Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions.
Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca(2+) signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca(2+)-specific) and structural (Ca(2+)- or Mg(2+)-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca(2+) binding using NMR and EF-hand mutants. Ca(2+) titration, as monitored by [(15)N,(1)H] heteronuclear single quantum coherence, suggests that Ca(2+) binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg(2+) binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca(2+)-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca(2+) and not Mg(2+). Ca(2+) binding induces conformational rearrangements in the protein by reversing Mg(2+)-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg(2+) with Ca(2+) reduces the Ca(2+)-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca(2+)-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg(2+)-bound NCS-1 are similar, the early unfolding transitions of Ca(2+)-bound NCS-1 are partially influenced in the presence of Mg(2+). This study demonstrates the importance of Mg(2+) as a modulator of calcium homeostasis and active-state behavior of NCS-1.
Aravind
Penmatsa
P
Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Chandra
Kousik
K
Reddy
Pasham Parameshwar
PP
Jeromin
Andreas
A
Chary
K V R
KV
Sharma
Yogendra
Y
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002151:Calorimetry; D002942:Circular Dichroism; D020832:EF Hand Motifs; D005453:Fluorescence; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism; D051580:Neuronal Calcium-Sensor Proteins / Q000737:chemistry* / Q000378:metabolism*; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism*; D009587:Nitrogen Isotopes; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D011522:Protons; D051381:Rats; D013816:Thermodynamics; D017186:Titrimetry
D050505:Mutant Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D009587:Nitrogen Isotopes; D011522:Protons; C101464:frequenin calcium sensor proteins; D019814:Myristic Acid; D008345:Manganese; D008274:Magnesium; D002118:Calcium