Available online at www.sciencedirect.com ScienceDirect Structure and function of adenylyl cyclases, key enzymes in cellular signaling 1,2,3 2,3 1,2,3 Basavraj Khannpnavar , Ved Mehta , Chao Qi and Volodymyr Korkhov1,2 The adenylyl cyclases (ACs) catalyze the production of the prokaryotes, the mammalian genomes encode only the ubiquitous second messenger, cAMP, which in turns acts on a class III ACs. Despite the profound differences in the number of effectors and thus regulates a plethora of cellular structures and domain organization, the six AC classes are functions. As the key enzymes in the highly evolutionarily functionally very similar, catalyzing the conversion of a conserved cAMP pathway, the ACs control the physiology of molecule of ATP into cAMP (Figure 1a). the cells, tissues, organs and organisms in health and disease. A comprehensive understanding of the specific role of the ACs Bacterial cAMP systems in these processes of life requires a deep mechanistic In bacteria, one of the most extensively studied roles of understanding of structure and mechanisms of action of these cAMP is in regulation of the Escherichia coli glucose enzymes. Here we highlight the exciting recent reports on the metabolism via the cAMP receptor protein CRP (or biochemistry and structure and higher order organization of the catabolite activator protein, CAP) [1]. In addition to its ACs and their signaling complexes. These studies have role in regulation of glucose metabolism, CRP can exert provided the glimpses into the principles of the AC-mediated profound influence on global gene expression in E. coli via homeostatic control of cellular physiology. its influence on more than 380 promoters and 70 transcrip- Addresses tion factors [2], contributing to the multiple roles of 1 Institute of Biochemistry, ETH Zurich, Switzerland cAMP in processes ranging from carbon metabolism to 2 Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen regulation of virulence phenotypes in pathogenic bacteria 5232, Switzerland [3–5]. The bacterial cAMP systems also play key roles in regulation of the cellular homeostasis, phototaxis, protein Corresponding author: Korkhov, Volodymyr ([email protected]) 3 secretion and virulence [6]. For example, in Pseudomonas Contributed equally. aeruginosa, cAMP pool generated by adenylyl cyclases Current Opinion in Structural Biology 2020, 63:34–41 CyaA and CyaB regulates the expression of the type-III secretion system (T3SS) and other important virulence This review comes from a themed issue on Membranes determinants via the cAMP-binding protein Vfr [7]. Edited by Beili Wu and Fei Sun Many of the bacterial species have evolved the ACs (or more broadly, nucleotidyl cyclases, NCs) as virulence https://doi.org/10.1016/j.sbi.2020.03.003 effectors or toxins [8]. These toxins are secreted by the pathogenic bacteria inside the host cell, hijacking the host’s 0959-440X/ã 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creative- cAMP system and aiding the pathogenic bacteria in invad- commons.org/licenses/by-nc-nd/4.0/). ing the host immune system. The anthrax edema factor (EF) from Bacillus anthracis is a prototypical and a very extensively studied AC toxin (REF). Other well-studied Introduction AC toxins include the Hemolysin-AC CyaA from Bordetella 0 0 pertussis, ExoY from P. aeruginosa, and MARTX ExoY-like Adenosine 3 ,5 -monophosphate (cAMP) is a ubiquitous edema factor from Vibrio species [9–12]. These nucleotidyl signaling molecule across all the domains of life. The cyclases toxins are active inside the host organisms and cAMP signaling pathway is a highly conserved regulatory depend on the host-specific binding partners such as cal- mechanism that plays a pivotal role in a wide range of modulin or actin for maximal catalytic activity (REF). The fundamental cellular process. Adenylyl cyclases (ACs) are cAMP pool generated by these ACs suppresses the host the enzymes that generate cAMP, and thus the key immune system, triggers the reorganization of the actin components of the cAMP signaling and regulation path- cytoskeleton causing bleb-niche formation, inter-endothe- ways. In this review we provide a brief general overview lial cell gap formation and increased vascular permeability. of the field, focusing on the recent breakthroughs in our This ultimately leads to tissue edema and injury, prevent- understanding of the structure and function of the ACs as ing the wound healing [8,13]. the key enzymes in cellular signaling. The ACs in all kingdoms of life include six distinct classes The most abundant class of the ACs, the class III (class I–VI). Although the six classes are present in enzymes are often coupled to domains that can be used Current Opinion in Structural Biology 2020, 63:34–41 www.sciencedirect.com Structure and function of adenylyl cyclases Khannpnavar et al. 35 Figure 1 (a) Class II CyaA: 1XFV (b) Class III AC10: 4CM0 (c) Class IV YpAC-IV: 3N0Y Current Opinion