Natsuko Goda

Structure of the N-terminal Domain of PEX1 AAA-ATPase: CHARACTERIZATION OF A PUTATIVE ADAPTOR-BINDING DOMAIN

Peroxisomes are responsible for several pathways in primary metabolism, including β-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-Å resolution, which clearly demonstrated that the domain belongs to the double-ψ-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-Å root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly

Significance Alzheimer’s disease (AD) involves neuron dysfunction and loss. This brain damage is thought to be caused by a small protein, the amyloid β-protein (Aβ), which forms aggregates that are neurotoxic. This neurotoxicity has been explained by multiple mechanisms. We reveal here a new neurotoxic mechanism that involves the interaction between patient-derived Aβ assemblies, termed amylospheroids, and the neuron-specific Na+/K+-ATPase α3 subunit. This interaction causes neurodegeneration through pre-synaptic calcium overload, which explains earlier observations that such neuronal hyperactivation is an early indicator of AD-related neurodegeneration. Importantly, amylospheroid concentrations correlate with disease severity and progression in AD patients. Amylospheroid:neuron-specific Na+/K+-ATPase α3 subunit interactions may be a useful therapeutic target for AD. Neurodegeneration correlates with Alzheimer’s disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na+/K+-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ–derived “thorns” responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn879 and Trp880 is essential for ASPD–NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD–NAKα3 interaction.

The PRESAT‐vector: Asymmetric T‐vector for high‐throughput screening of soluble protein domains for structural proteomics

A rapid unidirectional method for cloning PCR‐amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT‐vector cloning, is based on a T‐vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5′‐sequence of the “rear” PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.

A common substrate recognition mode conserved between katanin P60 and VPS4 governs microtubule severing and membrane skeleton reorganization

Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.

The common phospholipid‐binding activity of the N‐terminal domains of PEX1 and VCP/p97

PEX1 is a type II AAA‐ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as β‐oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N‐terminal domain and those of other membrane‐related AAA‐ATPases, such as valosin‐containing protein (p97). The N‐terminal domain of valosine‐containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N‐terminal domain of PEX1 remains unknown. Here we found that N‐terminal domains isolated from valosine‐containing protein, as well as from PEX1, bind phosphoinositides. The N‐terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3‐monophosphate and phosphatidylinositol 4‐monophosphate, whereas the N‐terminal domain of valosine‐containing protein displays broad and nonspecific lipid binding. Although N‐ethylmaleimide‐sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine‐containing protein, they displayed lipid specificity similar to that of the N‐terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine‐containing protein.

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

Background: Hef/FANCM participates in interstrand cross-link DNA repair. Results: The predicted intrinsically disordered region (IDR) in Hef was experimentally verified. Proliferating cell nuclear antigen and a RecJ-like protein interact with the IDR individually, but not simultaneously. Conclusion: The IDR in Hef interacts with multiple proteins. Significance: Hef may function in DNA repair by association of its IDR with multiple partners. Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.

Structure of the N-terminal domain of PEX1 AAA-ATPase

Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-A resolution, which clearly demonstrated that the domain belongs to the double-psi-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-A root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.

Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.

Structural difference of vasoactive intestinal peptide in two distinct membrane mimicking environments

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide which belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. Knowledge for the conformation of VIP bound to membrane is important because the receptor activation is initiated by membrane binding of VIP. We have previously observed that VIP-G (glycine-extended VIP) is unstructured in solution, as evidenced by the limited NMR chemical shift dispersion. In this study, we determined the three-dimensional structures of VIP-G in two distinct membrane-mimicking environments. Although these are basically similar structures composed of a disordered N-terminal region and a long α-helix, micelle-bound VIP-G has a curved α-helix. The side chains of residues Phe(6), Tyr(10), Leu(13), and Met(17) found at the concave face form a hydrophobic patch in the micelle-bound state. The structural differences in two distinct membrane-mimicking environments show that the micelle-bound VIP-G localized at the water-micelle boundary with these side chains toward micelle interior. In micelle-bound PACAP-38 (one of the glucagon/secretin superfamily peptide) structure, the identical hydrophobic residues form the micelle-binding interface. This result suggests that these residues play an important role for the membrane binding of VIP and PACAP.

Structure and Function of the N-terminal Nucleolin Binding Domain of Nuclear Valosin-containing Protein-like 2 (NVL2) Harboring a Nucleolar Localization Signal

The N-terminal regions of AAA-ATPases (ATPase associated with various cellular activities) often contain a domain that defines the distinct functions of the enzymes, such as substrate specificity and subcellular localization. As described herein, we have determined the solution structure of an N-terminal unique domain isolated from nuclear valosin-containing protein (VCP)-like protein 2 (NVL2UD). NVL2UD contains three α helices with an organization resembling that of a winged helix motif, whereas a pair of β-strands is missing. The structure is unique and distinct from those of other known type II AAA-ATPases, such as VCP. Consequently, we identified nucleolin from a HeLa cell extract as a binding partner of this domain. Nucleolin contains a long (∼300 amino acids) intrinsically unstructured region, followed by the four tandem RNA recognition motifs and the C-terminal glycine/arginine-rich domain. Binding analyses revealed that NVL2UD potentially binds to any of the combinations of two successive RNA binding domains in the presence of RNA. Furthermore, NVL2UD has a characteristic loop, in which the key basic residues RRKR are exposed to the solvent at the edge of the molecule. The mutation study showed that these residues are necessary and sufficient for nucleolin-RNA complex binding as well as nucleolar localization. Based on the observations presented above, we propose that NVL2 serves as an unfoldase for the nucleolin-RNA complex. As inferred from its RNA dependence and its ATPase activity, NVL2 might facilitate the dissociation and recycling of nucleolin, thereby promoting efficient ribosome biogenesis.