Tài liệu Báo cáo khoa học: Comparative studies on the functional roles of N- and C-terminal regions

Comparative studies on the functional roles of N- and

C-terminal regions of molluskan and vertebrate troponin-I

Hiroyuki Tanaka1

, Yuhei Takeya1

, Teppei Doi1

, Fumiaki Yumoto2,3, Masaru Tanokura3

,

Iwao Ohtsuki2

, Kiyoyoshi Nishita1 and Takao Ojima1

1 Laboratory of Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Japan

2 Laboratory of Physiology, The Jikei University School of Medicine, Tokyo, Japan

3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan

Troponin is a Ca2+-dependent regulatory protein com￾plex, which constitute thin filaments together with

actin and tropomyosin [1]. It is composed of three dis￾tinct subunits: troponin-C (TnC), which binds Ca2+,

troponin-T (TnT), which binds tropomyosin, and trop￾onin-I (TnI), which binds actin and inhibits actin–myo￾sin interaction [2–4]. In relaxed muscle, TnI binds to

actin and inhibits contraction. Upon muscle stimula￾tion, Ca2+ binds to TnC and induces the release of the

inhibition by TnI, resulting in muscle contraction. To

understand the molecular mechanisms of this Ca2+

switching, extensive studies of the structure, function,

and Ca2+-dependent conformational changes of tropo￾nin subunits have been carried out.

In vertebrate muscles, TnC has a dumbbell-like

shape with the N- and C-terminal globular domains

linked by a central helix [5,6]. Each domain contains

two EF-hand Ca2+-binding motifs [7], thus TnC has

four possible Ca2+-binding sites, sites I and II in the

N-domain and sites III and IV in the C-domain [8,9].

Keywords

invertebrate; mollusk; regulatory

mechanism; troponin; troponin-I

Correspondence

Takao Ojima, Laboratory of Biochemistry

and Biotechnology, Graduate School of

Fisheries Sciences, Hokkaido University,

Hakodate, Hokkaido 041–8611, Japan

Tel ⁄ Fax: +81 138 408800

E-mail: [email protected]

Note

The nucleotide sequences of cDNAs enco￾ding Akazara scallop 52K-TnI and 19K-TnI

are available in DDBJ ⁄ EMBL ⁄ GenBank

databases under accession numbers,

AB206837 and AB206838, respectively

(Received 24 March 2005, revised 13 June

2005, accepted 15 July 2005)

doi:10.1111/j.1742-4658.2005.04866.x

Vertebrate troponin regulates muscle contraction through alternative bind￾ing of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to

actin or troponin-C (TnC) in a Ca2+-dependent manner. To elucidate the

molecular mechanisms of this regulation by molluskan troponin, we com￾pared the functional properties of the recombinant fragments of Akazara

scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akaz￾ara scallop TnI (ATnI232)292), which contains the inhibitory region (resi￾dues 104–115 of rabbit TnI) and the regulatory TnC-binding site (residues

116–131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin

Mg-ATPase. However, it did not interact with TnC, even in the presence

of Ca2+. These results indicated that the mechanism involved in the alter￾native binding of this region was not observed in molluskan troponin. On

the other hand, ATnI130)252, which contains the structural TnC-binding site

(residues 1–30 of rabbit TnI) and the inhibitory region, bound strongly to

both actin and TnC. Moreover, the ternary complex consisting of this frag￾ment, troponin-T, and TnC activated the ATPase in a Ca2+-dependent

manner almost as effectively as intact Akazara scallop troponin. Therefore,

Akazara scallop troponin regulates the contraction through the activating

mechanisms that involve the region spanning from the structural TnC￾binding site to the inhibitory region of TnI. Together with the observation

that corresponding rabbit TnI-fragment (RTnI1)116) shows similar activa￾ting effects, these findings suggest the importance of the TnI N-terminal

region not only for maintaining the structural integrity of troponin com￾plex but also for Ca2+-dependent activation.

Abbreviations

TnC, troponin-C; TnI, troponin-I; TnT, troponin-T; IPTG, isopropyl-1-thio-b-D-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride.

FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4475