Cooperativity and halonium transfer in the ternary NCI···CH3I···-CN halogen-bonded complex: An ab initio gas phase study.

CONTEXT: The strength and nature of the two halogen bonds in the NCI···CH3I···-CN halogen-bonded ternary complex are studied in the gas phase via ab initio calculations. Different indicators of halogen bond strength were employed to examine the interactions including geometries, complexation energies, Natural Bond Order (NBO) Wiberg bond indices, and Atoms in Molecules (AIM)-based charge density topological properties. The results show that the halogen bond is strong and partly covalent in nature when CH3I donates the halogen bond, but weak and noncovalent in nature when CH3I accepts the halogen bond. Significant halogen bond cooperativity emerges in the ternary complex relative to the corresponding heterodimer complexes, NCI···CH3I and CH3I···-CN, respectively. For example, the CCSD(T) complexation energy of the ternary complex (-18.27 kcal/mol) is about twice the sum of the complexation energies of the component dimers (-9.54 kcal/mol). The halonium transfer reaction that converts the ternary complex into an equivalent one was also investigated. The electronic barrier for the halonium transfer was calculated to be 6.70 kcal/mol at the CCSD(T) level. Although the MP2 level underestimates and the MP3 overestimates the barrier, their calculated MP2.5 average barrier (6.44 kcal/mol) is close to that of the more robust CCSD(T) level. Insights on the halonium ion transfer reaction was obtained by examining the reaction energy and force profiles along the intrinsic reaction coordinate, IRC. The corresponding evolution of other properties such as bond lengths, Wiberg bond indices, and Mulliken charges provides specific insight on the extent of structural rearrangements and electronic redistribution throughout the entire IRC space. METHODS: The MP2 method was used for geometry optimizations. Energy calculations were performed using the CCSD(T) method. The aug-cc-pVTZ basis set was employed for all atoms other than iodine for which the aug-cc-pVTZ-PP basis set was used instead.

Hydrogenation Reactions with Heterobimetallic Complexes.

Hydrogenations are fundamentally and industrially important reactions that are atom economical paths to synthesize value-added products from feedstock chemicals. The cooperative effects of two or more metal centers in multimetallic active sites is a successful strategy to activate small molecules and facilitate catalytic reactions, and this strategy has been recently applied to catalytic hydrogenation reactions. Furthermore, heterobimetallic complexes have been well-documented to provide novel reaction pathways and improved selectivity, compared to their homo-bimetallic and monometallic analogues. This minireview provides a historical perspective on the development of heterobimetallic catalysts for the hydrogenation of unsaturated substrates and describes recent developments in this burgeoning research area.

α1C S1928 Phosphorylation of CaV1.2 Channel Controls Vascular Reactivity and Blood Pressure.

BACKGROUND: Increased vascular CaV1.2 channel function causes enhanced arterial tone during hypertension. This is mediated by elevations in angiotensin II/protein kinase C signaling. Yet, the mechanisms underlying these changes are unclear. We hypothesize that α1C phosphorylation at serine 1928 (S1928) is a key event mediating increased CaV1.2 channel function and vascular reactivity during angiotensin II signaling and hypertension. METHODS AND RESULTS: The hypothesis was examined in freshly isolated mesenteric arteries and arterial myocytes from control and angiotensin II-infused mice. Specific techniques include superresolution imaging, proximity ligation assay, patch-clamp electrophysiology, Ca2+ imaging, pressure myography, laser speckle imaging, and blood pressure telemetry. Hierarchical "nested" and appropriate parametric or nonparametric t test and ANOVAs were used to assess statistical differences. We found that angiotensin II redistributed the CaV1.2 pore-forming α1C subunit into larger clusters. This was correlated with elevated CaV1.2 channel activity and cooperativity, global intracellular Ca2+ and contraction of arterial myocytes, enhanced myogenic tone, and altered blood flow in wild-type mice. These angiotensin II-induced changes were prevented/ameliorated in cells/arteries from S1928 mutated to alanine knockin mice, which contain a negative modulation of the α1C S1928 phosphorylation site. In angiotensin II-induced hypertension, increased α1C clustering, CaV1.2 activity and cooperativity, myogenic tone, and blood pressure in wild-type cells/tissue/mice were averted/reduced in S1928 mutated to alanine samples. CONCLUSIONS: Results suggest an essential role for α1C S1928 phosphorylation in regulating channel distribution, activity and gating modality, and vascular function during angiotensin II signaling and hypertension. Phosphorylation of this single vascular α1C amino acid could be a risk factor for hypertension that may be targeted for therapeutic intervention.

