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4Ac6f0708394e259036cf237a15 International Journal of Molecular Sciences Article Expression, Functional Characterization, and Preliminary Crystallization of the Cochaperone Prefoldin from the Thermophilic Fungus Chaetomium thermophilum Kento Morita 1, Yohei Y. Yamamoto 1, Ayaka Hori 1, Tomohiro Obata 1, Yuko Uno 1, Kyosuke Shinohara 1, Keiichi Noguchi 2, Kentaro Noi 3,4, Teru Ogura 3,4, Kentaro Ishii 5, Koichi Kato 5 ID , Mahito Kikumoto 6, Rocio Arranz 7, Jose M. Valpuesta 7 ID and Masafumi Yohda 1,* 1 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Naka, Koganei, Tokyo 184-8588, Japan; [email protected] (K.M.); [email protected] (Y.Y.Y.); [email protected] (A.H.); [email protected] (T.O.); [email protected] (Y.U.); [email protected] (K.S.) 2 Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, Naka, Koganei, Tokyo 184-8588, Japan; [email protected] 3 Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.; [email protected] (K.N.); [email protected] (T.O.) 4 Core Research for Evolutional Science and Technology (CREST), JST, Kawaguchi, Saitama 332-0012, Japan 5 Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Myodaiji, Okazaki 444-8787, Japan; [email protected] (K.I.); [email protected] (K.K.) 6 Structural Biology Research Center, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-8601, Japan; [email protected] 7 Departamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología (CNB-CSIC), Madrid 28049, Spain; [email protected] (R.A.); [email protected] (J.M.V.) * Correspondence: [email protected]; Tel.: +81-42-388-7479 Received: 4 June 2018; Accepted: 15 August 2018; Published: 19 August 2018 Abstract: Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin–chaperonin system. Keywords: chaperone; chaperonin; Chaetomium thermophilum; proteostasis; interaction; folding Int. J. Mol. Sci. 2018, 19, 2452; doi:10.3390/ijms19082452 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 2452 2 of 13 1. Introduction ProteinInt. J. Mol. homeostasisSci. 2018, 19, x FOR is PEER maintained REVIEW by molecular chaperones [1]. Most molecular chaperones2 of 13 are heat shock proteins (Hsps) that are expressed under stress conditions to protect proteins from thermal 1. Introduction denaturation. Molecular chaperones recognize the hydrophobic surface of unfolded or misfolded polypeptides,Protein and homeostasis induce correct is maintained folding or by facilitate molecular degradation. chaperones [1]. Chaperonins, Most molecular also chaperones known as are Hsp60s, are theheat most shock important proteins (Hsps) and ubiquitous that are expressed chaperones. under Chaperoninsstress conditions exist to protect as two proteins stacked from rings, thermal which are composeddenaturation. of 7–9 subunitsMolecular of chaperones approximately recogn 60ize kDa the hydrophobic each. Chaperonin surface captures of unfolded an unfoldedor misfolded protein polypeptides, and induce correct folding or facilitate degradation. Chaperonins, also known as in the cavity of each ring and mediates protein folding in an ATP-dependent fashion. Chaperonins Hsp60s, are the most important and ubiquitous chaperones. Chaperonins exist as two stacked rings, are subdividedwhich are composed in two families, of 7–9 subunits group of I andapproximately group II chaperonins 60 kDa each. Chaperonin [2,3]. Group captures I chaperonins an unfolded exist in eubacteriaprotein and in eukaryoticthe cavity of organelles, each ring suchand mediates as mitochondria protein folding and chloroplasts. in an ATP-dependent Group II chaperoninsfashion. are foundChaperonins in archaea are andsubdivided eukaryotic in two cytosol. families, group I and group II chaperonins [2,3]. Group I Prefoldinchaperonins (PFD) exist in is eubacteria a cochaperone and eukaryotic of group organelles, II chaperonin. such as PFD, mitochondria originally and discovered chloroplasts. when screeningGroup yeast II chaperonins genes, is involvedare found inin microtubulearchaea