Nucleotide-binding Loop Research Articles

Disease-associated mutations in Drp1 have fundamentally different effects on the mitochondrial fission machinery.

Patient mutations have been identified throughout dynamin-related protein 1 (Drp1), the key mediator of mitochondrial fission. These mutations generally impact children and often result in severe neurological defects and, in some cases, death. Previously, the underlying functional defect leading to patient phenotypes has been speculated based on comparisons with synthetic mutations at similar sites. Drp1 is comprised by four domains, including a GTPase domain (G-domain) that regulates GTP binding and hydrolysis and a middle domain (MD) that mediates Drp1 self-assembly. We analyzed six mutations, four in the MD and two in the G-domain. All the MD mutants studied were found to be dimer-limited in solution, and three of these MD mutants were found to be impaired in self-assembly. However, one of the MD mutants retains oligomerization capability despite being located within this self-assembly region. Further, this mutant retains its ability to interact with a pre-formed lipid template but is unable to re-shape large, unilamellar vesicles (LUVs). This indicates that Drp1 dimers alone are sufficient to form a helical polymer on lipid template. Differences were also observed between the two G-domain mutations. One of the mutants is in a nucleotide-binding loop that is critical for functional hydrolysis. This mutant exhibited impaired GTP hydrolysis but can still oligomerize in solution and with a lipid template. In contrast, the other G-domain mutation is located relatively distant from the nucleotide binding site. This mutant poorly tubulates LUVs, highlighting the role of G-domain interactions in driving membrane curvature. Overall, the functional defects caused by mutations in Drp1 are highly variable even when the mutations occur within the same functional domain. This study provides a framework for characterizing additional Drp1 mutations to provide a comprehensive understanding of functional sites within this essential protein.

A Novel Autosomal Dominant JAK2R715T Mutation Causes Polycythemia Vera in 3 Generations

