Principal component analysis of data from NMR titration experiment of uniformly 15N labeled amyloid beta (1-42) peptide with osmolytes and phenolic compounds.

A simple NMR method to analyze the data obtained by NMR titration experiment of amyloid formation inhibitors against uniformly 15N-labeled amyloid-ß 1-42 peptide (Aß(1-42)) was described. By using solution nuclear magnetic resonance (NMR) measurement, the simplest method for monitoring the effects of Aß fibrilization inhibitors is the NMR chemical shift perturbation (CSP) experiment using 15N-labeled Aß(1-42). However, the flexible and dynamic nature of Aß(1-42) monomer may hamper the interpretation of CSP data. Here we introduced principal component analysis (PCA) for visualizing and analyzing NMR data of Aß(1-42) in the presence of amyloid inhibitors including high concentration osmolytes. We measured 1H-15N 2D spectra of Aß(1-42) at various temperatures as well as of Aß(1-42) with several inhibitors, and subjected all the data to PCA (PCA-HSQC). The PCA diagram succeeded in differentiating the various amyloid inhibitors, including epigallocatechin gallate (EGCg), rosmarinic acid (RA) and curcumin (CUR) from high concentration osmolytes. We hypothesized that the CSPs reflected the conformational equilibrium of intrinsically disordered Aß(1-42) induced by weak inhibitor binding rather than the specific molecular interactions.

Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1-42 in 1,1,1,3,3,3-hexafluoro-2-propanol.

Alzheimer's disease involves accumulation of senile plaques in which filamentous aggregates of amyloid beta (Aß) peptides are deposited. Recent studies demonstrate that oligomerization pathways of Aß peptides may be complicated. To understand the mechanisms of Aß(1-42) oligomer formation in more detail, we have established a method to produce (15)N-labeled Aß(1-42) suited for nuclear magnetic resonance (NMR) studies. For physicochemical studies, the starting protein material should be solely monomeric and all Aß aggregates must be removed. Here, we succeeded in fractionating a "precipitation-resistant" fraction of Aß(1-42) from an "aggregation-prone" fraction by high-performance liquid chromatography (HPLC), even from bacterially overexpressed Aß(1-42). However, both Aß(1-42) fractions after 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) treatment formed amyloid fibrils. This indicates that the "aggregation seed" was not completely monomerized during HFIP treatment. In addition, Aß(1-42) dissolved in HFIP was found to display a monomer-dimer equilibrium, as shown by two-dimensional (1)H-(15)N NMR. We demonstrated that the initial concentration of Aß during the HFIP pretreatment altered the kinetic profiles of Aß fibril formation in a thioflavin T fluorescence assay. The findings described here should ensure reproducible results when studying the Aß(1-42) peptide.

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 (NVL2(UD)). NVL2(UD) contains three α helices with an organization resembling that of a winged helix motif, whereas a pair of ß-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 NVL2(UD) potentially binds to any of the combinations of two successive RNA binding domains in the presence of RNA. Furthermore, NVL2(UD) 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.

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.

LBT/PTD dual tagged vector for purification, cellular protein delivery and visualization in living cells.

Cellular protein delivery is an emerging technique, by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an E. coli expression vector suited for protein cellular delivery experiments. The plasmid is designed to generate a C-terminal fusion with the 12 amino acid HIV-Tat peptide as a protein transduction domain (PTD), whereas the protein N-terminus is fused to an 17-residue peptide lanthanide-binding tag (LBT). LBT is used for both purification by affinity chromatography and fluorescent detection with Tb(3+) as a coordinating metal. We have employed the TA-cloning site between the two tags, LBT and PTD, according to the PRESAT-vector methodology [N. Goda, T. Tenno, H. Takasu, H. Hiroaki, M. Shirakawa, The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics, Protein Sci. 13 (2004) 652-658], which facilitates unidirectional cloning of any PCR-amplified DNA fragments corresponding to the protein of interest. A simple three-step protocol consisting of affinity purification of LBT/PTD dual-tagged proteins has also been developed, in which the proteins are purified by heparin-, then immobilized Ni(2+)-, and then heparin-affinity chromatography, in this order. The purified protein is ready for protein delivery experiment, and the delivered protein is visible by fluorescent microscopy. Our LBT/PTD dual-tagged PRESAT-vector provides a powerful research tool for exploring cellular functions of proteins in the post-genomic era.

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

¹H, ¹³C, and ¹5N resonance assignment of the SPFH domain of human stomatin.

Stomatin, a 288-residue protein, is a component of the membrane skeleton of red blood cells (RBCs), which helps to physically support the membrane and maintains its function. In RBCs, stomatin binds to the glucose transporter GLUT-1 and may regulate its function. Stomatin has a stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of its polypeptide chain. There are 12 SPFH domain-containing proteins, most of which are localized at the cellular or subcellular membranes. Although the molecular function of the SPFH domain has not yet been established, the domain may be involved in protein oligomerization. The SPFH domain of the archaeal stomatin homolog has been shown to form unique oligomers. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the SPFH domain of human stomatin [hSTOM(SPFH)]. These may help in determining the structure of hSTOM(SPFH) in solution as well as in clarifying its involvement in protein oligomerization.

Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism.

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.

Fine-tuning of protein domain boundary by minimizing potential coiled coil regions.

Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in (1)H-(15)N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer-dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by (1)H-(15)N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies.