title: Structural Insights into the Distinct Binding Mode of Cyclic Di-AMP with SaCpaA_RCK.
abstract: Cyclic di-AMP (c-di-AMP) is a relatively new member of the family of bacterial cyclic dinucleotide second messengers. It has attracted significant attention in recent years because of the abundant roles it plays in a variety of Gram-positive bacteria. The structural features that allow diverse bacterial proteins to bind c-di-AMP are not fully understood. Here we report the biophysical and structural studies of c-di-AMP in complex with a bacterial cation-proton antiporter (CpaA) RCK (regulator of the conductance of K(+)) protein from Staphylococcus aureus (Sa). The crystal structure of the SaCpaA_RCK C-terminal domain (CTD) in complex with c-di-AMP was determined to a resolution of 1.81 Å. This structure revealed two well-liganded water molecules, each interacting with one of the adenine bases by a unique H2Olp-π interaction to stabilize the complex. Sequence blasting using the SaCpaA_RCK primary sequence against the bacterial genome database returned many CpaA analogues, and alignment of these sequences revealed that the active site residues are all well-conserved, indicating a universal c-di-AMP binding mode for CpaA_RCK. A proteoliposome activity assay using the full-length SaCpaA membrane protein indicated that c-di-AMP binding alters its antiporter activity by approximately 40%. A comparison of this structure to all other reported c-di-AMP-receptor complex structures revealed that c-di-AMP binds to receptors in either a "U-shape" or "V-shape" mode. The two adenine rings are stabilized in the inner interaction zone by a variety of CH-π, cation-π, backbone-π, or H2Olp-π interaction, but more commonly in the outer interaction zone by hydrophobic CH-π or π-π interaction. The structures determined to date provide an understanding of the mechanisms by which a single c-di-AMP can interact with a variety of receptor proteins, and how c-di-AMP binds receptor proteins in a special way different from that of c-di-GMP.
authors.affiliation: †National Chung Hsing University Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Ko-Hsin
authors.initials: KH
authors.lastname: Chin
authors.affiliation: ‡Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Juin-Ming
authors.initials: JM
authors.lastname: Liang
authors.affiliation: ‡Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Jauo-Guey
authors.initials: JG
authors.lastname: Yang
authors.affiliation: ‡Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Min-Shao
authors.initials: MS
authors.lastname: Shih
authors.affiliation: ‡Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Zhi-Le
authors.initials: ZL
authors.lastname: Tu
authors.affiliation: ‡Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Yu-Chuang
authors.initials: YC
authors.lastname: Wang
authors.affiliation: ‡Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Xing-Han
authors.initials: XH
authors.lastname: Sun
authors.affiliation: ‡Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Nien-Jen
authors.initials: NJ
authors.lastname: Hu
authors.affiliation: §School of Biological Sciences, Nanyang Technological University, Singapore 637551.
authors.forename: Zhao-Xun
authors.initials: ZX
authors.lastname: Liang
authors.affiliation: ∥School of Microbiology, Biosciences Institute, University College Cork, Cork, Ireland.
authors.forename: J Maxwell
authors.initials: JM
authors.lastname: Dow
authors.affiliation: ⊥Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, U.K.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Ryan
authors.affiliation: †National Chung Hsing University Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan, ROC.|‡Institute of Biochemistry, National Chung Hsing University, Taichung 40227, Taiwan, ROC.
authors.forename: Shan-Ho
authors.initials: SH
authors.lastname: Chou
mesh_terms: D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D015226:Dinucleoside Phosphates / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000378:metabolism
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D015226:Dinucleoside Phosphates; C528998:cyclic diadenosine phosphate