Polycythemia vera (PV) has high hemoglobin (Hb) concentration caused by clonal proliferation of myeloid cells from a pluripotent stem cell (Prchal, N Engl J Med. 1974). Acquired somatic mutations of JAK2 gene, JAK2V617F and rarely exon 12, are present in virtually all PV patients (Ugo, Nature 2005, Scott N Engl J Med. 2007). The thromboses are the major causes of PV-related morbidity and mortality. A 38-year female suffered an ischemic stroke in 2017 at age 32 with negative thrombophilia work-up. She had elevated hemoglobin (Hb) 18 g/dL with intermittent elevation of leukocytes and normal platelets with borderline low erythropoietin (EPO) levels. JAK2V617F and exon 12 mutation analysis were negative. Her marrow was interpreted as compatible with PV and normal cytogenetics. Her hematologist treated her with phlebotomies. She was referred to us for an additional evaluation and management. Myeloid next generation sequencing (NGS) revealed a previously undescribed mutation JAK2R715T in the pseudokinase domain (JH2) with allelic burden ~50%. Some of her erythroid progenitors (BFU-Es) grew without extrinsic EPO, a hallmark of PV (Prchal, N Engl J Med. 1974). Phlebotomies were stopped; in two months, her Hb had risen to 17.4 g/dL and hematocrit had changed to 48.5% with normal leukocytes and platelets. We obtained JAK2 inhibitor fedratinib on compassionate basis from the manufacturer. On fedratinib, her Hb normalized to 15.2 g/dL with 42.8 % hematocrit. Her iron deficiency was corrected without increase of her Hb. The patient's germline mutation was confirmed by DNA sequencing of her nails. We encouraged her to have her only son and parents tested by NGS. The same JAK2R715T mutation was found in her 10-year-old son's blood sample. He also has elevated Hb at 16.2 g/dL (normal for age 11.5-15.5 g/dL), normal leukocytes and platelets were 400 K/µl. Her asymptomatic 63-year-old mother’ Hb was 17 g/dL, hematocrit 50.5%, and sequencing of her JAK2 also revealed JAK2R715T at ~50%. To assess how the R715T mutation may affect kinase activation, the JH1-JH2 domains of JAK2R715T and JAK2WT were modelled using AlphaFold 2.0 (Jumper, Nature 2021). The overall structure of JAK2R715T JH2 was more rigid than that of JAK2WT, in agreement with DynaMut in silico prediction (PMID: 29718330). -Helix C is shown in dark blue, Y570 and T555 in yellow, and ATP and T715 in red. Detailed comparison of JAK2R715T with JAK2WT JH2 (see Figure) indicated that the rigidification of the structure involved notably residues of the JH2-JH2 trans-dimerization domain centered around -Helix C. This has been suggested to mediate direct JH2-JH2 contacts (Wilmes, Science 2020) and a lengthening of the ß-sheet region surrounding the inhibitory phosphorylation site Tyr570 (Echarri, Mol Cell Biol. 2004). The R715 is in direct vicinity of the ATP binding pocket and promotes a closed lobe conformation through interaction with Thr555 of the nucleotide-binding loop (Bandaranayake, Nat Struct Mol Biol. 2012). We suggest that loss of this interaction may result in a more opened lobe conformation and disturbing of ATP binding to JH2, which regulates JAK2 kinase activity (Hammarén, Proc Natl Acad Sci USA 2015). To evaluate the effect of JAK2R715T in erythroid progenitor cells, the patient's erythroid progenitors derived from peripheral mononuclear cells were grown for 21 days with and without JAK inhibitor (ruxolitinib) following the previously published in vitro expansion protocol (Bruchova, Blood Cells Mol Dis, 2009). Compared to PVs and healthy controls, the patient cells showed increased proliferation. JAK inhibitor ruxolitinib treatment of patient cells induced decreased proliferation and differentiation compared to untreated cells. Assessment of STAT5 phosphorylation on patient's blood cells confirmed increased JAK2 activity to levels even higher than this of patients with confirmed JAK2V617F positive PV. X-chromosome based clonality studies of both females are ongoing. The EPO-independent colonies of her BFU-E is being evaluated for possible acquired conversion to homozygosity by uniparental disomy that is present in PV (Kralovics, Experimental Hematology, 2002). We conclude that this is the first example of dominantly inherited PV phenotype in 3 generations due to a novel germline JAK2R715T gain-of function mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DOCKING STUDIES ON IMIDAZOLIDINE ANALOGUES FOR MANAGEMENT OF DIABETES

Glycogen synthase kinase-3I² (GSK-3I²) has recently emerged, in the field of medicinal chemistry, as one of the most attractive therapeutic targets for type II diabetes. Phenylmethylene hydantoins (PMHs) forms strong interactions with the hinge region of GSK-3I²; carbonyl oxygen at position 2 form a H-bonding with backbone nitrogen of Val135 and the NH at position 3 to the carbonyl oxygen of Asp133. The hydantoin ring was sandwiched between Ala83, on top, and Leu188, on the bottom. The aromatic ring is rotated out of plane from the hydantoin plane, allowing extensive interactions with the nucleotide-binding loop. Furthermore, the substituted benzylidene ring system builds an H-bonding interaction with the guanidine moiety of Arg141. Targeting Arg141 is important to improve the activity in the process of designing new derivatives because it is considered the selectivity residue for GSK-3I².

Establishment of a mutation system in Tetrahymena outer arm dynein and P-loop functions of the alpha heavy chain (Dyh3p)

