Norihisa Yasui

Structural basis for semaphorin signalling through the plexin receptor

Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexin’s intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour. However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected ‘face-to-face’ homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected ‘head-on’ homodimer arrangement. In contrast, the structure of the Sema6A–PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo ‘partner exchange’. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding ‘signal’ is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.

Structure of a receptor-binding fragment of reelin and mutational analysis reveal a recognition mechanism similar to endocytic receptors

Reelin, a large secreted protein implicated in the cortical development of the mammalian brain, is composed of eight tandem concatenations of “reelin repeats” and binds to neuronal receptors belonging to the low-density lipoprotein receptor gene family. We found that both receptor-binding and subsequent Dab1 phosphorylation occur solely in the segment spanning the fifth and sixth reelin repeats (R5–6). Monomeric fragment exhibited a suboptimal level of signaling activity and artificial oligomerization resulted in a 10-fold increase in activity, indicating the critical importance of higher-order multimerization in physiological reelin. A 2.0-Å crystal structure from the R5–6 fragment revealed not only a unique domain arrangement wherein two repeats were aligned side by side with the same orientation, but also the unexpected presence of bound Zn ions. Structure-guided alanine mutagenesis of R5–6 revealed that two Lys residues (Lys-2360 and Lys-2467) constitute a central binding site for the low-density lipoprotein receptor class A module in the receptor, indicating a strong similarity to the ligand recognition mode shared among the endocytic lipoprotein receptors.

Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains

Significance Protein–protein interactions are essential for cellular regulation, but how changes in individual interactions influence cellular physiology or cause disease remains poorly characterized. Although selective and potent inhibitors of protein–protein interactions are powerful tools, developing such reagents is challenging. This is because signaling networks are composed of members of highly conserved protein domain families, with the Src-homology 2 (SH2) domain as an archetype. To address this challenge, we used protein design to successfully generate a set of reagents, termed monobodies, directed to the SH2 domains of SH2 domain-containing phosphatase 2 (SHP2), for which no specific inhibitors had been identified. These monobodies selectively and potently inhibit SHP2 function and demonstrate utility in dissecting the signaling networks of cancer cells. The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2–phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein–protein interactions in cancer cells.

Broad Ranges of Affinity and Specificity of Anti-Histone Antibodies Revealed by a Quantitative Peptide Immunoprecipitation Assay

Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method, we identified additional factors potentially affecting the interpretation of ChIP experiments.

Structure of a signaling-competent reelin fragment revealed by X-ray crystallography and electron tomography.

The large extracellular glycoprotein reelin directs neuronal migration during brain development and plays a fundamental role in layer formation. It is composed of eight tandem repeats of an ∼380‐residue unit, termed the reelin repeat, which has a central epidermal growth factor (EGF) module flanked by two homologous subrepeats with no obvious sequence similarity to proteins of known structure. The 2.05 Å crystal structure of the mouse reelin repeat 3 reveals that the subrepeat assumes a β‐jelly‐roll fold with unexpected structural similarity to carbohydrate‐binding domains. Despite the interruption by the EGF module, the two subdomains make direct contact, resulting in a compact overall structure. Electron micrographs of a four‐domain fragment encompassing repeats 3–6, which is capable of inducing Disabled‐1 phosphorylation in neurons, show a rod‐like shape. Furthermore, a three‐dimensional molecular envelope of the fragment obtained by single‐particle tomography can be fitted with four concatenated repeat 3 atomic structures, providing the first glimpse of the structural unit for this important signaling molecule.

Novel affinity tag system using structurally defined antibody-tag interaction: application to single-step protein purification

Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti‐peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18‐residue affinity tag with ideal solution binding kinetics, providing a low‐cost purification means when combined with nondenaturing elution by water‐miscible organic solvents. Three‐dimensional information provides a firm structural basis for the antibody–peptide interaction, opening opportunities for further improvements/modifications.

