Jha P K, Sarkar S
Department of Anatomy & Cellular Biology, Graduate Program in Cell, Molecular and Developmental Biology, Tufts University, Boston, Massachusetts 02111, USA.
Biochemistry. 1998 Sep 1;37(35):12253-60. doi: 10.1021/bi980025z.
Troponin T (TnT), a subunit of the heterotrimeric troponin (Tn) complex, is essential for the Ca2+ regulation of vertebrate striated muscle contraction both in vivo and in vitro. With the exception of bovine cardiac TnT, all known vertebrate TnT isoforms lack a thiol group, a property which makes the wild-type proteins unsuitable as cross-linking substrate. We generated a mutant human fast skeletal TnT in which Ser155 was changed to Cys (TnT-Cys155). Mutation of this residue in TnT as well as in vitro expression in Escherichia coli and purification of the recombinant mutant protein did not affect its biological properties in terms of in vitro binding to troponin I (TnI), troponin C (TnC), actin-tropomyosin (actin-Tm), and actomyosin ATPase activity. TnT-Cys155 was labeled with 4-maleimidobenzophenone (BP-TnT155) and photo-cross-linked to TnI, TnC, Tm, and all of the thin filament proteins. BP-TnT155 did not cross-link to Tm and showed weak Ca2+/Mg2+-independent cross-linking with TnI in the binary complex and in the presence of all thin filament protein components. BP-TnT155 showed Ca2+/Mg2+-dependent cross-linking with TnC in the binary and ternary complexes and Ca2+-favored cross-linking with TnI in the ternary complex. Thus, residue 155 of TnT is within 10 A (the length of cross-linker) of TnC in the presence or absence of Ca2+ and comes within 10 A of both TnI and TnC in the presence of Ca2+. TnT residue 155 is in close proximity to or may even partly encompass the Tm binding site. These results suggest that TnT, in association with TnI, may participate in the "information transfer" mediated by the Ca2+ binding signal from TnC to Tm and the region around TnT residue 155 probably acts as a linker between troponin and actin-Tm in this signal transmission process. Our results also suggest that TnT contains at least one Ca2+/Mg2+-dependent TnC binding region located between its Tm and TnI binding regions. A recombinant truncated fragment of TnI, TnI96-181, containing amino acid residues 96-181 and labeled with BP at Cys-133, failed to cross-link with TnT, indicating that the region around Cys-133 of TnI is not involved in binary interaction with TnT.
肌钙蛋白T（TnT）是异三聚体肌钙蛋白（Tn）复合物的一个亚基，在体内和体外对脊椎动物横纹肌收缩的Ca2+调节至关重要。除牛心肌TnT外，所有已知的脊椎动物TnT同工型都缺乏巯基，这一特性使得野生型蛋白不适于作为交联底物。我们构建了一个突变型人快肌TnT，其中Ser155被替换为Cys（TnT-Cys155）。TnT中该残基的突变以及在大肠杆菌中的体外表达和重组突变蛋白的纯化，在体外与肌钙蛋白I（TnI）、肌钙蛋白C（TnC）、肌动蛋白-原肌球蛋白（肌动蛋白-Tm）结合以及肌动球蛋白ATP酶活性方面，均未影响其生物学特性。TnT-Cys155用4-马来酰亚胺基二苯甲酮（BP-TnT155）标记，并与TnI、TnC、Tm以及所有细肌丝蛋白进行光交联。BP-TnT155未与Tm交联，在二元复合物中以及存在所有细肌丝蛋白组分时，与TnI呈现弱的不依赖Ca2+/Mg2+的交联。BP-TnT155在二元和三元复合物中与TnC呈现依赖Ca2+/Mg2+的交联，在三元复合物中与TnI呈现Ca2+偏好的交联。因此，在有或没有Ca2+存在时，TnT的第155位残基与TnC的距离在10埃（交联剂的长度）以内，在有Ca2+存在时与TnI和TnC的距离均在10埃以内。TnT的第155位残基紧邻甚至可能部分包含Tm结合位点。这些结果表明，TnT与TnI一起，可能参与由TnC的Ca2+结合信号介导至Tm的“信息传递”，并且TnT第155位残基周围区域可能在该信号传递过程中作为肌钙蛋白与肌动蛋白-Tm之间的连接物。我们的结果还表明，TnT在其Tm和TnI结合区域之间至少包含一个依赖Ca2+/Mg2+的TnC结合区域。一个含有氨基酸残基96 - 181并在Cys-133处用BP标记的TnI重组截短片段TnI96-181，未能与TnT交联，表明TnI的Cys-133周围区域不参与与TnT的二元相互作用。
