In this study, we utilize Protein Residue Networks (PRNs), constructed using Local Spatial Pattern (LSP) alignment, to explore the dynamic behavior of Catabolite Activator Protein (CAP) upon the sequential binding of cAMP. We employed the Degree Centrality of these PRNs to investigate protein dynamics on a sub-nanosecond time scale, hypothesizing that it would reflect changes in CAPs entropy related to its thermal motions. We show that the binding of the first cAMP led to an increase in stability in the Cyclic-Nucleotide Binding Domain A (CNBD-A) and destabilization in CNBD-B, agreeing with previous reports explaining the negative cooperativity of cAMP binding in terms of an entropy-driven allostery. LSP-based PRNs also allow for the study of Betweenness Centrality, another graph-theoretical characteristic of PRNs, providing insights into global residue connectivity within CAP. Using this approach, we were able to correctly identify amino acids that were shown to be critical in mediating allosteric interactions in CAP. The agreement between our studies and previous experimental reports validates our method, particularly with respect to the reliability of Degree Centrality as a proxy for entropy related to protein thermal dynamics. Because LSP-based PRNs can be easily extended to include dynamics of small organic molecules, polynucleotides, or other allosteric proteins, the methods presented here mark a significant advancement in the field, positioning them as vital tools for a fast, cost-effective, and accurate analysis of entropy-driven allostery and identification of allosteric hotspots.

Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs.

Mutations in the human leucine rich repeat protein kinase-2 (LRRK2) create risk factors for Parkinson's disease, and pathological functions of LRRK2 are often correlated with aberrant kinase activity. Past research has focused on developing selective LRRK2 kinase inhibitors. In this study, we combined enhanced sampling simulations with HDX-MS to characterize the inhibitor-induced dynamic changes and the allosteric communications within the C-terminal domains of LRRK2, LRRK2^RCKW. We find that the binding of MLi-2 (a type I kinase inhibitor) stabilizes a closed kinase conformation and reduces the global dynamics of LRRK2^RCKW, leading to a more compact LRRK2^RCKW structure. In contrast, the binding of Rebastinib (a type II kinase inhibitor) stabilizes an open kinase conformation, which promotes a more extended LRRK2^RCKW structure. By probing the distinct effects of the type I and type II inhibitors, key interdomain interactions are found to regulate the communication between the kinase domain and the GTPase domain. The intermediate states revealed in our simulations facilitate the efforts toward in silico design of allosteric modulators that control LRRK2 conformations and potentially mediate the oligomeric states of LRRK2 and its interactions with other proteins.

Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCβ-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

LRRK2 is a multi-domain protein with three catalytically inert N-terminal domains (NtDs) and four C-terminal domains, including a kinase and a GTPase domain. LRRK2 mutations are linked to Parkinson's Disease. Recent structures of LRRK2RCKW and a full-length inactive LRRK2 (fl-LRRK2INACT) monomer revealed that the kinase domain drives LRRK2 activation. The LRR domain and also an ordered LRR- COR linker, wrap around the C-lobe of the kinase domain and sterically block the substrate binding surface in fl-LRRK2INACT. Here we focus on the crosstalk between domains. Our biochemical studies of GTPase and kinase activities of fl-LRRK2 and LRRK2RCKW reveal how mutations influence this crosstalk differently depending on the domain borders investigated. Furthermore, we demonstrate that removing the NtDs leads to altered intramolecular regulation. To further investigate the crosstalk, we used Hydrogen-Deuterium exchange Mass Spectrometry (HDX-MS) to characterize the conformation of LRRK2RCKW   and Gaussian Accelerated Molecular Dynamics (GaMD) to create dynamic portraits of fl-LRRK2 and LRRK2RCKW. These models allowed us to investigate the dynamic changes in wild type and mutant LRRK2s. Our data show that the a3ROC helix, the Switch II motif in the ROC domain, and the LRR-ROC linker play crucial roles in mediating local and global conformational changes. We demonstrate how these regions are affected by other domains in fl-LRRK2 and LRRK2RCKW and show how unleashing of the NtDs as well as PD mutations lead to changes in conformation and dynamics of the ROC and kinase domains which ultimately impact kinase and GTPase activities. These allosteric sites are potential therapeutic targets.

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIβ, RIIα, RIIβ), RIα is most essential for regulating PKA activity in cells. Our 2 RIα₂C₂ holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg²⁺ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIβ, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A-N3A' motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIβ holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

Mutations of the human leucine-rich repeat kinase 2 (LRRK2) have been associated with both, idiopathic and familial Parkinson's disease (PD). Most of these pathogenic mutations are located in the kinase domain (KD) or GTPase domain of LRRK2. In this study we describe a mechanism in which protein kinase activity can be modulated by reversible oxidation or reduction, involving a unique pair of adjacent cysteines, the "CC" motif. Among all human protein kinases, only LRRK2 contains this "CC" motif (C2024 and C2025) in the Activation Segment (AS) of the kinase domain. In an approach combining site-directed mutagenesis, biochemical analyses, cell-based assays, and Gaussian accelerated Molecular Dynamics (GaMD) simulations we could attribute a role for each of those cysteines. We employed reducing and oxidizing agents with potential clinical relevance to investigate effects on kinase activity and microtubule docking. We find that each cysteine gives a distinct contribution: the first cysteine, C2024, is essential for LRRK2 protein kinase activity, while the adjacent cysteine, C2025, contributes significantly to redox sensitivity. Implementing thiolates (R-S^-) in GaMD simulations allowed us to analyse how each of the cysteines in the "CC" motif interacts with its surrounding residues depending on its oxidation state. From our studies we conclude that oxidizing agents can downregulate kinase activity of hyperactive LRRK2 PD mutations and may provide promising tools for therapeutic strategies.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2_RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2_RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.