Structural Basis for Variant-Specific Neuroligin-Binding by α-Neurexin

Neurexins (Nrxs) are presynaptic membrane proteins with a single membrane-spanning domain that mediate asymmetric trans-synaptic cell adhesion by binding to their postsynaptic receptor neuroligins. α-Nrx has a large extracellular region comprised of multiple copies of laminin, neurexin, sex-hormone-binding globulin (LNS) domains and epidermal growth factor (EGF) modules, while that of β-Nrx has but a single LNS domain. It has long been known that the larger α-Nrx and the shorter β-Nrx show distinct binding behaviors toward different isoforms/variants of neuroligins, although the underlying mechanism has yet to be elucidated. Here, we describe the crystal structure of a fragment corresponding to the C-terminal one-third of the Nrx1α ectodomain, consisting of LNS5-EGF3-LNS6. The 2.3 Å-resolution structure revealed the presence of a domain configuration that was rigidified by inter-domain contacts, as opposed to the more common flexible “beads-on-a-string” arrangement. Although the neuroligin-binding site on the LNS6 domain was completely exposed, the location of the α-Nrx specific LNS5-EGF3 segment proved incompatible with the loop segment inserted in the B+ neuroligin variant, which explains the variant-specific neuroligin recognition capability observed in α-Nrx. This, combined with a low-resolution molecular envelope obtained by a single particle reconstruction performed on negatively stained full-length Nrx1α sample, allowed us to derive a structural model of the α-Nrx ectodomain. This model will help us understand not only how the large α-Nrx ectodomain is accommodated in the synaptic cleft, but also how the trans-synaptic adhesion mediated by α- and β-Nrxs could differentially affect synaptic structure and function.

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region

Background: Reelin is a large glycoprotein critical in brain development and functions as a form of multimer. Results: Disulfide-bonded homodimer through Cys2101 is the functional unit of biologically active reelin protein. Conclusion: An intact higher order architecture of reelin multimer is essential for exerting its full biological activity. Significance: Ultrastructural and biochemical characterization of gigantic reelin protein is crucial for mechanistic understanding of reelin signaling. Reelin is a 3461-residue secreted glycoprotein that plays a critical role in brain development through its action on target neurons. Although it is known that functional reelin protein exists as multimer formed by interchain disulfide bond(s) as well as through non-covalent interactions, the chemical nature of the multimer assembly has been elusive. In the present study, we identified, among 122 cysteines present in full-length reelin, the single critical cysteine residue (Cys2101) responsible for the covalent multimerization. C2101A mutant reelin failed to assemble into disulfide-bonded multimers, whereas it still exhibited non-covalently associated high molecular weight oligomeric states in solution. Detailed analysis of tryptic fragments produced from the purified reelin proteins revealed that the minimum unit of the multimer is a homodimeric reelin linked via Cys2101 present in the central region and that this cysteine does not connect to the N-terminal region of reelin, which had been postulated as the primary oligomerization domain. A surface plasmon resonance binding assay confirmed that C2101A mutant reelin retained binding capability toward two neuronal receptors apolipoprotein E receptor 2 and very low density lipoprotein receptor. However, it failed to show signaling activity in the assay using the cultured neurons. These results indicate that an intact higher order architecture of reelin multimer maintained by both Cys2101-mediated homodimerization and other non-covalent association present elsewhere in the reelin primary structure are essential for exerting its full biological activity.

Detection of endogenous LRP6 expressed on human cells by monoclonal antibodies specific for the native conformation

LRP6 is a cell surface molecule that plays a critical role in the Wnt signaling pathway, and is implicated in numerous human diseases. Studies of cellular signaling mediated by LRP6 have relied on overexpression experiments, due to the lack of good monoclonal antibodies (mAbs) reactive with native LRP6 ectodomain. By using native recombinant LRP6 ectodomain fragment produced in mammalian expression system, we succeeded in developing a panel of anti-human LRP6 mAbs. Selected mAbs were capable of staining endogenous LRP6 on cell surface by using flow cytometry and immunofluorescence microscopy, and enriching detergent-solubilized LRP6 from cell lysate by immunoprecipitation.

Structure of the F-spondin reeler domain reveals a unique β-sandwich fold with a deformable disulfide-bonded loop

F-spondin is a secreted and extracellular matrix-attached protein that has been implicated in axonal pathfinding during neural development as well as in vascular remodelling in adult tissues. F-spondin is composed of a reeler, a spondin and six thrombospondin type 1 repeat domains. The reeler domain shares homology with the amino-terminal domain of reelin, a large secreted glycoprotein that guides migrating neurons during cortical development. Crystal structures of the F-spondin reeler domain were determined at 1.45 and 2.70 A resolution. The structure revealed a nine-stranded antiparallel beta-sandwich fold similar to the immunoglobulin or fibronectin type III domains, but with a unique extra beta-hairpin. Moreover, an amino-terminal extension which is anchored at its beginning via a conserved disulfide bond loosely packs against one face of the beta-sandwich, making a major contribution to the surface features of the domain. Structural comparison among the different molecules contained in two different crystals reveals an unusual conformational plasticity of the amino-terminal loop, suggesting its role in molecular interactions.