Terukazu Nogi

Structural basis for recognition of acidic-cluster dileucine sequence by GGA1.

GGAs (Golgi-localizing, γ-adaptin ear homology domain, ARF-interacting proteins) are critical for the transport of soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes by means of interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF), and clathrin. The amino-terminal VHS domains of GGAs form complexes with the cytoplasmic domains of sorting receptors by recognizing acidic-cluster dileucine (ACLL) sequences. Here we report the X-ray structure of the GGA1 VHS domain alone, and in complex with the carboxy-terminal peptide of cation-independent mannose 6-phosphate receptor containing an ACLL sequence. The VHS domain forms a super helix with eight α-helices, similar to the VHS domains of TOM1 and Hrs. Unidirectional movements of helices α6 and α8, and some of their side chains, create a set of electrostatic and hydrophobic interactions for correct recognition of the ACLL peptide. This recognition mechanism provides the basis for regulation of protein transport from the TGN to endosomes/lysosomes, which is shared by sortilin and low-density lipoprotein receptor-related protein.

Crystal structure of α5β1 integrin ectodomain: Atomic details of the fibronectin receptor

The crystal structure of the α5β1 integrin reveals conformational changes and amino acids important for ligand binding.

Molecular Mechanism of Membrane Recruitment of Gga by Arf in Lysosomal Protein Transport

GGAs are critical for trafficking soluble proteins from the trans-Golgi network (TGN) to endosomes/lysosomes through interactions with TGN-sorting receptors, ADP-ribosylation factor (ARF) and clathrin. ARF–GTP bound to TGN membranes recruits its effector GGA by binding to the GAT domain, thus facilitating recognition of GGA for cargo-loaded receptors. Here we report the X-ray crystal structures of the human GGA1-GAT domain and the complex between ARF1–GTP and the N-terminal region of the GAT domain. When unbound, the GAT domain forms an elongated bundle of three a-helices with a hydrophobic core. Structurally, this domain, combined with the preceding VHS domain, resembles CALM, an AP180 homolog involved in endocytosis. In the complex with ARF1–GTP, a helix-loop-helix of the N-terminal part of GGA1-GAT interacts with the switches 1 and 2 of ARF1 predominantly in a hydrophobic manner. These data reveal a molecular mechanism underlying membrane recruitment of adaptor proteins by ARF–GTP.

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.

Structural basis for the accessory protein recruitment by the γ -adaptin ear domain

The adaptor proteins AP-1 and GGA regulate membrane traffic between the trans-Golgi network (TGN) and endosomes/lysosomes through ARF-regulated membrane association, recognition of sorting signals, and recruitment of clathrin and accessory proteins. The γ1-adaptin subunits of AP-1 and GGA possess homologous ear domains involved in the recruitment of accessory proteins, γ-synergin and Rabaptin-5. The crystal structure of the human γ1-adaptin ear domain consists solely of an immunoglobulin-like fold, unlike the α-adaptin ear domain. Structure-based mutational analyses reveal a binding site for the accessory proteins that is composed of conserved basic residues, indicating that the recruitment mechanism in γ1-adaptin and GGA is distinct from that in α-adaptin.

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.

Higher-Order Architecture of Cell Adhesion Mediated by Polymorphic Synaptic Adhesion Molecules Neurexin and Neuroligin

Polymorphic adhesion molecules neurexin and neuroligin (NL) mediate asymmetric trans-synaptic adhesion, which is crucial for synapse development and function. It is not known whether or how individual synapse function is controlled by the interactions between variants and isoforms of these molecules with differing ectodomain regions. At a physiological concentration of Ca(2+), the ectodomain complex of neurexin-1 β isoform (Nrx1β) and NL1 spontaneously assembled into crystals of a lateral sheet-like superstructure topologically compatible with transcellular adhesion. Correlative light-electron microscopy confirmed extracellular sheet formation at the junctions between Nrx1β- and NL1-expressing non-neuronal cells, mimicking the close, parallel synaptic membrane apposition. The same NL1-expressing cells, however, did not form this higher-order architecture with cells expressing the much longer neurexin-1 α isoform, suggesting a functional discrimination mechanism between synaptic contacts made by different isoforms of neurexin variants.

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.

Ultrahigh-resolution structure of high-potential iron-sulfur protein from Thermochromatium tepidum.

Crystals of the high-potential iron-sulfur protein (HiPIP) from Thermochromatium tepidum diffract X-rays to 0.80 A using synchrotron radiation at 100 K. The crystal structure of this HiPIP was refined at this ultrahigh resolution with anisotropic temperature factors for all atoms to conventional crystallographic R factors of 0.092 and 0.101 for F(o) > 4sigma(F(o)) and all reflections, respectively. The present structure provides a more precise picture than the previous 1.5 A structure and allows location of the positions of most H atoms. The structure revealed a partly hydrophobic cavity near the main hydrophobic area and a much larger inter-cluster approach distance (23.454 A, the c constant of the unit cell) in the crystal packing than other types of HiPIPs. The structural features involved in the electron-transfer reaction of HiPIP are discussed.

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.