Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases.

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

LRRK2 dynamics analysis identifies allosteric control of the crosstalk between its catalytic domains.

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.

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2.

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 LRRK2RCKW 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 LRRK2RCKW 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.

PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain.

Leucine-rich-repeat-kinase 1 (LRRK1) and its homolog LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinson's disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKCα unexpectedly does not activate LRRK1 by phosphorylating the kinase domain, but instead phosphorylates a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain of the GTPase domain. These residues are positioned at the equivalent region of the LRRK2 DK helix reported to stabilize the kinase domain αC-helix in the active conformation. Thr1075 represents an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation of Ser1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ∼3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 activates LRRK1 by promoting interaction and stabilization of the αC-helix on the kinase domain. This study provides new fundamental insights into the mechanism controlling LRRK1 activity and reveals a novel unexpected activation mechanism.

Conformational plasticity of the ULK3 kinase domain.

The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

Trypsiligase-Catalyzed Labeling of Proteins on Living Cells.

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins' native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.

Lessons from LIMK1 enzymology and their impact on inhibitor design.

LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique 'rock-and-poke' mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding.

Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1.

Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure-affinity relationships for a series of Bromosporine-based bivalent bromodomain ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1). Experimental techniques-Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance-were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein-ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro, due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.

Melanin is not required for turgor generation but enhances cell-wall rigidity in appressoria of the corn pathogen Colletotrichum graminicola.

The ascomycete and causative agent of maize anthracnose and stem rot, Colletotrichum graminicola, differentiates melanized infection cells called appressoria that are indispensable for breaching the plant cell wall. High concentrations of osmolytes accumulate within the appressorium, and the internal turgor pressure of up to 5.4 MPa provides sufficient force to penetrate the leaf epidermis directly. In order to assess the function of melanin in C. graminicola appressoria, we identified and characterized the polyketide synthase 1 (CgPKS1) gene which displayed high similarity to fungal polyketide synthases (PKS) involved in synthesis of 1,3,6,8-tetrahydronaphthalene, the first intermediate in melanin biosynthesis. Cgpks1 albino mutants created by targeted gene disruption were unable to penetrate intact leaves and ruptured frequently but, surprisingly, were able to penetrate ultrathin polytetrafluoroethylene membranes mimicking the plant surface. Nonmelanized Cgpks1 appressoria were sensitive to externally applied cell-wall-degrading enzymes whereas melanized appressoria were not affected. Expression studies using a truncated CgPKS1 fused to green fluorescent protein revealed fluorescence in immature appressoria and in setae, which is in agreement with transcript data obtained by RNA-Seq and quantitative polymerase chain reaction. Unexpectedly, surface scans of mutant and wild-type appressoria revealed considerable differences in cell-wall morphology. Melanization of appressoria is indispensable for successful infection of intact leaves. However, cell collapse experiments and analysis of the appressorial osmolyte content by Mach-Zehnder interferometry convincingly showed that melanin is not required for solute accumulation and turgor generation, thus questioning the role of melanin as a barrier for osmolytes in appressoria of C. graminicola.

Mechanism of human PINK1 activation at the TOM complex in a reconstituted system.

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear. Here, we report that co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit toward PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modeling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These findings will aid in the development of small-molecule activators of PINK1 as a therapeutic strategy for PD.

Tetrahydropyridine LIMK inhibitors: Structure activity studies and biological characterization.

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation.

Inhibition of Parkinson's disease-related LRRK2 by type I and type II kinase inhibitors: Activity and structures.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD) and a risk factor for the sporadic form. Increased kinase activity was shown in patients with both familial and sporadic PD, making LRRK2 kinase inhibitors a major focus of drug development efforts. Although much progress has been made in understanding the structural biology of LRRK2, there are no available structures of LRRK2 inhibitor complexes. To this end, we solved cryo-electron microscopy structures of LRRK2, wild-type and PD-linked mutants, bound to the LRRK2-specific type I inhibitor MLi-2 and the broad-spectrum type II inhibitor GZD-824. Our structures revealed an active-like LRRK2 kinase in the type I inhibitor complex, and an inactive DYG-out in the type II inhibitor complex. Our structural analysis also showed how inhibitor-induced conformational changes in LRRK2 are affected by its autoinhibitory N-terminal repeats. The structures provide a template for the rational development of LRRK2 kinase inhibitors covering both canonical inhibitor binding modes.

Structure of LRRK1 and mechanisms of autoinhibition and activation.