Amplifying Colossal Thermal Expansion of a Martensitic Molecular Crystal through Interlayer Shear-induced Side-chain Liberation.

Molecular crystals capable of colossal thermal expansion (TE) are fascinating owing to their substantial and continuous volume changes and reasonably linear responses to temperature. This makes them promising candidates for micromachine applications. Macroscopic motion is driven by subtle yet cooperative movements of molecules that respond to the thermal motions of dynamic functional units. The study of p-TIPS-DSB presented here offers a compelling case highlighting the relationship between the degree of dynamicity of functional units and TE behavior. In its α-phase, the p-TIPS-DSB crystal undergoes an irreversible martensitic transition to the ß-phase, accompanied by significant cooperative interlayer shear. This process substantially enhances the mobility of the side-chains driven by the increased free volume surrounding them. This nearly doubles the volumetric TE coefficient from 255.3 (10) to 444.9 (32) MK-1, particularly in the actuation direction from 175.0 (7) to 291.7 (20) MK-1, enabling about 4.5% elongation/contraction. As demonstrated here, p-TIPS-DSB exhibits a decent force density (> 1.4 × 107 N m-3) and precise motion control capabilities due to its hysteresis-free and non-abrupt TE nature. Furthermore, we demonstrated the limited operating distance of colossal TE materials can be amplified by utilizing levers, highlighting the high potential of these materials for use in micromachines.

Transition metal pincer catalysts for formic acid dehydrogenation: a mechanistic perspective.

The storage and transportation of hydrogen gas, a non-polluting alternative to carbon-based fuels, have always been challenging due to its extreme flammability. In this regard, formic acid (FA) is a promising liquid organic hydrogen carrier (LOHC), and over the past decades, significant progress has been made in dehydrogenating FA through transition metal catalysis. In this review, our goal is to provide a detailed insight into the existing processes to expose various mechanistic challenges associated with FA dehydrogenation (FAD). Specifically, methodologies catalyzed by pincer-ligated metal complexes were chosen. Pincer ligands are preferred as they provide structural rigidity to the complexes, making the isolation and analysis of reaction intermediates less challenging and consequently providing a better mechanistic understanding. In this perspective, the catalytic activity of the reported pincer complexes in FAD was overviewed, and more importantly, the catalytic cycles were examined in detail. Further attention was given to the structural modifications, role of additives, reaction medium, and their crucial effects on the outcome.

High Nucleotide Skew Palindromic DNA Sequences Function as Potential Replication Origins due to their Unzipping Propensity.

Locations of DNA replication initiation in prokaryotes, called "origins of replication", are well-characterized. However, a mechanistic understanding of the sequence dependence of the local unzipping of double-stranded DNA, the first step towards replication initiation, is lacking. Here, utilizing a Markov chain model that was created to address the directional nature of DNA unzipping and replication, we model the sequence dependence of local melting of double-stranded linear DNA segments. We show that generalized palindromic sequences with high nucleotide skews have a low kinetic barrier for local melting near melting temperatures. This allows for such sequences to function as potential replication origins. We support our claim with evidence for high-skew palindromic sequences within the replication origins of mitochondrial DNA, bacteria, archaea and plasmids.