Axonemal dyneins are large AAA+ type motor proteins that exhibit unique motor properties during ciliary beating. This study established a mutation system for Tetrahymena outer arm dynein and characterized four nucleotide-binding loops (P-loops; P1-P4) in the alpha heavy chain (Dyh3p). Macronuclear transformation of the mutant DYH3 genes in DYH3-knockout (KO-DYH3) cells enabled P-loop mutations that abolish the ability of nucleotide binding to be stably maintained in the polyploid genome. This mutation system revealed that the P3 and P4 mutant dyneins rescued lethality in macronuclear KO-DYH3 cells and exhibited normal ciliary localization. Intriguingly, however, an in vitro motility assay showed that the P3 mutation abolished the motor activity of Dyh3p, whereas the P4 mutation did not affect the gliding velocity or gliding index of Dyh3p. In contrast, no P1 or P2 mutant cells were isolated from the KO-DYH3 cells, which suggests that nucleotide binding at the P1 and P2 sites is required for the intracellular function of Dyh3p. This mutation system will be useful for further molecular studies of diverse axonemal dyneins and ciliary motility.

The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase

The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.

Artocarpus altilis CG-901 alters critical nodes in the JH1-kinase domain of Janus kinase 2 affecting upstream JAK/STAT3 signaling.

As a key step in achieving low-cost, easily accessible anti-cancer therapy for low- and middle-income countries, we recently established the scientific basis for the folkloric use of Artocarpus altilis for the treatment of cancer by investigating the geranyl dihydrochalcone (CG-901) content and its interference with signal transducer and activator of transcription 3 (STAT3) phosphorylation and blockage of further downstream signaling. In the current study, the CG-901 upstream target was queried by chemical fingerprinting similarity assessment, semi-empirical (PM6ESCF) QMMM and molecular dynamics (MD) simulation. Moderate (∼0.4) to high (∼0.7) Tanimoto scores were found when the CG-901 scaffold was compared to ligands co-crystallized with Janus kinases (JAK) 1-3. High negative energy values were obtained when the CG-901 was treated semi-empirically (PM6ESCF) within the classical field of JAK (1-3). Multiple nanosecond MD simulations showed that CG-901 did not cause any large structural perturbations in the nucleotide-binding, activation and catalytic loops within the kinase (JH1) domain of JAK (1-3); however, it reduced the energy required to attain metastability along the path to energy minima conformation. In comparison to JAK1 and Apo-state JAK2, JAK2-bound CG-901 exhibited a highly re-organized key intra-domain protein network; indicating atomic level interference with inter-residue communication. In conclusion, CG-901 isolated from A. altilis represents a broad-spectrum JAK inhibitor, which may underlie the mechanism of STAT3 phosphorylation blockage. Graphical abstract Upper panel Janus kinase 2 upstream signaling pathway. Lower panel Apo-JAK2 (left) and CG-901-bound JAK2 (right).

Structure and function of ADCK3, an ancient mitochondrial protein involved in coenzyme Q biosynthesis

Coenzyme Q (Q) is an essential lipid best known for its role as an electron carrier in the electron transport chain. Although required for human health, Q biosynthesis is not completely understood and the functions of many Q biosynthetic proteins remain unclear. The atypical mitochondrial kinase, ADCK3, is a Q biosynthetic protein of unknown function belonging to the UbiB protein kinase‐like family. Here, we determine a crystal structure of ADCK3, the first crystal structure of a UbiB protein. We characterize the unique alanine‐rich nucleotide binding loop that dictates an unusual binding preference for ADP over ATP. We find that this preference can be reversed with a single alanine to glycine mutation, which also enables autophosphorylation while inhibiting Q biosynthesis in vivo. Furthermore, we identify an N‐terminal domain that blocks the substrate binding pocket—another inhibitory feature. This work serves as a foundation for further structure‐function analyses aimed at defining the role of ADCK3 in Q biosynthesis as part of a larger effort to treat human Q deficiency.

