Human antibody C10 neutralizes by diminishing Zika but enhancing dengue virus dynamics.

The human monoclonal antibody (HmAb) C10 potently cross-neutralizes Zika virus (ZIKV) and dengue virus. Analysis of antibody fragment (Fab) C10 interactions with ZIKV and dengue virus serotype 2 (DENV2) particles by cryoelectron microscopy (cryo-EM) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS) shows that Fab C10 binding decreases overall ZIKV particle dynamics, whereas with DENV2, the same Fab causes increased dynamics. Testing of different Fab C10:DENV2 E protein molar ratios revealed that, at higher Fab ratios, especially at saturated concentrations, the Fab enhanced viral dynamics (detected by HDXMS), and observation under cryo-EM showed increased numbers of distorted particles. Our results suggest that Fab C10 stabilizes ZIKV but that with DENV2 particles, high Fab C10 occupancy promotes E protein dimer conformational changes leading to overall increased particle dynamics and distortion of the viral surface. This is the first instance of a broadly neutralizing antibody eliciting virus-specific increases in whole virus particle dynamics.

The ganglioside GM1a functions as a coreceptor/attachment factor for dengue virus during infection.

Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routes by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. We studied the interaction of the virus with GM1a using fluorescence correlation spectroscopy, fluorescence crosscorrelation spectroscopy, imaging fluorescence correlation spectroscopy, amide hydrogen/deuterium exchange mass spectrometry, and isothermal titration calorimetry. Additionally, we explored the effect of this interaction on infectivity and movement of the virus during infection was explored using plaque assay and fluorescence-based imaging and single particle tracking. GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a 10-fold and 7-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems, respectively. We determined that the interaction of the virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection. Collectively, our results suggest that GM1a functions as a coreceptor/attachment factor for DENV during infection in mammalian systems.

Antibody affinity versus dengue morphology influences neutralization.

Different strains within a dengue serotype (DENV1-4) can have smooth, or "bumpy" surface morphologies with different antigenic characteristics at average body temperature (37°C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes.

Dengue virus 2 capsid protein chaperones the strand displacement of 5'-3' cyclization sequences.

By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5'-3' panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5'-3' panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Dengue virus strain 2 capsid protein switches the annealing pathway and reduces intrinsic dynamics of the conserved 5' untranslated region.

The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.

Adenylate control in cAMP signaling: implications for adaptation in signalosomes.

In cAMP-Protein Kinase A (PKA) signaling, A-kinase anchoring protein scaffolds assemble PKA in close proximity to phosphodiesterases (PDE), kinase-substrates to form signaling islands or 'signalosomes'. In its basal state, inactive PKA holoenzyme (R2:C2) is activated by binding of cAMP to regulatory (R)-subunits leading to dissociation of active catalytic (C)-subunits. PDEs hydrolyze cAMP-bound to the R-subunits to generate 5'-AMP for termination and resetting the cAMP signaling. Mechanistic basis for cAMP signaling has been derived primarily by focusing on the proteins in isolation. Here, we set out to simulate cAMP signaling activation-termination cycles in a signalosome-like environment with PDEs and PKA subunits in close proximity to each other. Using a combination of fluorescence polarization and amide hydrogen exchange mass spectrometry with regulatory (RIα), C-subunit (Cα) and PDE8 catalytic domain, we have tracked movement of cAMP through activation-termination cycles. cAMP signaling operates as a continuum of four phases: (1) Activation and dissociation of PKA into R- and C-subunits by cAMP and facilitated by substrate (2) PDE recruitment to R-subunits (3) Hydrolysis of cAMP to 5'-AMP (4) Reassociation of C-subunit to 5'-AMP-bound-RIα in the presence of excess ATP to reset cAMP signaling to form the inactive PKA holoenzyme. Our results demonstrate that 5'-AMP is not merely a passive hydrolysis end-product of PDE action. A 'ligand-free' state R subunit does not exist in signalosomes as previously assumed. Instead the R-subunit toggles between cAMP- or 5'-AMP bound forms. This highlights, for the first time, the importance of 5'-AMP in promoting adaptation and uncovers adenylate control in cAMP signaling.

Development of Phosphodiesterase-Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems-cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG, and dual-specificity RegA-RD complexes-and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

Synergistic Allostery in Multiligand-Protein Interactions.

