to show that the LN domain region of netrin Gs contains the binding site for NGLs to which they bind with 1:1 stoichiometry and sub-micromolar affinity. Netrin Gs are alternatively spliced in their LE domain regions, but the binding region, the LN domain, is identical in all splice forms. We determined the crystal structure for a fragment comprising the LN domain and domain LE1 of netrin G2 by sulfur single-wavelength anomalous diffraction phasing and refined it to 1.8 Å resolution. The structure reveals an overall architecture similar to that of laminin α chain LN domains but includes significant differences including a Ca²⁺ binding site in the LN domain. These results reveal the minimal binding unit for interaction of netrin Gs with NGLs, define structural features specific to netrin Gs, and suggest that netrin G alternative splicing is not involved in NGL recognition.     

Auxiliary Ca2+ binding sites can influence the structure of CIB1

Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca²⁺ ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (K_d, 10⁻²M) Ca²⁺ binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca²⁺ crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca²⁺ binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca²⁺ concentrations.     

Common domain structure of Ca2+ and lipid-binding proteins

The phospholipase A2 inhibitor, lipocortin, shares common sequences with three abundant Ca2+-regulated membrane binding proteins of unknown function which are present in many cell and tissue types. A two-domain model for the structure of lipocortin is described and it is suggested that the new Ca2+-regulated proteins each contain at least one lipocortin domain. The structural and biochemical properties of each protein indicate that they all directly interact with phospholipids. Potential sites of interaction with the lipocortin domain are identified on the basis of homology with phospholipid transfer proteins and phospholipase A2.     

Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS.     

Crystal structure of the LG3 domain of endorepellin, an angiogenesis inhibitor

Endorepellin, the C-terminal region of perlecan, inhibits angiogenesis by disrupting actin cytoskeleton and focal adhesions. The C-terminal laminin-like globular domain (LG3) of endorepellin directs most of this antiangiogenic activity. To investigate the angiostatic mechanism and to identify structural determinants, we have solved crystal structures of the LG3 domain in both apo- and calcium-bound forms at resolutions of 1.5 Å and 2.8 Å, respectively. The conserved core has the jellyroll fold characteristic of LG domains. The calcium-induced structural changes seem very restricted, and the calcium binding site appears to be preformed, suggesting that the bound calcium ion, rather than structural rearrangements, contributes to antiangiogenesis. We have identified H4268 on the EF loop as a key residue for the biochemical function of LG3, since its mutation abolishes antiangiogenic activity, and mutant LG3 can no longer form a direct interaction with integrin. Taken together, we propose that these two distinct structural elements contribute to the angiostatic effect of endorepellin.     

The cPLA2 C2alpha domain in solution: structure and dynamics of its Ca2+-activated and cation-free states

Cytosolic phospholipase A2 is involved in several signal transduction pathways where it catalyses release of arachidonic acid from intracellular lipid membranes. Its membrane insertion is facilitated by its independently folding C2alpha domain, which is activated by the binding of two intracellular Ca2+ ions. However, the details of its membrane-insertion mechanism, including its Ca2+-activation mechanism, are not understood. There are several unresolved issues, including the following. There are two experimentally resolved structures of the Ca2+-activated state of its isolated C2alpha domain, one determined using x-ray crystallography and the other determined using NMR spectroscopy, which differ from each other significantly in the spatial region that inserts into the membrane. This by itself adds to ambiguities associated with investigations targeting its mechanism of membrane insertion. Furthermore, there is no experimentally determined structure of its cation-free state, which hinders investigations associated with its cation-activation mechanism. In this work, we generate several unrestrained molecular dynamics trajectories of its isolated C2alpha domain in solution (equivalent to approximately 60 ns) and investigate these issues. Our main results are as follows: a), the Ca2+ coordination scheme of the domain is consistent with the x-ray structure and with previous mutagenesis studies; b), the helical segment of the Ca2+-binding loop, CBL-I, undergoes nanosecond timescale flexing (but not an unwinding), as can be inferred from physiological temperature NMR data and in contrast to low temperature x-ray data; and c), removal of the two activating Ca2+ ions from their binding pockets does not alter the backbone structure of the domain, a result consistent with electron paramagnetic resonance data.