Structures reveal details of small molecule binding to cardiac troponin.
In cardiac and skeletal muscle, the troponin complex turns muscle contraction on and off in a calcium-dependent manner. Many small molecules are known to bind to the troponin complex to modulate its calcium binding affinity, and this may be useful in a broad range of conditions in which striated muscle function is compromised, such as congestive heart failure. As a tool for developing drugs specific for the cardiac isoform of troponin, we have designed a chimeric construct (cChimera) consisting of the regulatory N-terminal domain of cardiac troponin C (cNTnC) fused to the switch region of cardiac troponin I (cTnI), mimicking the key binding event that turns on muscle contraction. We demonstrate by solution NMR spectroscopy that cChimera faithfully reproduces the native interface between cTnI and cNTnC. We determined that small molecules based on diphenylamine can bind to cChimera with a KDas low as 10μM. Solution NMR structures show that minimal structural perturbations in cChimera are needed to accommodate 3-methyldiphenylamine (3-mDPA), which is probably why it binds with higher affinity than previously studied compounds like bepridil, despite its significantly smaller size. The unsubstituted aromatic ring of 3-mDPA binds to an inner hydrophobic pocket adjacent to the central beta sheet of cNTnC. However, the methyl-substituted ring is able to bind in two different orientations, either inserting into the cNTnC-cTnI interface or "flipping out" to form contacts primarily with helix C of cNTnC. Our work suggests that preservation of the native interaction between cNTnC and cTnI is key to the development of a high affinity cardiac troponin-specific drug.
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Fangze
F
Cai
Department of Medicine, University of Alberta, Edmonton, AB, Canada.
Monica X
MX
Li
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Sandra E
SE
Pineda-Sanabria
Department of Chemistry, University of Alberta, Edmonton, AB, Canada.
Shorena
S
Gelozia
Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA.
Steffen
S
Lindert
Department of Chemistry, University of Alberta, Edmonton, AB, Canada.
Frederick
F
West
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Brian D
BD
Sykes
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada; Department of Medicine, University of Alberta, Edmonton, AB, Canada. Electronic address: phwang1@ualberta.ca.
Peter M
PM
Hwang
D000818:Animals; D001665:Binding Sites; D055808:Drug Discovery; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism*; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I
Calcium sensitizer; Cardiac troponin activator; Drug development; Nuclear magnetic resonance spectroscopy; Systolic heart failure