Intersite communication in dimeric enzymes highlighted by structural and thermodynamic analysis of didansyltyrosine binding to thymidylate synthases.

Intersite communication in dimeric enzymes, triggered by ligand binding, represents both a challenge and an opportunity in enzyme inhibition strategy. Though often understestimated, it can impact on the in vivo biological mechansim of an inhibitor and on its pharmacokinetics. Thymidylate synthase (TS) is a homodimeric enzyme present in almost all living organisms that plays a crucial role in DNA synthesis and cell replication. While its inhibition is a valid strategy in the therapy of several human cancers, designing specific inhibitors of bacterial TSs poses a challenge to the development of new anti-infective agents. N,O-didansyl-l-tyrosine (DDT) inhibits both Escherichia coli TS (EcTS) and Lactobacillus casei TS (LcTS). The available X-ray structure of the DDT:dUMP:EcTS ternary complex indicated an unexpected binding mode for DDT to EcTS, involving a rearrangement of the protein and addressing the matter of communication between the two active sites of an enzyme dimer. Combining molecular-level information on DDT binding to EcTS and LcTS extracted from structural and FRET-based fluorometric evidence with a thermodynamic characterization of these events obtained by fluorometric and calorimetric titrations, this study unveiled a negative cooperativity between the DDT bindings to the two monomers of each enzyme dimer. This result, complemented by the species-specific thermodynamic signatures of the binding events, implied that communication across the protein dimer was triggered by the first DDT binding. These findings could challenge the conventional understanding of TS inhibition and open the way for the development of novel TS inhibitors with a different mechanism of action and enhanced efficacy and specificity.

All-body concept and quantified limits of cooperativity and related effects in homodromic cyclic water clusters from a molecular-wide and electron density-based approach.

We strongly advocate distinguishing cooperativity from cooperativity-induced effects. From the MOWeD-based approach, the origin of all-body cooperativity is synonymous with physics- and quantum-based processes of electron (e) delocalization throughout water clusters. To this effect, over 10 atom-pairs contribute to the total e-density at a BCP(H,O) between water molecules in a tetramer. Intermolecular all-body e-delocalization, that is, cooperativity, is an energy-minimizing process that fully explains non-additive increase in stability of a water molecule in clusters with an increase in their size. A non-linear change in cooperativity and cooperativity-induced effects, such as (i) structural (e.g., a change in d(O,O)) or topological intra- and intermolecular properties in water clusters (e.g., electron density or potential energy density at bond critical points) is theoretically reproduced by the proposed expression. It predicted the limiting value of delocalized electrons by a H2O molecule in homodromic cyclic clusters to be 1.58e. O-atoms provide the vast majority of electrons that "travel throughout a cluster predominantly on a privileged exchange quantum density highway" (⋅⋅⋅O-H⋅⋅⋅O-H⋅⋅⋅O-H⋅⋅⋅) using Bader's classical bond paths as density bridges linking water molecules. There are, however, additional electron exchange channels that are not seen on molecular graphs as bond paths. A 3D visual representation of the "privileged" and "additional" exchange channels as well as detailed intra- and inter-molecular patterns of e-sharing and (de)localizing is presented. The energy stabilizing contribution made by three O-atoms of neighboring water molecules was found to be large (-597 kcal/mol in cyclic hexamer) and 5 times more significant than that of a classical O-H⋅⋅⋅O intermolecular H-bond.

5-Oxo-7,7-Dimethyl-5,6,7,8-Tetrahydro-4-H-Chromene Bearing N, N-Dimethylaniline as Turn-Off Fluorescent Chemosensor for Picric Acid in Real Water Sample.

