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.

Cyclic‐di‐AMPsynthesis by the diadenylate cyclaseCdaAis modulated by the peptidoglycan biosynthesis enzymeGlmMinLactococcus lactis

“…Effectors that bind c-di-AMP have been identified including the transcription factor DarR from Mycobacterium smegmatis ; the K + transport-gating protein KtrA, ion transporter CpaA and PII-like signal transduction protein PstA from Staphylococcus aureus ; the ydaO riboswitch class in Actinobacteria, Bacillales, Clostridia and Cyanobacteria (Nelson et al, 2013); pyruvate carboxylase, CbpA, CbpB, NrdR, PstA; and several other proteins that may bind c-di-AMP indirectly in L. monocytogenes (Sureka et al, 2014). Crystal structures of pyruvate carboxylase (Sureka et al, 2014), PIIlike signal transduction proteins (Campeotto et al, 2015;Choi et al, 2015;Gundlach et al, 2015a;Muller et al, 2015), regulator of conductance of K + (RCK) domains of KtrA and CpaA (Chin et al, 2015;Kim et al, 2015), and the ydaO riboswitch (Gao and Serganov, 2014;Jones and Ferre-D'Amare, 2014;Ren and Patel, 2014) have provided insights into c-di-AMP binding sites. The first structure of a CdaA-type DAC has also been recently reported .…”

Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation inStaphylococcus aureusbut dispensable for viability in anaerobic conditions

“…Several c-di-AMP target proteins have been identified. In S. aureus, these include the proteins KtrA, PstA, KdpD, CpaA and OpuCA (24)(25)(26)(27)(28)(29). KtrA is the regulatory gating component of the constitutively expressed Ktr potassium transport system and KdpD forms part of a two-component system required for the regulation of the Kdp transporter, a second potassium uptake system (25,30).…”

“…PstA is a PII-like signal transduction protein, the cellular function of which is still unknown, even though several crystal structures are available (27,31,32). CpaA is a predicted cation/proton antiporter and OpuCA is the ATPase component of the carnitine ABC transporter OpuC (24,26,28). Interestingly, none of these uncovered target proteins were found to be essential for the growth of S. aureus under standard laboratory conditions (24)(25)(26)(27)33).…”

Identification of the main glutamine and glutamate transporters inStaphylococcus aureusand their impact on c-di-AMP production

“…Over the last decade, considerable evidence has emerged that c-di-AMP plays a major role in osmotic regulation, primarily by positively regulating potassium export or negatively regulating potassium and osmolyte uptake (Rocha et al ., 2019, Quintana et al ., 2019, Kim et al ., 2015, Corrigan et al ., 2013, Moscoso et al ., 2015, Chin et al ., 2015, Huynh et al ., 2016, Schuster et al ., 2016, Pham & Turner, 2019, Pham et al ., 2018, Devaux et al ., 2018, Zarrella et al ., 2018, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Gundlach et al ., 2017c, Gundlach et al ., 2019). However, individual c-di-AMP target proteins identified thus far are themselves not essential.…”

“…There is now considerable evidence that one such messenger, cyclic di-adenosine monophosphate (c-di-AMP) plays a significant role in osmoregulation in bacteria (Pham et al ., 2018, Pham & Turner, 2019, Quintana et al ., 2019, Zarrella et al ., 2018, Teh et al ., 2019, Fahmi et al ., 2019, Devaux et al ., 2018, Bai et al ., 2014, Zeden et al ., 2018, Corrigan et al ., 2011, Rocha et al ., 2019, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Witte et al ., 2013, Whiteley et al ., 2015, Whiteley et al ., 2017). c-di-AMP binds to and negatively regulates a number of different potassium and osmolyte importers (Rocha et al ., 2019, Quintana et al ., 2019, Kim et al ., 2015, Corrigan et al ., 2013, Moscoso et al ., 2015, Chin et al ., 2015, Huynh et al ., 2016, Schuster et al ., 2016, Pham & Turner, 2019, Pham et al ., 2018, Devaux et al ., 2018, Zarrella et al ., 2018, Gundlach et al ., 2017b, Gundlach et al ., 2017a, Gundlach et al ., 2017c). c-di-AMP is essential for bacterial growth under standard growth conditions but it is also toxic at high levels in many Firmicutes, hence its cellular levels must be tightly regulated (Gundlach et al ., 2015b, Mehne et al ., 2013, Corrigan et al ., 2011, Corrigan et al ., 2015, Woodward et al ., 2010, Witte et al ., 2013).…”