Abstract:Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. For signaling enzymes, substrate recognition, binding, and product release are often rate-determining steps in which enthalpy-entropy compensation plays a crucial role. While the nature of enthalpic interactions can be inferred from structural data, the molecular origin and role of entropy in enzyme catalysis remains poorly understood. Using thermocalorimetry, NMR, and MD simulations, we studie… Show more
“…Perhaps more importantly, many of these studies have shown how disruptions in these pathways act to change function. Taken with our previous studies, nucleotide/substrate-binding cooperativity emerges as a critical function in protein kinase A that arises from the structural changes of multiple domains (i.e., communities) that must be coordinated and synchronized to ensure a cooperative binding response 18 , 20 . Like other tumorigenic transcriptomes that result in a fully active PKA-C, it is likely that alterations in the allosteric network impart defective binding cooperativity and may play a role in their aberrant functions 35 , 36 , 53 .…”
Section: Discussionsupporting
confidence: 72%
“…3a ). These correlations constitute central allosteric nodes necessary for binding cooperativity 18 , 20 , 31 . In addition, allosteric cross-talk exists between residues in the C-terminal tail and residues in the αA-helix (K21, K28), αE-helix (R144, A147, L152), activation loop (R190, V191, G193), and αF-helix (G214, G225).…”
Section: Resultsmentioning
confidence: 99%
“…A prominent feature of PKA-C is the alternation of positive and negative binding cooperativity that drives the enzymatic cycle 18 . Positive binding cooperativity induced by ATP-binding enhances the affinity for the substrate, whereas negative binding cooperativity of ADP facilitates the release of the phosphorylated product.…”
An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.
“…Perhaps more importantly, many of these studies have shown how disruptions in these pathways act to change function. Taken with our previous studies, nucleotide/substrate-binding cooperativity emerges as a critical function in protein kinase A that arises from the structural changes of multiple domains (i.e., communities) that must be coordinated and synchronized to ensure a cooperative binding response 18 , 20 . Like other tumorigenic transcriptomes that result in a fully active PKA-C, it is likely that alterations in the allosteric network impart defective binding cooperativity and may play a role in their aberrant functions 35 , 36 , 53 .…”
Section: Discussionsupporting
confidence: 72%
“…3a ). These correlations constitute central allosteric nodes necessary for binding cooperativity 18 , 20 , 31 . In addition, allosteric cross-talk exists between residues in the C-terminal tail and residues in the αA-helix (K21, K28), αE-helix (R144, A147, L152), activation loop (R190, V191, G193), and αF-helix (G214, G225).…”
Section: Resultsmentioning
confidence: 99%
“…A prominent feature of PKA-C is the alternation of positive and negative binding cooperativity that drives the enzymatic cycle 18 . Positive binding cooperativity induced by ATP-binding enhances the affinity for the substrate, whereas negative binding cooperativity of ADP facilitates the release of the phosphorylated product.…”
An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.
“…In comparison, the physiological substrate PLN in complex with PKA exhibits a committed open-close conformational landscape, with little of the tertiary or higher-order substates observed in the PKS complex. This result agrees well with the synchronous global process measurable by NMR relaxation dispersion only for PKA:PLN but not for PKA:PKS (56). The observed free-energy landscape of the PKA:PLN complex most resembles the PKA:RIIβ complex, highlighting that both substrates are native phospho-acceptor sequences.…”
Section: Dynamic Signatures Of the Kinase:nucleotide:inhibitor Ternarysupporting
A dense interplay between structure and dynamics underlies the working of proteins, especially enzymes. Protein kinases are molecular switches that are optimized for their regulation rather than catalytic turnover rates. Using long-simulations dynamic allostery analysis, this study describes an exploration of the dynamic kinase:peptide complex. We have used protein kinase A (PKA) as a model system as a generic prototype of the protein kinase superfamily of signaling enzymes. Our results explain the role of dynamic coupling of active-site residues that must work in coherence to provide for a successful activation or inhibition response from the kinase. Amino acid networks-based community analysis allows us to ponder the conformational entropy of the kinase:nucleotide:peptide ternary complex. We use a combination of 7 peptides that include 3 types of PKA-binding partners: Substrates, products, and inhibitors. The substrate peptides provide for dynamic insights into the enzyme:substrate complex, while the product phospho-peptide allows for accessing modes of enzyme:product release. Mapping of allosteric communities onto the PKA structure allows us to locate the more unvarying and flexible dynamic regions of the kinase. These distributions, when correlated with the structural elements of the kinase core, allow for a detailed exploration of key dynamics-based signatures that could affect peptide recognition and binding at the kinase active site. These studies provide a unique dynamic allostery-based perspective to kinase:peptide complexes that have previously been explored only in a structural or thermodynamic context.
“…Thus, there is a need to perform studies that addresses the internal motions for both binding partners. Furthermore, many of the previously published NMR ps-ns dynamics studies on protein binding have mostly focused on backbone motions, but it has been demonstrated that it is crucial to also investigate side chain dynamics, which can correspond to substantial conformational entropy, are heterogeneous, are involved in allosteric phenomena, and can have a significant response upon binding 1,4,6,11,26,27 . However, studies in which the side chain fast dynamics have been investigated remain a minority among the experimental studies on protein dynamics by NMR.Figure 1The 3D structure of the TAZ1/CTAD-HIF-1α complex 16 (pdb code 1L8C).…”
The interaction between the C-terminal transactivation domain of HIF-1α (CTAD-HIF-1α) and the transcriptional adapter zinc binding 1 (TAZ1) domain of CREB binding protein participate in the initiation of gene transcription during hypoxia. Unbound CTAD-HIF-1α is disordered but undergoes a disorder-to-order transition upon binding to TAZ1. We have here performed NMR side chain and backbone relaxation studies on TAZ1 and side chain relaxation measurements on CTAD-HIF-1α in order to investigate the role of picosecond to nanosecond dynamics. We find that the internal motions are significantly affected upon binding, both on the side chain and the backbone level. The dynamic response corresponds to a conformational entropy change that contributes substantially to the binding thermodynamics for both binding partners. Furthermore, the conformational entropy change for the well-folded TAZ1 varies upon binding to different IDP targets. We further identify a cluster consisting of side chains in bound TAZ1 and CTAD-HIF-1α that experience extensive dynamics and are part of the binding region that involves the N-terminal end of the LPQL motif in CTAD-HIF-1α; a feature that might have an important role in the termination of the hypoxic response.
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