We have synthesized a one-pot, three-component pyran-based fluorescence chemosensor using onion extract as a green catalyst. The confirmed structure of the 1:2 binding of receptor SPR-2-picric acid adduct revealed that the pyran-based receptor accommodated two guest picric acid molecules through non-covalent interactions. UV-Vis and fluorescence spectroscopy show high selectivity and sensitivity towards picric acid. The 1D/2D NMR and Job's plot analysis show the complexation and stoichiometric binding of the receptor SPR-2 with picric acid are 1:2. The 1H NMR spectral studies confirm that the formation of receptor SPR-2-picric acid adduct via weak hydrogen bonding. The cooperativity of the receptor SPR-2-picric acid adduct shows negative cooperativity due to the weak hydrogen bonding of receptor SPR-2 and picric acid. Further, the density functional theory (DFT) confirmed the molecular level interaction of the SPR-2 and receptor SPR-2-Picric acid adduct. The receptor was effectively used to assess picric acid concentrations in real water samples.

Self-Assembly of Thienopyrrole-Fused Thiadiazoles Containing an Amide Linker: Control of Supramolecular Polymerization Mechanism and Chiroptical Properties by Trialkoxy Side Chains.

Three thienopyrrole-fused thiadiazole (TPT) fluorescent dyes featuring a common amide linker and different alkoxy substituents on peripheral trialkoxybenzene moieties were synthesized, and their self-assembly behavior in solution was investigated. The obtained results revealed a substantial steric effect of the alkoxy substituents on the supramolecular polymerization mechanism, which results from a combination of π-stacking and hydrogen (H)-bonding interactions. Detailed spectroscopic measurements revealed that with increasing steric demand of the substituents, the supramolecular polymerization processes in pure methylcyclohexane (MCH) or a mixture of MCH and toluene become temperature-sensitive and enthalpically favorable, resulting in a change from the isodesmic assembly mechanism to the cooperative mechanism. Theoretical calculations suggested that in TPTs with bulky substituents, steric hindrance causes the H-bonding array of the amide moieties to be aligned along the stacking axis of the π-systems; thus, the H-bonding interactions are strengthened compared to those in TPTs with less bulky substituents, compensating for the weakened π-stacking interactions. A chiral TPT derivative with (S) stereogenic centers was found to form homochiral helical supramolecular assemblies that generate discernible circularly polarized luminescence. Achiral TPTs also generate helical assemblies to which preferential helicity can be imparted through the external chiral bias of the solvents (R)- and (S)-limonene.

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro.

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

Structural Variations and Rearrangements in Bacterial Type II Toxin-Antitoxin Systems.

Bacteria encode a wide range of survival and immunity systems, including CRISPR-Cas, restriction-modification systems, and toxin-antitoxin systems involved in defence against bacteriophages, as well as survival during challenging growth conditions or exposure to antibiotics. Toxin-antitoxin (TA) systems are small two- or three-gene cassettes consisting of a metabolic regulator (the "toxin") and its associated antidote (the "antitoxin"), which also often functions as a transcriptional regulator. TA systems are widespread in the genomes of pathogens but are also present in commensal bacterial species and on plasmids. For mobile elements such as plasmids, TA systems play a role in maintenance, and increasing evidence now points to roles of chromosomal toxin-antitoxin systems in anti-phage defence. Moreover, the widespread occurrence of toxin-antitoxin systems in the genomes of pathogens has been suggested to relate to survival during host infection as well as in persistence during antibiotic treatment. Upon repeated exposure to antibiotics, TA systems have been shown to acquire point mutations as well as more dramatic rearrangements such as in-frame deletions with potential relevance for bacterial survival and pathogenesis. In this review, we present an overview of the known functional and structural consequences of mutations and rearrangements arising in bacterial toxin-antitoxin systems and discuss their relevance for survival and persistence of pathogenic species.

Signal integration and adaptive sensory diversity tuning in Escherichia coli chemotaxis.

In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal's ambient concentration increases. However, among competitive ligands, the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefits E. coli populations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.

Delineating redox cooperativity in water-soluble and membrane multiheme cytochromes through protein design.

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to delineate the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.

Systematic dissection of sequence features affecting binding specificity of a pioneer factor reveals binding synergy between FOXA1 and AP-1.

