Abstract
Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca(2+)-depletion sensor STIM1. This is followed by a fast Ca(2+)·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp(76) of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.
Highlights
Calmodulin (CaM) binds the calmodulin binding domain (CMBD) of Orai1 causing Ca2ϩ·CaM-dependent inhibition (CDI)
Unique Binding Mode of Orai1-CMBD to CaM—We show a unique binding mode of CaM to the target peptide Orai1CMBD: binding of one peptide to each lobe of CaM (Fig. 10A)
Based on the number of residues of CMBDs that are critical for interaction with CaM-N and CaM-C, CaM target peptides can be divided into different groups: 1–7, 1–10, 1–14, 1–16, and 1–18 (Fig. 10B)(29, 30)
Summary
Calmodulin (CaM) binds the calmodulin binding domain (CMBD) of Orai causing Ca2ϩ·CaM-dependent inhibition (CDI). Orai is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca2؉-depletion sensor STIM1 This is followed by a fast Ca2؉1⁄7calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai region called the calmodulin binding domain (CMBD). Lewis and colleagues [13] showed that calmodulin (CaM) and a cytosolic domain of STIM1 are important for CDI They further showed that binding of CaM to an N-terminal fragment of Orai adjacent to its first transmembrane helix is calcium-dependent and critical for CDI. We carried out structural studies on the CaM1⁄7Orai1-CMBD complex, hoping to 1) understand how CaM facilitates CDI of Orai1; 2) add to our knowledge of the interaction modes of CaM with its target proteins. Further biochemical analyses and mutational studies indicate that in solution CaM interacts with Orai1-CMBDs in a 1:2 binding mode, with CaM-N and CaM-C each binding one Orai1-CMBD
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