Molecular dynamic simulations of the tubulin–human gamma synuclein complex: structural insight into the regulatory mechanism involved in inducing resistance against Taxol

Members of the synuclein family (α, β and γ synucleins) are intrinsically disordered in nature and play a crucial role in the progression of various neurodegenerative disorders and cancers. The association of γSyn with both BubR1 as well as microtubule subunits renders resistance against various anti-cancer drugs. However, the structural aspects underlying drug resistance have not been explored. In this study, the mechanism involved in the association between γSyn and microtubule subunits (αβTub) was investigated and the results reveal a strong interaction between γSyn and the tail regions of αβTub. Complexation of γSyn induces conformational rearrangements in the nucleotide binding loops (NBL), interdomain and tail regions of both α and βTub. Moreover, in βTub, the massive displacement observed in M and S loops significantly alters the binding site of microtubule targeting drugs like Taxol. The resulting weak association between Taxol and βTub of the γSyn-αβTub complex was confirmed by molecular dynamic simulation studies. In addition, the effect of Taxol on NBL, M and S loops of αβTub, is reversed in the presence of γSyn. These results clearly indicate that the presence of γSyn annulled the allosteric regulation imposed by Taxol on the αβTub complex as well as preventing the binding of microtubule targeting drugs, which eventually leads to the development of resistance against these drugs in cancer cells.

Location of FLT3-ITDs within the beta1-Sheet of FLT3 Kinase Confers Resistance to Tyrosine Kinase Inhibitors (TKI) in Vitro.

Abstract 2440 Introduction:In AML, the recently described tyrosine kinase domain-1 (TKD1)-ITDs of FLT3 (Breitenbuecher et al., Blood 2009, Kayser-S et al., Blood 2009) are located within the beta1-sheet, nucleotide binding loop and beta2-sheet of tyrosine kinase domain 1 (TKD1), respectively. Multivariate analysis of clinical data revealed that location of FLT3-ITDs within the beta1-sheet of TKD1 is an unfavorable prognostic factor (Kayser-S et al., Blood 2009). Recently, we uncovered a novel mechanism of primary resistance to FLT3 tyrosine kinase inhibitors (TKIs) in a patient displaying an atypical localization within the beta2-sheet-ITD (A627E). Here, we characterized in-vitro sensitivity to FLT3-TKI in growth factor dependent hematopoietic cell lines expressing a representative panel of FLT3-ITDs isolated from patient material. In particular, we compared sensitivity of beta1-sheet ITDs with typical ITDs located in the juxtamembrane domain of FLT3. Methods:FLT3-ITDs isolated from patient material, were sequenced, subcloned in parallel into two expression vectors (pAL and pMSCV-Puromycin-IRES-GFP), and stably expressed in growth-factor dependent hematopoietic Ba/F3 cells and in parallel in 32D cells. Constitutive phosphorylation of FLT3-ITD receptors and of downstream signaling pathways was analyzed by Western-blotting. Transformation of Ba/F3 and 32D cells was investigated by colony formation assays and by withdrawal of IL-3. Induction of apoptosis in response to various concentrations of FLT3-kinase inhibitors midostaurin (PKC412) and quizartinib (AC220) was measured by flow-cytometry at 24h and 48h, respectively. For statistical analysis of replicates t-test was employed. Results:Biological characteristics of FLT3-ITD mutations located in two different structural domains of FLT3-kinase were characterized: (1) beta1-sheet-ITDs E611V(96nt) and Q613E(99nt) and (2) nucleotide binding loop-ITD A620V(84nt). (Our nomenclature used for description of ITDs indicates position of amino-acid where ITD is located, possible exchange of amino-acid residues at this position and nucleotide length of ITD) Hematopoietic cells (32D and Ba/F3) expressing these ITD mutations showed colony formation in methylcellulose medium, growth-factor independent proliferation upon IL-3 withdrawal and constitutive phosphorylation of FLT3 signaling. Three ‘typical' juxtamembrane domain (JMD) ITDs were used as controls; ITD598/599(36nt), ITD598/599(66nt) and ITDK602R(21nt). As compared to these JM-ITDs, we observed significantly less apoptosis of beta1-sheet-ITDs at all concentrations of FLT3-kinase inhibitors applied. However, this difference in sensitivity gradually decreased when incubating with midostaurin or quizartinib for longer periods of time as 48h. Of note, length of FLT3-ITD mutations did not appear to influence sensitivity to TKI treatment. Conclusion:Investigating representative FLT3-ITD-mutations located within the TKD1 revealed that beta1-sheet-ITDs mediate constitutive activation of FLT3 receptors leading to transformation of hematopoietic cell lines 32D and Ba/F3. In comparison to typical JMD-ITDs, beta1-sheet-ITDs analyzed here revealed resistance to FLT3-inhibitors as midostaurin and quizartinib across two cellular reconstitution models (32D and Ba/F3) and using two different expression vectors (pAL and pMSCV). Our results indicate that differential sensitivity is rather an effect of ITD-localization within a functional domain of FLT3 and not necessarily conferred by the length of ITDs. Taken together, our data provide a rationale to prospectively analyze not only the FLT3-ITD mutation status or FLT3-ITD allelic ratio but also location of ITD-mutations in ongoing clinical trials as this may have direct impact on response to therapy (tyrosine kinase inhibitors, chemotherapy, allogeneic stem cell transplantation) in FLT3-ITD positive AML. Disclosures:Heidel:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fischer:Novartis: Honoraria.