Leucine Rich Repeat Kinase 1 and 2 (LRRK1 and LRRK2) are homologs in the ROCO family of proteins in humans. Despite their shared domain architecture and involvement in intracellular trafficking, their disease associations are strikingly different: LRRK2 is involved in familial Parkinson's disease while LRRK1 is linked to bone diseases. Furthermore, Parkinson's disease-linked mutations in LRRK2 are typically autosomal dominant gain-of-function while those in LRRK1 are autosomal recessive loss-of-function. Here, to understand these differences, we solved cryo-EM structures of LRRK1 in its monomeric and dimeric forms. Both differ from the corresponding LRRK2 structures. Unlike LRRK2, which is sterically autoinhibited as a monomer, LRRK1 is sterically autoinhibited in a dimer-dependent manner. LRRK1 has an additional level of autoinhibition that prevents activation of the kinase and is absent in LRRK2. Finally, we place the structural signatures of LRRK1 and LRRK2 in the context of the evolution of the LRRK family of proteins.

Sfp-type 4'-phosphopantetheinyl transferase is indispensable for fungal pathogenicity.

In filamentous fungi, Sfp-type 4'-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary (alpha-aminoadipate reductase [AAR]) and secondary (polyketide synthases and nonribosomal peptide synthetases) metabolism. We cloned the PPTase gene PPT1 of the maize anthracnose fungus Colletotrichum graminicola and generated PPTase-deficient mutants (Deltappt1). Deltappt1 strains were auxotrophic for Lys, unable to synthesize siderophores, hypersensitive to reactive oxygen species, and unable to synthesize polyketides (PKs). A differential analysis of secondary metabolites produced by wild-type and Deltappt1 strains led to the identification of six novel PKs. Infection-related morphogenesis was affected in Deltappt1 strains. Rarely formed appressoria of Deltappt1 strains were nonmelanized and ruptured on intact plant. The hyphae of Deltappt1 strains colonized wounded maize (Zea mays) leaves but failed to generate necrotic anthracnose disease symptoms and were defective in asexual sporulation. To analyze the pleiotropic pathogenicity phenotype, we generated AAR-deficient mutants (Deltaaar1) and employed a melanin-deficient mutant (M1.502). Results indicated that PPT1 activates enzymes required at defined stages of infection. Melanization is required for cell wall rigidity and appressorium function, and Lys supplied by the AAR1 pathway is essential for necrotrophic development. As PPTase-deficient mutants of Magnaporthe oryzea were also nonpathogenic, we conclude that PPTases represent a novel fungal pathogenicity factor.

Enabling pseudokinases as potential drug targets.

Pseudokinases play significant roles in disease development. Similar to active kinases, their cellular functions can be targeted pharmacologically. But notably, instead of inhibiting an enzymatic activity, drug-like molecules act by stabilizing distinct pseudokinase conformations, by interfering with protein interactions, or by inducing proteasomal degradation. Herein, we describe our approach of enabling particular pseudokinases as potential drug targets. The method starts with obtaining recombinant proteins for assay development and for biochemical evaluation. The next step is to probe the pseudoactive site as a binding pocket for small molecules, providing initial insight into binding modes and even candidate chemotypes. Finally, structural features of pseudokinase:inhibitor complexes are explored. Taken together, we provide detailed method descriptions for essential inhibitor development technologies.

Structural Aspects of LIMK Regulation and Pharmacology.

Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.

Identification of Pyrimidine-Based Lead Compounds for Understudied Kinases Implicated in Driving Neurodegeneration.

The pyrimidine core has been utilized extensively to construct kinase inhibitors, including eight FDA-approved drugs. Because the pyrimidine hinge-binding motif is accommodated by many human kinases, kinome-wide selectivity of resultant molecules can be poor. This liability was seen as an advantage since it is well tolerated by many understudied kinases. We hypothesized that nonexemplified aminopyrimidines bearing side chains from well-annotated pyrimidine-based inhibitors with off-target activity on understudied kinases would provide us with useful inhibitors of these lesser studied kinases. Our strategy paired mixing and matching the side chains from the 2- and 4-positions of the parent compounds with modifications at the 5-position of the pyrimidine core, which is situated near the gatekeeper residue of the binding pocket. Utilizing this approach, we imparted improved kinome-wide selectivity to most members of the resultant library. Importantly, we also identified potent biochemical and cell-active lead compounds for understudied kinases like DRAK1, BMP2K, and MARK3/4.