Calcium and myosin S1 binding cause structural rearrangement of the C-terminus of human cardiac troponin T.

Abstract

High resolution structure for the C-terminus of cardiac troponin T (cTnT) remains elusive, due to its predicted flexibility. Multiple recent cryo-EM studies were unable to successfully resolve the C-terminal tail of cTnT, residues 273-288. This region contains a mutational hotspot, R278, and other mutations with varying degrees of pathogenicity and penetrance. To structurally resolve the flexible cTnT C-terminus, time resolved fluorescence resonance energy transfer (TR-FRET) was performed in the reconstituted cardiac thin filament (CTF). cTnT-V274C or V283C were labeled with the donor IAEDANS and cardiac troponin C (cTnC) T53C (N-lobe) or G125C (C-lobe) were labeled with the acceptor DABMI. Measurements were taken in low calcium, high calcium (pCa3), and high calcium plus myosin S1. In low calcium cTnT-V274C was 45Å from cTnC-T53C and this remained unchanged in high calcium, however with calcium and S1 this decreased to 38Å. This was accompanied by a significant increase in order, as measured by a decrease in the full width at half-maximum (FWHM), from 34Å to 14Å. In low calcium, cTnT-V274C was 53Å from cTnC-G125C, this decreased to 47Å in high calcium and 49Å with calcium and S1. Similarly, this was also accompanied by an increase in order from 37Å in low calcium, 31Å in high calcium, and 6Å in high calcium plus S1. Interestingly, while the average distances from cTnT-V283C to cTnC-T53C and cTnC-G125C were 49Å and 58Å, respectively in low calcium, this remained unchanged in high calcium and high calcium plus S1. Taken together, these data suggest the distal C-terminal tail may have a stable interaction, most likely with actin, that is insensitive to calcium and S1 binding, while the proximal tail moves closer to cTnC and becomes more rigid with calcium and S1 binding.