The KdpC subunit of the Escherichia coli K+‐transporting KdpB P‐type ATPase acts as a catalytic chaperone

In Bacteria and Archaea, high-affinity potassium uptake is mediated by the ATP-driven KdpFABC complex. On the basis of the biochemical properties of the ATP-hydrolyzing subunit KdpB, the transport complex is classified as type IA P-type ATPase. However, the KdpA subunit, which promotes K(+) transport, clearly resembles a potassium channel, such that the KdpFABC complex represents a chimera of ion pumps and ion channels. In the present study, we demonstrate that the blending of these two groups of transporters in KdpFABC also entails a nucleotide-binding mechanism in which the KdpC subunit acts as a catalytic chaperone. This mechanism is found neither in P-type ATPases nor in ion channels, although parallels are found in ABC transporters. In the latter, the ATP nucleotide is coordinated by the LSGGQ signature motif via double hydrogen bonds at a conserved glutamine residue, which is also present in KdpC. High-affinity nucleotide binding to the KdpFABC complex was dependent on the presence of this conserved glutamine residue in KdpC. In addition, both ATP binding to KdpC and ATP hydrolysis activity of KdpFABC were sensitive to the accessibility, presence or absence of the hydroxyl groups at the ribose moiety of the nucleotide. Furthermore, the KdpC subunit was shown to interact with the nucleotide-binding loop of KdpB in an ATP-dependent manner around the ATP-binding pocket, thereby increasing the ATP-binding affinity by the formation of a transient KdpB/KdpC/ATP ternary complex.

Bacterial Tubulin Distinct Loop Sequences and Primitive Assembly Properties Support Its Origin from a Eukaryotic Tubulin Ancestor

The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic αβ-tubulin but, strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either α- or β-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide-binding loops are more conserved, and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic cytosolic chaperonin, which is absent from the Prosthecobacter dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than αβ-tubulin, forming pairs of protofilaments that coalesce into bundles instead of microtubules, and it lacks the ability to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like β-tubulin. BtubA/B chimera bearing the β-tubulin loops M, H1-S2, and S9-S10 in BtubB fold, assemble, and have reduced GTPase activity. However, introduction of the α-tubulin loop S9-S10 with its unique eight-residue insertion impaired folding. From the sequence analyses, its primitive assembly features, and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding α- and β-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution.

Crystal Structures of Two Aminoglycoside Kinases Bound with a Eukaryotic Protein Kinase Inhibitor

Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3′)-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3′)-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eurkaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.

Different FLT3-ITD Integration Sites Are Associated with Differential Sensitivity to Tyrosine Kinase Inhibitors (TKI) In Vitro.

