Crystal Structure of Cardiac Troponin C Regulatory Domain in Complex with Cadmium and Deoxycholic Acid Reveals Novel Conformation

Abstract

Summary The amino-terminal regulatory domain of cardiac troponin C (cNTnC) plays an important role as the calcium sensor for the troponin complex. Calcium binding to cNTnC results in conformational changes that trigger a cascade of events that leads to cardiac muscle contraction. Cardiac NTnC consists of two EF-hand calcium binding motifs, one of which is dysfunctional in binding calcium. Nevertheless, the defunct EF-hand still maintains a role in cNTnC function. For its structural analysis by X-ray crystallography, human cNTnC with wild-type primary sequence was crystallized in a novel crystallization condition. The crystal structure was solved from single wavelength anomalous dispersion method and refined to 2.2 Å resolution. The structure displays several novel features. Firstly, both EF-hand motifs coordinate cadmium ions derived from the crystallization milieu. Secondly, the ion coordination in the defunct EF-hand motif accompanies unusual changes in the protein conformation. Thirdly, deoxycholic acid, also derived from the crystallization milieu, is bound in the central hydrophobic cavity. This is reminiscent of the interactions observed for cardiac calcium sensitizer drugs that bind to the same core region and maintain the ‘open’ conformational state of calcium bound cNTnC. The cadmium ion coordination in the defunct EF-hand indicates that this vestigial calcium binding site retains the structural and functional elements that allow it to coordinate a cadmium ion. However, it is a result of, or concomitant with, large and unusual structural changes in cNTnC.

The structure of cardiac troponin C regulatory domain with bound Cd2+reveals a closed conformation and unique ion coordination

“…Here, we present a 1.4 Å resolution crystal structure of WT human cNTnC that coordinates Cd 2+ in both the EF1 and EF2 loops, including the EF1 vestigial site. The structure has a similar overall conformation to that observed in Ca 2+ -bound NMR structures (Spyracopoulos et al, 1997), but differs significantly from our previous crystal structure of Cd 2+ -bound cNTnC in complex with deoxycholic acid (Li et al, 2011). Analysis of the ion coordination in both EF1 and EF2 reveals differences from what has previously been observed in cTnC.…”

“…The previously described plasmid pET21a_WT_cNTnC_ 1-89 (Li et al, 2011) was transformed into Escherichia coli expression host BL21 (DE3) competent cells. The expressed protein (cNTnC) is 89 residues in length (residues 1-89 of human troponin C; UniProt accession No.…”

“…Although the structure of the human cNTnC domain has been studied extensively by NMR (Spyracopoulos et al, 1997;Li et al, 1999;Robertson et al, 2010;Wang et al, 2002), few crystal structures of cNTnC are available owing to difficulty in forming ordered crystals. We have recently found that the presence of Cd 2+ in the crystallization conditions results in cNTnC crystals that diffract to high resolution (Li et al, 2011). Cd 2+ is similar to Ca 2+ in terms of ionic radius (Cd 2+ , 0.97 Å ; Ca 2+ , 0.99 Å ).…”

Comparative Transcriptomic Study of Muscle Provides New Insights into the Growth Superiority of a Novel Grouper Hybrid

“…Troponin (Tn) is a complex of three regulatory proteins (troponin C, troponin T and troponin I) that is integral to muscle contraction and development [46, 47]. Troponin C (TnC), containing four Ca 2+ -binding EF hands [42, 43], can directly bind to and sense Ca 2+ [37, 44, 45], which was released into the cytosol by RyRs cellular calcium channels [38, 39]. We thus tested whether troponins and genes involved in the calcium signaling pathway were also up-regulated in the hybrid grouper.…”

“…Ca 2+ release mediated by ryanodine receptors (RyRs) from the sarcoplasmic reticulum (SR) into the cytosol causes a very rapid and dramatic increase in the cytoplasmic calcium concentration, which has been widely exploited for signal transduction [38–41]. Troponin C (TnC), which contains four calcium-binding EF hands [42, 43], is one of the proteins that can directly bind to and sense Ca 2+ [37, 44, 45]. Together with troponin I (TnI) and troponin T (TnT), TnC forms a troponin complex that binds to G-actin and tropomyosin synchronously then plays a major role in Ca 2+ dependent regulation of muscle contraction [46, 47].…”