and eukaryotic formation cytosol. and acts in an actin-folding complex [4,5], and is alsoPrefoldin found in (PFD) archaea. is a Archaealcochaperone PFDs of aregroup hexamers II chaperonin. composed PFD, of originally two types discovered of subunits: when Two α subunitsscreening and four yeastβ genes,subunits. is involved Archaeal in PFDsmicrotubule are molecular formation chaperones and acts in capable an actin of-folding stabilizing complex a range of nonnative[4,5], and proteins, is also found and in releasing archaea. themArchaeal for PFDs subsequent are hexamers group compos II chaperonin-assisteded of two types of subunits: folding [ 6–8]. Two α subunits and four β subunits. Archaeal PFDs are molecular chaperones capable of stabilizing Eukaryotic PFD interacts specifically and transfers substrates to a eukaryotic group II chaperonin a range of nonnative proteins, and releasing them for subsequent group II chaperonin-assisted (chaperoninfolding [6 containing–8]. Eukaryotic TCP-1 PFD (CCT); interacts also specifically called TCP-1 and transfers ring complex substrates (TRiC)). to a eukaryotic Eukaryotic group PFDs II are composedchaperonin of six (chaperonin different subunits: containing two TCPα-1-like (CCT); subunits also called (PFD3 TCP- and1 ring PFD5) complex and (TRiC)). four β Eukaryotic-like subunits (PFD1,PFDs PFD2, are PFD4,composed and of PFD6). six different subunits: two α-like subunits (PFD3 and PFD5) and four β-like Thesubunits crystal (PFD1, structures PFD2, PFD4, of archaeal and PFD6). PFDs from Methanobacterium thermoautotrophicum and Pyrococcus horikoshii OT3The havecrystal been structures determined of archaeal to 2.3 andPFDs 3.0 from Å resolution, Methanobacterium respectively thermoautotrophicum [9,10]. PFD resembles and a jellyfish:Pyrococcus Its body horikoshii consists OT3 of ahave double-barrel been determined assembly to 2.3 with and six 3.0 longÅ resolution, tentacle-like respectively coiled coils [9,10]. protruding PFD fromresembles it (Figure a1 ).jellyfish: Each of Its the bodyα consistsand β subunits of a double contains-barrel assembly two or one with central six longβ -hairpin,tentacle-like respectively; coiled thesecoils are N-protruding and C-terminally from it (Figure flanked 1). Each by of coiled-coil the α and β helices.subunits Thecontains coiled-coil two or one helices central of β each-hairpin, subunit respectively; these are N- and C-terminally flanked by coiled-coil helices. The coiled-coil helices of assemble into an antiparallel conformation. The detailed interaction mechanism between archaeal PFD each subunit assemble into an antiparallel conformation. The detailed interaction mechanism and groupbetween II chaperoninarchaeal PFD has and been group studied II chaperonin (Figure has1)[ been11–13 studied]. (Figure 1) [11–13]. FigureFigure 1. The 1. The schematic schematic model model for for thethe cooperation between between archaeal archaeal prefoldin prefoldin (PFD) (PFD) and group and groupII chaperonin. Crystal structures of Pyrococcus PFD (PDB ID: 2ZDI), and Methanococcus group II II chaperonin. Crystal structures of Pyrococcus PFD (PDB ID: 2ZDI), and Methanococcus group II chaperonins (PDB IDs: 3LOS (closed), 3IYF (open)) are illustrated by PyMOL (Schrödinger Ltd., chaperonins (PDB IDs: 3LOS (closed), 3IYF (open)) are illustrated by PyMOL (Schrödinger Ltd., Cambridge, MA, USA). The α subunit and β subunit of PFD are shown in red and blue, respectively. α β Cambridge,The complex MA, USA).is also illustrated The subunit by PyMOL. and PFDsubunit captures of PFD an unfolded are shown protein in red by and six blue,tentacles, respectively. and The complextransfers it is toalso a group illustrated II chaperonin by PyMOL. in the open PFD conformation. captures an unfolded protein by six tentacles, and transfers it to a group II chaperonin in the open conformation. Int. J. Mol. Sci. 2018, 19, 2452 3 of 13 Archaeal group II chaperonins consist of
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