Báo cáo khoa học: Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from

Roles of conserved arginines in ATP-binding domains

of AAA+ chaperone ClpB from Thermus thermophilus

Takashi Yamasaki1

, Yosuke Nakazaki1

, Masasuke Yoshida2 and Yo-hei Watanabe1

1 Department of Biology, Faculty of Science and Engineering, Konan University, Okamoto, Kobe, Japan

2 Department of Molecular Biosciences, Kyoto Sangyo University, Motoyama-Kamigamo, Japan

Introduction

The expanded superfamily of ATPases associated with

diverse cellular activities (AAA+) are involved in a

variety of cellular activities, including membrane

fusion, DNA replication, protein degradation, and dis￾aggregation. Members of the AAA+ family contain

one or more highly conserved AAA+ modules, and

Keywords

AAA+; aggregation; arginine finger;

chaperone; ClpB

Correspondence

Y-h. Watanabe, Department of Biology,

Faculty of Science and Engineering, Konan

University, Okamoto 8-9-1, Kobe 658-8501,

Japan

Fax ⁄ Tel: +81 78 435 2514

E-mail: [email protected]

(Received 7 March 2011, revised 30 April

2011, accepted 6 May 2011)

doi:10.1111/j.1742-4658.2011.08167.x

ClpB, a member of the expanded superfamily of ATPases associated with

diverse cellular activities (AAA+), forms a ring-shaped hexamer and coop￾erates with the DnaK chaperone system to reactivate aggregated proteins

in an ATP-dependent manner. The ClpB protomer consists of an N-termi￾nal domain, an AAA+ module (AAA-1), a middle domain, and a second

AAA+ module (AAA-2). Each AAA+ module contains highly conserved

WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine

(AAA-2). Here, we investigated the roles of these arginines (Arg322,

Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase

cycle and chaperone function by alanine substitution. These mutations did

not affect nucleotide binding, but did inhibit the hydrolysis of the bound

ATP and slow the threading of the denatured protein through the central

pore of the T. thermophilus ClpB ring, which severely impaired the chaper￾one functions. Previously, it was demonstrated that ATP binding to the

AAA-1 module induced motion of the middle domain and stabilized the

ClpB hexamer. However, the arginine mutations of the AAA-1 module

destabilized the ClpB hexamer, even though ATP-induced motion of the

middle domain was not affected. These results indicated that the three argi￾nines are crucial for ATP hydrolysis and chaperone activity, but not for

ATP binding. In addition, the two arginines in AAA-1 and the ATP￾induced motion of the middle domain independently contribute to the sta￾bilization of the hexamer.

Structured digital abstract

l TClpB binds to TClpB by molecular sieving (View Interaction 1, 2)

Abbreviations

AAA, ATPase associated with diverse cellular activities; AAA+, expanded superfamily of ATPases associated with diverse cellular activities;

ABD-F, 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide; AMP-PNP, adenosine 5¢-(b,c-imido)triphosphate; ATPcS, adenosine

5¢-O-(thiotriphosphate); FITC, fluorescein isothiocyanate; G6PDH, glucose-6-phosphate dehydrogenase; Mant-ADP, 2¢(3¢)-O-N¢-methylaniloyl￾aminoadenosine-5¢-diphosphate; P1, position 1; P2, position 2; TBAP, Thermus thermophilus BAP; TCEP, tris-(2-carboxyethyl)phosphine

hydrochloride; TClpA, Thermus thermophilus ClpA; TClpB, Thermus thermophilus ClpB; TClpP, Thermus thermophilus ClpP; T DnaJ,

Thermus thermophilus DnaJ; TDnaK, Thermus thermophilus DnaK; TGrpE, Thermus thermophilus GrpE.

FEBS Journal 278 (2011) 2395–2403 ª 2011 The Authors Journal compilation ª 2011 FEBS 2395