Mapping the Interacting Regions between Troponins T and C: BINDING OF TnT AND TnI PEPTIDES TO TnC AND NMR MAPPING OF THE TnT-BINDING SITE ON TnC

Tharin M.A Blumenschein,Brian P Tripet,Robert S Hodges,Brian D Sykes

doi:10.1074/jbc.m105130200

Tharin M.A Blumenschein, Brian P Tripet + Show 2 more

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https://doi.org/10.1074/jbc.m105130200

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Journal: Journal of Biological Chemistry	Publication Date: Sep 1, 2001
Citations: 29	License type: cc-by

Affiliation: University of Alberta

Abstract

Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex. The interactions among the components of the troponin complex (troponins T, C, and I) are essential to understanding the regulation of muscle contraction. While the structure of TnC is well known, and a model for the binary TnC.TnI complex has been recently published (Tung, C.-S., Wall, M. E., Gallagher, S. C., and Trewhella, J. (2000) Protein Sci. 9, 1312-1326), very little is known about TnT. Using non-denaturing gels and NMR spectroscopy, we have analyzed the interactions between TnC and five peptides from TnT as well as how three TnI peptides affect these interactions. Rabbit fast skeletal muscle peptide TnT-(160-193) binds to TnC with a dissociation constant of 30 +/- 6 microm. This binding still occurs in the presence of TnI-(1-40) but is prevented by the presence of TnI-(56-115) or TnI-(96-139), both containing the primary inhibitory region of TnI. TnT-(228-260) also binds TnC. The binding site for TnT-(160-193) is located on the C-terminal domain of TnC and was mapped to the surface of TnC using NMR chemical shift mapping techniques. In the context of the model for the TnC.TnI complex, we discuss the interactions between TnT and the other troponin subunits.

Highlights

Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex
The troponin complex is constituted by a Ca2ϩbinding subunit, troponin C (TnC),1 an inhibitory subunit, troponin I (TnI), and a tropomyosin-binding subunit, troponin T (TnT)
Despite the amount of information on the interactions between TnC and TnI that has emerged in the last few years, not much is known about the structure of TnT, either isolated or interacting with another troponin subunit

Summary

Introduction

Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex. Rabbit fast skeletal muscle peptide TnT-(160 –193) binds to TnC with a dissociation constant of 30 ؎ 6 ␮M. Skeletal muscle contraction is regulated by troponin and tropomyosin located in the thin filament. Contraction begins with the binding of Ca2ϩ to the troponin complex, triggering conformational changes that are propagated to tropomyosin and actin. The interactions among the three components in the troponin complex as well as how they are affected by the presence of calcium are essential for understanding the regulation of muscle contraction. A model for the structure of the skeletal binary TnC1⁄7TnI complex was recently built based on a number of binding and activity assays such as cross-linking, fluorescent resonance energy transfer (FRET), and neutron scattering data as well as the available structural information [15]. Direct interaction of TnT with TnC is responsible for this calcium-sensitizing effect [17]

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