Amide hydrogen-deuterium exchange mass spectrometry is powerful for describing combinatorial coupling effects of a cooperative ligand pair binding at noncontiguous sites: adenosine at the ATP-pocket and a docking peptide (PIFtide) at the PIF-pocket, on a model protein kinase PDK1. Binding of two ligands to PDK1 reveal multiple hotspots of synergistic allostery with cumulative effects greater than the sum of individual effects mediated by each ligand. We quantified this synergism and ranked these hotspots using a difference in deuteration-based approach, which showed that the strongest synergistic effects were observed at three of the critical catalytic loci of kinases: the αB-αC helices, and HRD-motif loop, and DFG-motif. Additionally, we observed weaker synergistic effects at a distal GHI-subdomain locus. Synergistic changes in deuterium exchange observed at a distal site but not at the intermediate sites of the large lobe of the kinase reveals allosteric propagation in proteins to operate through two modes. Direct electrostatic interactions between polar and charged amino acids that mediate targeted relay of allosteric signals, and diffused relay of allosteric signals through soft matter-like hydrophobic core amino acids. Furthermore, we provide evidence that the conserved ß-3 strand lysine of protein kinases (Lys111 of PDK1) functions as an integrator node to coordinate allosteric coupling of the two ligand-binding sites. It maintains indirect interactions with the ATP-pocket and mediates a critical salt bridge with a glutamate (Glu130) of αC helix, which is conserved across all kinases. In summary, allosteric propagation in cooperative, dual-liganded enzyme targets is bidirectional and synergistic and offers a strategy for combinatorial drug development.

Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays.

Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation.

Dissecting Orthosteric Contacts for a Reverse-Fragment-Based Ligand Design.

Orthosteric sites on proteins are formed typically from noncontiguous interacting sites in three-dimensional space where the composite binding interaction of a biological ligand is mediated by multiple synergistic interactions of its constituent functional groups. Through these multiple interactions, ligands stabilize both the ligand binding site and the local secondary structure. However, relative energetic contributions of the individual contacts in these protein-ligand interactions are difficult to resolve. Deconvolution of the contributions of these various functional groups in natural inhibitors/ligand would greatly aid in iterative fragment-based drug discovery (FBDD). In this study, we describe an approach of progressive unfolding of a target protein using a gradient of denaturant urea to reveal the individual energetic contributions of various ligand-functional groups to the affinity of the entire ligand. Through calibrated unfolding of two protein-ligand systems: cAMP-bound regulatory subunit of Protein Kinase A (RIα) and IBMX-bound phosphodiesterase8 (PDE8), monitored by amide hydrogen-deuterium exchange mass spectrometry, we show progressive disruption of individual orthosteric contacts in the ligand binding sites, allowing us to rank the energetic contributions of these individual interactions. In the two cAMP-binding sites of RIα, exocyclic phosphate oxygens of cAMP were identified to mediate stronger interactions than ribose 2'-OH in both the RIα-cAMP binding interfaces. Further, we have also ranked the relative contributions of the different functional groups of IBMX based on their interactions with the orthosteric residues of PDE8. This strategy for deconstruction of individual binding sites and identification of the strongest functional group interaction in enzyme orthosteric sites offers a rational starting point for FBDD.

Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design.

A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the µM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD).

Dipteran larvae and microbes facilitate nutrient sequestration in the Nepenthes gracilis pitcher plant host.

The fluid-containing traps of Nepenthes carnivorous pitcher plants (Nepenthaceae) are often inhabited by organisms known as inquilines. Dipteran larvae are key components of such communities and are thought to facilitate pitcher nitrogen sequestration by converting prey protein into inorganic nitrogen, although this has never been demonstrated in Nepenthes Pitcher fluids are also inhabited by microbes, although the relationship(s) between these and the plant is still unclear. In this study, we examined the hypothesis of digestive mutualism between N. gracilis pitchers and both dipteran larvae and fluid microbes. Using dipteran larvae, prey and fluid volumes mimicking in situ pitcher conditions, we conducted in vitro experiments and measured changes in available fluid nitrogen in response to dipteran larvae and microbe presence. We showed that the presence of dipteran larvae resulted in significantly higher and faster releases of ammonium and soluble protein into fluids in artificial pitchers, and that the presence of fluid microbes did likewise for ammonium. We showed also that niche segregation occurs between phorid and culicid larvae, with the former fragmenting prey carcasses and the latter suppressing fluid microbe levels. These results clarify the relationships between several key pitcher-dwelling organisms, and show that pitcher communities facilitate nutrient sequestration in their host.

