Structure and regulation of full-length human leucine-rich repeat kinase 1.

The human leucine-rich repeat kinases (LRRKs), LRRK1 and LRRK2 are large and unusually complex multi-domain kinases, which regulate fundamental cellular processes and have been implicated in human disease. Structures of LRRK2 have recently been determined, but the structure and molecular mechanisms regulating the activity of the LRRK1 as well as differences in the regulation of LRRK1 and LRRK2 remain unclear. Here, we report a cryo-EM structure of the LRRK1 monomer and a lower-resolution cryo-EM map of the LRRK1 dimer. The monomer structure, in which the kinase is in an inactive conformation, reveals key interdomain interfaces that control kinase activity as we validate experimentally. Both the LRRK1 monomer and dimer are structurally distinct compared to LRRK2. Overall, our results provide structural insights into the activation of the human LRRKs, which advance our understanding of their physiological and pathological roles.

Biochemical Discovery, Intracellular Evaluation, and Crystallographic Characterization of Synthetic and Natural Product Adenosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase A (PKA) Inhibitors.

The recent demonstration that adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) plays an oncogenic role in a number of important cancers has led to a renaissance in drug development interest targeting this kinase. We therefore have established a suite of biochemical, cell-based, and structural biology assays for identifying and evaluating new pharmacophores for PKA inhibition. This discovery process started with a 384-well high-throughput screen of more than 200,000 substances, including fractionated natural product extracts. Identified active compounds were further prioritized in biochemical, biophysical, and cell-based assays. Priority lead compounds were assessed in detail to fully characterize several previously unrecognized PKA pharmacophores including the generation of new X-ray crystallography structures demonstrating unique interactions between PKA and bound inhibitor molecules.

Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding.

RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease.

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and 19F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.

Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα.

Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAcα, a kinase fusion chimera of the J domain of DnaJB1 with PKAcα, the catalytic subunit of protein kinase A (PKA). Here we report the crystal structures of the chimeric fusion RIα2:J-PKAcα2 holoenzyme formed by J-PKAcα and the PKA regulatory (R) subunit RIα, and the wild-type (WT) RIα2:PKAcα2 holoenzyme. The chimeric and WT RIα holoenzymes have quaternary structures different from the previously solved WT RIß and RIIß holoenzymes. The WT RIα holoenzyme showed the same configuration as the chimeric RIα2:J-PKAcα2 holoenzyme and a distinct second conformation. The J domains are positioned away from the symmetrical interface between the two RIα:J-PKAcα heterodimers in the chimeric fusion holoenzyme and are highly dynamic. The structural and dynamic features of these holoenzymes enhance our understanding of the fusion chimera protein J-PKAcα that drives FLHCC as well as the isoform specificity of PKA.