Naoko Iwaya

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.

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.

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 Ca2+ ions inhibit the MT‐severing activity of p60‐katanin, whereas the MT‐binding activity is preserved in the presence of Ca2+. In detail, the basal ATPase activity of p60‐katanin is stimulated twofold by both MTs and the C‐terminal domain of adaptor‐p80, whereas Ca2+ reduces elevated ATPase activity to the basal level. We identify the Ca2+‐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 Ca2+ regulates both severing and ATP hydrolysis activity, because the Ca2+‐binding site on the N‐terminal domain moves close to the AAA domain during MT severing.

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 15N, 13C, and 1H 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.