High-resolution cryo-EM structure of the junction region of the native cardiac thin filament in relaxed state

Abstract

Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca²⁺ levels. Cardiac thin filament (cTF) consists of two strands composed of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes forming regulatory units. 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 comprised 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-the region that connects adjacent regulatory units and confers to cTF internal cooperativity. Numerous studies have predicted interactions among actin, Tm, and TnT1 within the junction region, although a direct structural description of the cTF junction region awaited completion. Here, we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at relaxing (pCa 8) Ca²⁺ conditions. We provide novel insights into the "head-to-tail" interactions between adjacent Tm molecules and interactions between the Tm junction with F-actin. We demonstrate how TnT1 stabilizes the Tm overlap region via its interactions with the Tm C- and N-termini and actin. Our data show that TnT1 works as a joint that anchors the Tm overlap region to actin, which stabilizes the relaxed state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.