The structure of the junction region of the native cardiac thin filament.

Abstract

Cardiac contraction depends on an intricate rearrangement of molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca2+ levels. Cardiac thin filament (cTF) consists of two strands comprised of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes. Tn binds Ca2+ to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation. The Tn complex has three subunits: Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. Tm strand is composed of adjacent Tm molecules that overlap “head-to-tail” along the actin filament. The N-terminus of TnT (e.g. TnT1) binds to the Tm overlap region to form the cTF junction region. Numerous studies have shown the crucial role of TnT1 in the Ca2+-dependent regulation of the cTF. Moreover, TnT1 is a hot spot for pathogenic mutations leading to cardiomyopathies in humans. However, a detailed structural description of the cTF junction region and, particularly, TnT1 interactions with Tm and actin remain unknown. Here we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at relaxing (pCa=8) Ca2+ conditions. We show that TnT1 stabilizes the Tm overlap region via mostly ionic interactions with the Tm C- and N-termini. Our data shows that TnT1 works as a joint that anchors the Tm overlap region to actin which stabilizes the off (e.g. Ca2+-free) state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.