The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm.

Two-component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZ(c)). Increasing osmolality decreased deuterium exchange localized to the four-helix bundle containing the autophosphorylation site (His(243)). EnvZ(c) exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His(243), suggesting that these two events are linked. In-vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H-bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli.

Parallel Allostery by cAMP and PDE Coordinates Activation and Termination Phases in cAMP Signaling.

The second messenger molecule cAMP regulates the activation phase of the cAMP signaling pathway through high-affinity interactions with the cytosolic cAMP receptor, the protein kinase A regulatory subunit (PKAR). Phosphodiesterases (PDEs) are enzymes responsible for catalyzing hydrolysis of cAMP to 5' AMP. It was recently shown that PDEs interact with PKAR to initiate the termination phase of the cAMP signaling pathway. While the steps in the activation phase are well understood, steps in the termination pathway are unknown. Specifically, the binding and allosteric networks that regulate the dynamic interplay between PKAR, PDE, and cAMP are unclear. In this study, PKAR and PDE from Dictyostelium discoideum (RD and RegA, respectively) were used as a model system to monitor complex formation in the presence and absence of cAMP. Amide hydrogen/deuterium exchange mass spectrometry was used to monitor slow conformational transitions in RD, using disordered regions as conformational probes. Our results reveal that RD regulates its interactions with cAMP and RegA at distinct loci by undergoing slow conformational transitions between two metastable states. In the presence of cAMP, RD and RegA form a stable ternary complex, while in the absence of cAMP they maintain transient interactions. RegA and cAMP each bind at orthogonal sites on RD with resultant contrasting effects on its dynamics through parallel allosteric relays at multiple important loci. RD thus serves as an integrative node in cAMP termination by coordinating multiple allosteric relays and governing the output signal response.

Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling.

Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.

Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: capturing transient ternary complexes by HDXMS.

cAMP signaling is a fundamental cellular process necessary for mediating responses to hormonal stimuli. In contrast to cAMP-dependent activation of protein kinase A (PKA), an important cellular target, far less is known on termination in cAMP signaling, specifically how phosphodiesterases (PDEs) facilitate dissociation and hydrolysis of bound cAMP. In this study, we have probed the dynamics of a ternary complex of PKA and a PDE-RegA with an excess of a PDE-nonhydrolyzable cAMP analog, Sp-cAMPS by amide hydrogen/deuterium exchange mass spectrometry (HDXMS). Our results highlight how HDXMS can be used to monitor reactions together with mapping conformational dynamics of transient signaling complexes. Our results confirm a two-state model for active RegA-mediated dissociation of bound cAMP. Further, our results reveal that Sp-cAMPS and RegA mediate mutually exclusive interactions with the same region of PKA and at specific concentrations of Sp-cAMPS, RegA is capable of blocking Sp-cAMPS reassociation to PKA. This provides a molecular basis for how PDEs modulate levels of intracellular cAMP so that PKA is better suited to responding to fluxes rather than constant levels of cAMP. This study underscores how HDXMS can be a powerful tool for monitoring reactions together with mapping conformational dynamics in signaling proteins. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Dynamics, allostery, and stabilities of whole virus particles by amide hydrogen/deuterium exchange mass spectrometry (HDXMS).

X-ray crystallography and cryo-electron microscopy have enabled the determination of structures of numerous viruses at high resolution and have greatly advanced the field of structural virology. These structures represent only a subset of snapshot end-state conformations, without describing all conformational transitions that virus particles undergo. Allostery plays a critical role in relaying the effects of varied perturbations both on the surface through environmental changes and protein (receptor/antibody) interactions into the genomic core of the virus. Correspondingly, allostery carries implications for communicating changes in genome packaging to the overall stability of the virus particle. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) of whole viruses is a powerful probe for uncovering virus allostery. Here we critically discuss advancements in understanding virus dynamics by HDXMS with single particle cryo-EM and computational approaches.

Cyclic AMP-induced conformational changes in mycobacterial protein acetyltransferases.

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.