Explore the vast range of titles available.

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 (LRRK2 RCKW ). 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.

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.

Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction. The irradiation and chemotherapy drugs that are used to destroy cancer cells also damage healthy cells. Germ cells – from which egg cells and sperm cells develop – are particularly vulnerable as they contain sensitive quality control mechanisms that kill any cell that contain damaged DNA. Consequently, after surviving cancer many patients are confronted with infertility. A protein called p63, which is closely related to another protein that suppresses the formation of tumors, plays an essential role in detecting and responding to DNA damage. In immature egg cells (also known as oocytes), p63 mostly exists in an inactive form. The protein then switches to an active form when DNA damage is detected to trigger the process of cell self-destruction. Now, Coutandin, Osterburg et al. have performed a range of biochemical, biophysical and cell culture experiments to study how p63 is kept in its inactive form in the oocytes of mice. The experiments showed that in the inactive form, the two ends of the protein form a sheet that closes a key site on the protein and prevents it from changing into its active form. However, this closed form can be thought of as being like a spring-loaded trap – it doesn’t take much energy to spring the trap and open the protein into its active form. Once this change has occurred, it is irreversible. Coutandin, Osterburg et al. also found that the oocytes of mice already contain all the proteins necessary to activate p63. This means that once the switch to the active form is triggered there is no delay waiting for other proteins to be made, which makes oocytes extremely sensitive to DNA damage. Further work is now needed to investigate the exact molecular mechanisms behind the activation of p63.

[This corrects the article DOI: 10.1371/journal.pbio.3001427.].

Protein kinases and GTPases are the two major molecular switches that regulate much of biology, and both of these domains are embedded within the large multi-domain Leucine-Rich Repeat Kinase 2 (LRRK2). Mutations in LRRK2 are the most common cause of familial Parkinson’s Disease (PD) and are also implicated in Crohn’s disease (Hui et al., 2018). The recent Cryo Electron Microscopy (Cryo-EM) structure of the four C-terminal domains (RCKW) of LRRK2 includes both of the catalytic domains (Deniston et al., 2020). Although the important allosteric N-terminal domains are missing in the Cryo-EM structure this structure allows us to not only explore the conserved features of the kinase domain, which is trapped in an inactive and open conformation, but also to observe the direct allosteric cross talk between the two domains. To define the unique features of the kinase domain and to better understand the dynamic switch mechanism that allows LRRK2 to toggle between its inactive and active conformations, we have compared the LRRK2 kinase domain to Src, BRaf, and PKA. We also compare and contrast the two canonical glycine-rich loop motifs in LRRK2 that anchor the nucleotide: the G-Loop in protein kinases that anchors ATP and the P-Loop in GTPases that anchors GTP. The GTPase domain of LRRK2 belongs to the Roco protein family and plays an important role as an allosteric effector domain (Bosgraaf et al., 2003, Marin et al., 2008). The RCKW structure also provides a template for the cross-talk between the kinase and GTPase domains and brings new mechanistic insights into the physiological function of LRRK2 and how the kinase domain, along with key phosphorylation sites, can serve as an allosteric hub for mediating conformational changes.

E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo . Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.

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.

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. Reimer et al. used cryo-EM and cellular assays to reveal the structural and regulatory features that distinguish LRRK1 from LRRK2, and placed these features in the context of the evolution of the LRRK family of proteins.

Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens , the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins.

Members of the p53 tumor-suppressor family are expressed as multiple isoforms. Isoforms with an N-terminal transactivation domain are transcriptionally active, while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63 α inhibits the tumor-suppressor function of TAp73 β . This can in principle be due to blocking of the promoter or by direct interaction between both proteins. p63 and p73 can hetero-oligomerize through their tetramerization domains and a hetero-tetramer consisting of two p63 and two p73 molecules is thermodynamically more stable than both homo-tetramers. Here we show that cells expressing both p63 and p73 exist in mouse epidermis and hair follicle and that hetero-tetramer complexes can be detected by immunoprecipitation in differentiating keratinocytes. Through structure determination of the hetero-tetramer, we reveal why this hetero-tetramer is the thermodynamically preferred species. We have created mutants that exclusively form either hetero-tetramers or homo-tetramers, allowing to investigate the function of these p63/p73 hetero-tetramers. Using these tools, we show that inhibition of TAp73 β in squamous cell carcinomas is due to promoter squelching and not direct interaction.