AbstractAbstract 1709 Introduction:Currently, small molecule FLT3 tyrosine kinase inhibitors (TKIs) are promising therapeutic approaches to overcome the dismal prognosis of AML patients harbouring FLT3-ITD mutations. However, up to 30% of these patients show primary resistance to FLT3-TKIs. Recently, we uncovered a novel mechanism of primary resistance to FLT3 TKIs in a patient displaying an atypical integration site of ITD within the beta2-sheet (ITD_A627E). The data suggested that atypical integration sites of ITDs within the tyrosine kinase domain-1 (TKD1) of FLT3 (beta1-sheet, nucleotide binding loop and beta2-sheet) are associated with rewired signaling and differential responsiveness to TKIs. Here, we characterized response to FLT3-TKIs employing various TKD1-ITDs of the beta1-sheet and nucleotide binding loop, respectively in a cellular reconstitution model. Methods:FLT3 TKD1-ITDs isolated from patient material, were sequenced, subcloned and stably transfected into growth-factor dependent hematopoietic Ba/F3 cells. Constitutive FLT3 phosphorylation and activation of downstream signaling was analyzed by Western-blotting. Transformation potential was analyzed by colony formation in methylcellulose medium and by withdrawal of IL-3. Induction of apoptosis in response to TKI (midostaurin/sorafenib) was measured by FACS analysis. Results:Biological characteristics of FLT3-ITDs from two structural domains of FLT3-TKD1 were characterized: (1) beta1-sheet-ITDs E611V (96nt) and Q613E (99nt) and (2) nucleotide binding loop-ITD A620V (84nt). Ba/F3 cells expressing these ITDs showed colony formation in methylcellulose assays and growth-factor independent proliferation upon IL-3 withdrawal indicating transformation. Western-blotting revealed constitutive phosphorylation of FLT3 and of downstream signaling nodes (STAT5/AKT/ERK). As compared to a typical juxtamembrane domain (JMD) ITD (ITD598, 36nt), we observed significantly less induction of apoptosis in TKD1-ITDs investigated at 24h of incubation across all concentrations of midostaurin used. Similar results were observed when TKD1-ITDs were treated with different concentrations of sorafenib for 24h. However, this difference in sensitivity gradually decreased when incubating with midostaurin or sorafenib for longer periods of time as 36h and 48h. Currently, additional FLT3-TKIs are being investigated and results will be presented. Conclusion:Our results employing representative TKD1-ITDs from beta1-sheet and nucleotide binding loop revealed that TKD1-ITDs mediate constitutive activation of FLT3 receptors leading to transformation of hematopoietic cells. In comparison to a typical JMD-ITD, TKD1-ITDs analyzed revealed differential responsiveness in induction of apoptosis to midostaurin and sorafenib. Thus, our in vitro data suggest that different ITD integration sites may be associated with differential sensitivity to midostaurin and sorafenib in vivo and provides a rationale to prospectively analyze not only the FLT3-ITD mutation status but also the ITD integration site in ongoing/future clinical trials using FLT3-TKIs. Disclosures:No relevant conflicts of interest to declare.

Mapping Flexibility and the Assembly Switch of Cell Division Protein FtsZ by Computational and Mutational Approaches

The molecular switch for nucleotide-regulated assembly and disassembly of the main prokaryotic cell division protein FtsZ is unknown despite the numerous crystal structures that are available. We have characterized the functional motions in FtsZ with a computational consensus of essential dynamics, structural comparisons, sequence conservation, and networks of co-evolving residues. Employing this information, we have constructed 17 mutants, which alter the FtsZ functional cycle at different stages, to modify FtsZ flexibility. The mutant phenotypes ranged from benign to total inactivation and included increased GTPase, reduced assembly, and stabilized assembly. Six mutations clustering at the long cleft between the C-terminal beta-sheet and core helix H7 deviated FtsZ assembly into curved filaments with inhibited GTPase, which still polymerize cooperatively. These mutations may perturb the predicted closure of the C-terminal domain onto H7 required for switching between curved and straight association modes and for GTPase activation. By mapping the FtsZ assembly switch, this work also gives insight into FtsZ druggability because the curved mutations delineate the putative binding site of the promising antibacterial FtsZ inhibitor PC190723.

