In this work, we have defined the unique mechanism by which mycobacteria couple cAMP concentration to protein lysine acetylation. Our structural and biochemical analysis defines two distinct functional states of the acetyltransferase, Mt-PatA. Dramatic structural changes required for cAMP binding are coupled to exposure of the catalytic site. In the auto-inhibited state, cAMP-binding site is blocked by the C-terminal helix from the catalytic domain. In addition, the substrate-mimicking lid in the catalytic domain buries the acetyl donor and a conserved surface that forms the protein-substrate-binding site. While excluding the C-terminus, cAMP stabilizes the rotation of the regulatory domain as well as tertiary shifts that are incompatible with the closed lid. These shifts release the steric double latch created by the lid and the C-terminal extension of the PAT domain (). The lid rearranges in the active state, creating a large binding surface for protein substrates.
A steric double-latch regulates Mt-PatA
Our results disfavor a sequential model in which cAMP binding initiates a specific mechanical pathway for the conformational change. Importantly, there is no site for cAMP to bind before the enzyme switches conformations (), and once cAMP stabilizes the active form, the lid does not readily fluctuate back to the inhibited conformation (, , ; Supplementary Figs. 3, 4
). In addition, C-terminal mutations designed to destabilize the latch at the regulatory site activate the enzyme in the absence of cAMP (). This gain of function shows that cAMP is not needed to induce the active conformation in the mutants. These findings support a two-state model for activation mediated by steric incompatibility of the alternate conformations. In this model, the relative stability of the two globally different conformations mediates the communication between the regulatory and catalytic sites. Consistent with changes in hydrogen exchange in peptides throughout the protein upon cAMP binding 26
, the widespread changes in rotamers detected using Ringer
and the effects of diverse mutations on activation provide evidence that changes in interactions all over the enzyme determine the relative stability of the two states.
Diverse proteins containing a cAMP-binding module--including CAP, hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels, and the regulatory subunit of protein kinase A--have been structurally characterized. Although these proteins mediate different biological functions, they all contain a conserved module for cAMP binding that allosterically controls activity 27,28
. The cAMP-binding modules have similar architectures, but show significant structural variations in the helical subdomain and the loop between strands 4 and 5 (using Mt-PatA numbering; Supplementary Fig. 2
). The core β-subdomain is structurally similar (rmsd = 0.73-1.00 Å for ~70 Cα atoms of Mt-PatA (Supplementary Fig. 1a
), and the key residues that interact with cAMP also are conserved among cAMP-binding modules (Supplementary Fig. 1b
Cyclic-AMP binding to Mt-PatA requires a >10 Å movement of helix F (residues 127-142) in the regulatory domain. Similarly in E. coli
, cAMP stabilizes a restructuring in the helix bearing cAMP-binding residues, linked to a coil-to-helix transition with many residues moving >10 Å (Supplementary Figs. 6a-c
). These changes result in steric clashes that are resolved by flipping out the CAP DNA-binding domains. The solution structure of apo-CAP determined using NMR shows that the cAMP binding site is not present in the major conformer in the absence of activator 28
. Like the cryptic cAMP site of Mt-PatA, this suggests that the conformation switches before the nucleotide binds. Moreover, NMR measurements demonstrate that two constitutively activating CAP mutations switch the population toward the active conformation in the absence of activator 28
, as embodied in the design of C-terminal mutants of Mt-PatA (). In contrast, the cAMP binding site is better formed in the crystal structure of apo-CAP, and the crystallographic results have been interpreted in terms of a sequential activation model 30
. Nonetheless, genetic, biochemical and NMR studies of E. coli
indicate that the two-state model for Mt-PatA activation captures general features of cAMP-regulated systems.
Large structural shifts also occur in the RIα inhibitory subunit of human cAMP-dependent protein kinase, where cAMP binding stabilizes dramatic conformational changes through the connecting helix (αB/C) that release the regulatory subunit 31
. These examples emphasize the general role of the cAMP binding domain in regulating large conformational changes, not subtle shifts 32
. By modulating the position of the helical subdomain, cAMP binding favors significant structural changes that must be accommodated in the attached functional domains by changes that are commensurate in magnitude 32
. While cAMP mediates large shifts, the structural responses in regulated domains are distinct in different systems (Supplementary Figs. 6d-g
The selective pressures that shaped the evolution of cAMP regulation of Mt-PatA remain to be defined. Bacterial pathogens including M. tuberculosis
commonly utilize cAMP as an important regulator of gene expression and host signaling to promote survival in changing environmental conditions 15,33
. The cellular dynamics of cAMP are determined by the interplay of synthetic adenylyl cyclases (ACs) and hydrolytic cyclic nucleotide phosphodiesterases (PDEs). The M. tuberculosis
genome encodes 10 predicted cAMP-binding proteins, one PDE, as well as an unusually large number of ACs (15 and 17 in strains H37Rv and CDC1551, respectively) 34
In contrast, other bacterial pathogens such as E. coli
have only one AC gene. This expansion of AC sequences suggests that Mt-PatA forms the nexus of multiple signaling pathways that modulate cAMP levels. While the CAP transcription-factor family mediates many of the effects of cAMP, the cAMP-signaling cascade in mycobacteria is remodeled to mediate direct post-translational regulation of protein lysine acetylation without the requirement for new gene expression 18
. M. tuberculosis
is thought to persist in a relatively quiescent state during infection 35,36
, and dormant mycobacteria may produce little new mRNA or protein 37
. Moreover, rapid changes in metabolism may be required to tolerate host immune defenses. Thus, the structure of the Mt-PatA we describe may be specialized to allow rapid regulation under growth-limiting conditions. The conservation of the residues that contact cAMP (Supplementary Fig. 1b
), however, may hamper efforts to develop selective inhibitors that target this site in Mt-PatA in preference to human cAMP-binding domains. Rather, control of the Mt-PatA latching mechanisms by stabilizing the auto-inhibited form may afford a more promising strategy for therapeutic development.
While the evolutionary pathway cannot be appreciated experimentally, structural comparisons afford clues to the molecular features that can enable the emergence of this allosteric control. In Mt-PatA, two sequence elements in the catalytic domain and the three-residue linker between the domains harness the conformational switch in the cAMP regulatory module. The lid and the C-terminal helix inserted in the PAT domain do not make direct contacts, but instead interact indirectly through the cAMP-binding module. No regulation is possible without the lid, so this was likely the first regulatory element to be incorporated. The archaeal PAT from Sulfolobus solfataricus
provides an example of an isolated PAT domain with an auto-inhibitory lid 38
. Without a regulatory domain, the lid in this archaeal PAT may respond directly to growth conditions. Consistent with the Kuriyan and Eisenberg model 6
, fusion of the cAMP binding domain in Mt-PatA and the evolution of interdomain interactions with the lid in the unbound form would provide an evolutionary pathway for metabolites to regulate auto-inhibition. The effects of C-terminal mutations in Mt-PatA show that the addition of the C-terminal helix provides a latch that stabilizes the auto-inhibited form relative to the active form, effectively increasing the cAMP concentration needed for activation. The interdomain linker tightly tethers the domains, preventing the regulatory and PAT domains from diffusing apart. This short covalent linkage restricts the relative motions of the domains to a rotation that functions to reveal cooperatively the cAMP binding site and the active site. This cooperativity of sterically incompatible conformations creates a two-state mechanism of long-range communication that couples regulatory and catalytic sites separated by over 32 Å. Overall, this switch in Mt-PatA affords insights into the structural features that can be exploited in the evolution of allosteric regulation through domain fusion.