in Structural Biology Representative structures of ACs. (a) A crystal structure of a class II AC anthrax edema factor showing the key residues involved in binding and catalysis of ATP into cAMP. (b) Active site architecture of human soluble ACs (AC10) in the presence of an ATP analogue and divalent metal ions. (c) X-ray structure of a class IV AC from Yersinia pestis, bound to an ATP analogue. Despite the significant differences in structural folds, these ACs are able to maintain similar active site configurations involving acidic residues for coordination of divalent metal ions and basic residues for stabilization of negatively charged ATP in the catalytic pocket. to sense and respond to a variety of stimuli. Because of metabolites, and so on [18]. Some of these proteins have this feature, the class III ACs can directly receive, amplify been assigned a role in pathogenicity of the mycobacte- and transmit the information [14]. For example, the rium. This is the case, for example, for Rv0386, which has adenylate cyclase CyaB responsible for major cAMP pool been shown to be critical for macrophage infection [19]. in P. aeruginosa, is a class III AC coupled to a membrane- anchored MASE2 sensor domain, which enables the Mammalian ACs direct regulation of the expression of T3SS and other All mammalian ACs are class III enzymes, with nine virulence determinants by the environmental stimuli membrane-integral isoforms participating in the GPCR À (CO2/HCO3 ) [15–17]. The genome of Mycobacterium and G protein-mediated signaling pathway (AC1-9), and tuberculosis encodes 16 ACs which facilitate regulation one soluble AC (AC10 or sAC) that is not directly linked of gene expression, metabolism, and virulence in to the GPCR signaling. The most conserved regions of response to extracellular stimuli such as pH, bicarbonate, these enzymes are the class III catalytic domains. The www.sciencedirect.com Current Opinion in Structural Biology 2020, 63:34–41 36 Membranes membrane ACs also share the architectural similarity light [30 ,35]. The structure of the catalytic domain of a ˚ (detailed in section 3 below): the 12 transmembrane light-sensitive rhodopsin-cyclase was reported at 2.3 A (TM) domains, and two ‘helical domains’ that link resolution and depicted similar homodimeric structural TM6 and TM12 to the catalytic domains 1 and 2 (C1a arrangement seen in Class III ACs with relatively and C2a, respectively). The less conserved C1b and C2b conserved catalytic residues [30 ]. Although it remains domains are predicted to play an isoform-specific role in to be determined how the different domains interact regulating the activity or the assembly of ACs and their with and regulate the light-sensitive ACs (and GCs), complexes. The role played by the TM region in the understanding how these enzymes operate and how membrane ACs, apart from membrane anchoring and cyclic nucleotide production can be controlled by light trafficking [20] or oligomerization of these enzymes under various conditions will enable new optogenetic [21], is currently not clear. applications in biology and medicine. Membrane bound cyclases have been shown to play Structure of the soluble domain of Rv1625c, the crucial role in mammalian physiology. AC1 and AC8 have evolutionary ancestor of the membrane ACs been shown to be involved in learning and memory [22]. One of the M. tuberculosis ACs, the membrane-integral AC1 has been implicated in neuropathic and inflamma- Rv1625c (Cya), has been suggested to be an evolutionary tory pain, playing a critical role in nerve injury-induced ancestortothemammalianmembraneACs[36].Theprecise plasticity in regions of the brain responsible for pain role of Rv1625c in mycobacteria is currently unknown, perception and has been considered as a potential target although the protein has been shown to be involved in for pain [23]. Both AC1 and AC8 knock out mice showed mycobacterial cholesterol metabolism [37,38 ] as well as in reduced inflammatory, neuropathic and muscle pain [23]. response to heat stress [39]. The sequence of Rv1625c AC3 has been linked to obesity and diabetes [24,25]. The includes six TM helices and a catalytic soluble domain Gbg and Gas subunits released upon receptor activation [36]. The functional unit of the protein is a dimer. Whereas are responsible for activating AC2 and AC4; this contrib- mammalian ACs are regulated by G proteins, the factors that utes to the excitatory effects of chronic opioid use and to regulate the Rv1625c/Cya in mycobacteria are unknown. A the development of tolerance [26]. The role of AC5 and recent study suggested that the TM domain of Rv1625c may 6 has been extensively studied in cardiac function [27,28]. be a receptor for a yet unknown signal, however this remains Mutations in AC5 have been linked to a movement to be experimentally verified [40]. disorder, familial dyskinesia and facial myokymia (FDFM) [29]. The X-ray structure of the soluble domain of the M.