Discovery of novel fibroblast growth factor receptor 1 kinase inhibitors by structure-based virtual screening.

Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and 16, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC(50) values of 23 and 50 microM. Initial optimization of 16 led to the more unsaturated 40, which has significantly enhanced potency, 1.9 microM. The core structures represent new structural motifs for FGFR1 kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek selectivity, and hit identification.

EHD4 and CDH23 Are Interacting Partners in Cochlear Hair Cells

Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner.

A novel FIP1L1-PDGFRA mutant destabilizing the inactive conformation of the kinase domain in chronic eosinophilic leukemia/hypereosinophilic syndrome

The Fip1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) gene fusion is a common cause of chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES), and patients suffering from this particular subgroup of CEL/HES respond to low-dose imatinib therapy. However, some patients may develop imatinib resistance because of an acquired T674I mutation, which is believed to prevent drug binding through steric hindrance. In an imatinib resistant FIP1L1-PDGFRA positive patient, we analyzed the molecular structure of the fusion gene and analyzed the effect of several kinase inhibitors on FIP1L1-PDGFRA-mediated proliferative responses in vitro. Sequencing of the FIP1L1-PDGFRA fusion gene revealed the occurrence of a S601P mutation, which is located within the nucleotide binding loop. In agreement with the clinical observations, imatinib did not inhibit the proliferation of S601P mutant FIP1L1-PDGFRA-transduced Ba/F3 cells. Moreover, sorafenib, which has been described to inhibit T674I mutant FIP1L1-PDGFRA, failed to block S601P mutant FIP1L1-PDGFRA. Structural modeling revealed that the newly identified S601P mutated form of PDGFRA destabilizes the inactive conformation of the kinase domain that is necessary to bind imatinib as well as sorafenib. We identified a novel mutation in FIP1L1-PDGFRA resulting in both imatinib and sorafenib resistance. The identification of novel drug-resistant FIP1L1-PDGFRA variants may help to develop the next generation of target-directed compounds for CEL/HES and other leukemias.

Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS

We have used unbiased phosphoproteomic approaches, based on quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC), to identify tyrosine phosphorylated proteins in isogenic human bronchial epithelial cells (HBECs) and human lung adenocarcinoma cell lines, expressing either of the two mutant alleles of EGFR (L858R and Del E746-A750), or a mutant KRAS allele, which are common in human lung adenocarcinomas. Tyrosine phosphorylation of signaling molecules was greater in HBECs expressing the mutant EGFRs than in cells expressing WT EGFR or mutant KRAS. Receptor tyrosine kinases (such as EGFR, ERBB2, MET, and IGF1R), and Mig-6, an inhibitor of EGFR signaling, were more phosphorylated in HBECs expressing mutant EGFR than in cells expressing WT EGFR or mutant RAS. Phosphorylation of some proteins differed in the two EGFR mutant-expressing cells; for example, some cell junction proteins (beta-catenin, plakoglobin, and E-cadherin) were more phosphorylated in HBECs expressing L858R EGFR than in cells expressing Del EGFR. There were also differences in degree of phosphorylation at individual tyrosine sites within a protein; for example, a previously uncharacterized phosphorylation site in the nucleotide-binding loop of the kinase domains of EGFR (Y727), ERBB2 (Y735), or ERBB4 (Y733), is phosphorylated significantly more in HBECs expressing the deletion mutant than in cells expressing the wild type or L858R EGFR. Signaling molecules not previously implicated in ERBB signaling, such as polymerase transcript release factor (PTRF), were also phosphorylated in cells expressing mutant EGFR. Bayesian network analysis of these and other datasets revealed that PTRF might be a potentially important component of the ERBB signaling network.

