Moonlighting Crypto-Enzymes and Domains as Ancient and Versatile Signaling Devices

Abstract

Increasing numbers of reports have revealed novel catalytically active cryptic guanylate cyclases (GCs) and adenylate cyclases (ACs) operating within complex proteins in prokaryotes and eukaryotes. Here we review the structural and functional aspects of some of these cyclases and provide examples that illustrate their roles in the regulation of the intramolecular functions of complex proteins, such as the phytosulfokine receptor (PSKR), and reassess their contribution to signal generation and tuning. Another multidomain protein, Arabidopsis thaliana K⁺ uptake permease (AtKUP5), also harbors multiple catalytically active sites including an N-terminal AC and C-terminal phosphodiesterase (PDE) with an abscisic acid-binding site. We argue that this architecture may enable the fine-tuning and/or sensing of K⁺ flux and integrate hormone responses to cAMP homeostasis. We also discuss how searches with motifs based on conserved amino acids in catalytic centers led to the discovery of GCs and ACs and propose how this approach can be applied to discover hitherto masked active sites in bacterial, fungal, and animal proteomes. Finally, we show that motif searches are a promising approach to discover ancient biological functions such as hormone or gas binding.

Keywords: H-NOX; abscisic acid (ABA); adenylate cyclase; crypto-domains; crypto-enzymes; guanylate cyclase; heme-proteins; phosphodiesterase; plant hormones; proteomes.

Figure 1

Domain organization of the phytosulfokine receptor (PSKR). In this leucine-rich transmembrane (TM) receptor kinase, the sulfonated ligand (S residues underlined in red) will, upon binding of the leucine-rich region (LRR), activate the guanylate cyclase (GC) nested within the cytosolic domain of the transmembrane (TM) receptor. PM stands for plasma membrane. The letters in square brackets [] represent different amino acids allowed in a position of the motif.

Figure 2

Mechanistic models of regulation of cryptic guanylate cyclase (GC) activity. (A) Several plant cryptic GCs are found embedded in kinase domains of leucine-rich repeat (LRR) receptors, where, in state 1, the GC is inactive while the kinase is active. In state 2, auto-generation of cGMP reduces kinase activity that is further inhibited by Ca²⁺ that subsequently activates the GC. Reducing cGMP by the action of phosphodiesterases (PDEs) and decreased cytoplasmic Ca²⁺ levels return the cryptic GC to state 1. (B) The phosphorylation state contributes to kinase and GC activities. Hyperphosphorylation at serine residues stimulates kinase activity while inhibiting GC activity, whereas altering tyrosine phosphorylation using permanently on or off mimetics inhibits kinase activity without affecting GC activity (for details see text and also [70]).

Figure 3

Dual crypto-enzymes. (A) Schematic of the architecture of AtKUP5 showing the intracellular N-terminal AC center that is modulated by K⁺ ions to generate cAMP and the PDE center that uses cAMP as its substrate and is activated by Ca²⁺-CaM binding to the CaM binding domain. The green arrow marks enhancing activities and the grey arrow points to a putative ABA binding site in the PDE domain, (B) Amino acid sequence of an A. thaliana F-box protein (At3g44080.1). The three domains that regulate the cyclic mononucleotide content in the cellular microenvironment are the AC, the GC, and the PDE. All three domains were identified with amino acid motifs based on functionally assigned common residues in the respective catalytic centers.

Figure 4

Schematic representation of spliceosome assembly and activation. Spliceosomal proteins and accessory splicing factors regulated by cAMP and cGMP are colored in different shades of red and blue, respectively.