Calmodulin binding to Arabidopsis cyclic nucleotide gated ion channels

Abstract

Recently we have reported that the αC-helix in the cyclic nucleotide binding domain (CNBD) is required for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis. A mutation at arginine 557 to cysteine (R557C) in the αC-helix of the CNBD caused an alteration in channel regulation. Protein sequence alignments revealed that R557 is located in a region that is important for calmodulin (CaM) binding. It has been hypothesized that CaM negatively regulates plant CNGCs similar to their counter parts in animals. However, only a handful of studies has been published so far and we still do not have much information about the regulation of CNGCs by CaM. Here, we conducted in silico binding prediction of CaM and Arabidopsis CNGC12 (AtCNGC12) to further study the role of R557. Our analysis revealed that R557 forms salt bridges with both D79 and E83 in AtCaM1. Interestingly, a mutation of R557 to C causes the loss of these salt bridges. Our data further suggests that this alteration in CaM binding causes the observed altered channel regulation and that R557 plays an important role in CaM binding.

Figure 1

Computational structural modeling of CaM binding with AtCNGC12 and AtCNGC12:R557C. Modeling of the tertiary structure of AtCaM1, and the αC-helix of AtCNGC12 and AtCNGC12:R557 was conducted using the crystallized structures of the potato CaM, PCM 6 (PDB# 1RFJ) and the cytoplasmic C-terminus of the invertebrate CNGC, SpIH (Flynn et al. 2007, PDB# 2PTM), respectively, as templates. The protein fold recognition server (Phyre) was used to model these proteins. The binding modeling was performed using an algorithm for molecular docking (PatchDock). All the images were generated using PyMOL. CaM is colored in cyan and the αC-helix is shown in magenta. Left part: overall binding model between AtCaM1 and AtCNGC12, Center part: close up of the boxed area of the left part in AtCNGC12, Right part: the same area in AtCNGC12:R557C. M73, M52 and M37 of AtCaM1 create a hydrophobic pocket together with F562 and I564 of AtCNGC12, which is a typical binding configuration between CaM and target proteins. R557 creates salt bridges with both D79 and E83 (center part). These salt bridges are no longer seen between AtCaM1 and AtCNGC12:R557C (right part).