Despite the unique ability of pioneer factors (PFs) to target nucleosomal sites in closed chromatin, they only bind a small fraction of their genomic motifs. The underlying mechanism of this selectivity is not well understood. Here, we design a high-throughput assay called chromatin immunoprecipitation with integrated synthetic oligonucleotides (ChIP-ISO) to systematically dissect sequence features affecting the binding specificity of a classic PF, FOXA1, in human A549 cells. Combining ChIP-ISO with in vitro and neural network analyses, we find that (1) FOXA1 binding is strongly affected by co-binding transcription factors (TFs) AP-1 and CEBPB; (2) FOXA1 and AP-1 show binding cooperativity in vitro; (3) FOXA1's binding is determined more by local sequences than chromatin context, including eu-/heterochromatin; and (4) AP-1 is partially responsible for differential binding of FOXA1 in different cell types. Our study presents a framework for elucidating genetic rules underlying PF binding specificity and reveals a mechanism for context-specific regulation of its binding.

Structural basis for transcription activation through cooperative recruitment of MntR.

The manganese transport regulator (MntR) from B. subtilis is a dual regulatory protein that responds to heightened Mn2+ availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn2+ exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the mneP promoter. Our structures, along with solution studies including mass photometry and in vivo transcription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

Molecular basis of CRX/DNA recognition and stoichiometry at the Ret4 response element.

Two retinal transcription factors, cone-rod homeobox (CRX) and neural retina leucine zipper (NRL), cooperate functionally and physically to control photoreceptor development and homeostasis. Mutations in CRX and NRL cause severe retinal diseases. Despite the roles of NRL and CRX, insight into their functions at the molecular level is lacking. Here, we have solved the crystal structure of the CRX homeodomain in complex with its cognate response element (Ret4) from the rhodopsin proximal promoter region. The structure reveals an unexpected 2:1 stoichiometry of CRX/Ret4 and unique orientation of CRX molecules on DNA, and it explains the mechanisms of pathogenic mutations in CRX. Mutations R41Q and E42K disrupt the CRX protein-protein contacts based on the structure and reduce the CRX/Ret4 binding stoichiometry, suggesting a novel disease mechanism. Furthermore, we show that NRL alters the stoichiometry and increases affinity of CRX binding at the rhodopsin promoter, which may enhance transcription of rod-specific genes and suppress transcription of cone-specific genes.

Coordination of bilayer properties by an inward-rectifier K+ channel is a cooperative process driven by protein-lipid interaction.

Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is difficult to assess due to reciprocal effects between membrane proteins, individual lipids, and membrane architecture. Using solid-state NMR (SSNMR) we previously showed that KirBac1.1, a bacterial Inward-Rectifier K+ channel, nucleates bilayer ordering and microdomain formation through tethering anionic lipids. Conversely, these lipids cooperatively bind cationic residues to activate the channel and initiate K+ flux. The mechanistic details governing the relationship between cooperative lipid loading and bilayer ordering are, however, unknown. To investigate, we generated KirBac1.1 samples with different concentrations of 13C-lableded phosphatidyl glycerol (PG) lipids and acquired a full suite of SSNMR 1D temperature series experiments using the ordered all-trans (AT) and disordered trans-gauche (TG) acyl conformations as markers of bilayer dynamics. We observed increased AT ordered signal, decreased TG disordered signal, and increased bilayer melting temperature with increased PG concentration. Further, we identified cooperativity between ordering and direct binding of PG lipids, indicating KirBac1.1-driven bilayer ordering and microdomain formation is a classically cooperative Hill-type process driven by and predicated upon direct binding of PG lipids. Our results provide unique mechanistic insight into how proteins and lipids in tandem contribute to supramolecular bilayer heterogeneity in the lipid membrane.

Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase.

Regulatory cystathionine ß-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.

The αC-ß4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-ß4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-ß4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-ß4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.