Regulation of TNF‐α‐activated PKC‐ζ signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase

Human biliverdin reductase (hBVR) is a dual function enzyme: a catalyst for bilirubin formation and a S/T/Y kinase that shares activators with protein kinase C (PKC) -zeta, including cytokines, insulin, and reactive oxygen species (ROS). Presently, we show that hBVR increases PKC-zeta autophosphorylation, stimulation by TNF-alpha, as well as cytokine stimulation of NF-kappaB DNA binding and promoter activity. S149 in hBVR S/T kinase domain and S230 in YLS230F in hBVR's docking site for the SH2 domain of signaling proteins are phosphorylation targets of PKC-zeta. Two hBVR-based peptides, KRNRYLS230F (#1) and KKRILHC281 (#2), but not their S-->A or C-->A derivatives, respectively, blocked PKC-zeta stimulation by TNF-alpha and its membrane translocation. The C-terminal-based peptide KYCCSRK296 (#3), enhanced PKC-zeta stimulation by TNF-alpha; for this, Lys296 was essential. In metabolically 32P-labeled HEK293 cells transfected with hBVR or PKC-zeta, TNF-alpha increased hBVR phosphorylation. TNF-alpha did not stimulate PKC-zeta in cells infected with small interfering RNA for hBVR or transfected with hBVR with a point mutation in the nucleotide-binding loop (G17), S149, or S230; this was similar to the response of "kinase-dead" PKC-zeta(K281R). We suggest peptide #1 blocks PKC-zeta-docking site interaction, peptide #2 disrupts function of the PKC-zeta C1 domain, and peptide #3 alters ATP presentation to the kinase. The findings are of potential significance for development of modulators of PKC-zeta activity and cellular response to cytokines.

Virulent Protein Kinase A

The Yersinia Gram-negative bacteria are pathogenic to humans causing such diseases as bubonic plague. Through the action of a type III protein secretion system, Yersinia injects bacterial proteins necessary to subvert the host cell's normal processes and to promote bacterial survival and replication. Navarro et al . report that, in addition to the previously known inhibitory activity toward guanosine triphosphatases (GTPases) Rac1 and RhoA, Yersinia protein kinase A (YpkA) also inhibits G protein (heterotrimeric guanine nucleotide-binding protein) signaling by phosphorylation of the Gα subunit. The actin cytoskeleton disruption in cells expressing either the full-length YpkA or a C-terminal fragment was prevented if the cells also expressed a constitutively active form of RhoA, suggesting that YpkA acted upstream of this GTPase. G protein-coupled receptors can stimulate reorganization of the actin cytoskeleton through the activation of RhoA. Exposure of cells transfected with the muscarinic receptor to carbachol stimulated Gα q signaling and promoted the formation of stress fibers. Transfection of YpkA, but not a catalytically inactive version, inhibited this Gα q -induced reorganization of the cytoskeleton. YpkA coimmunoprecipitated with Gα q (both GDP-bound and GTP-bound forms) and Gα q was phosphorylated when immunoprecipitated from cells expressing catalytically active YpkA. The phosphorylation site was mapped to a serine within the consensus guanine nucleotide-binding loop by liquid chromatography tandem mass spectrometry. Phosphorylation of this residue appeared to disrupt activation of the G protein as substitution of the serine with aspartate (to mimic phosphorylation) prevented GTP loading. Not only was signaling from the muscarinic receptor to the cytoskeleton blocked by expression of YpkA, so was nuclear translocation of tubby, a transcriptional regulator released from the plasma membrane in response to Gα q -mediated stimulation of phospholipase C. In cells infected with Yersinia , both wild-type and catalytically inactive YpkA disrupted the actin cytoskeleton, presumably because the protein contains two functional domains that can inhibit the actin cytoskeleton: the C-terminal guanine nucleotide dissociation inhibitor region that directly targets RhoA and the serine-threonine kinase activity of the N-terminal region that inhibits G protein signaling to the cytoskeleton. L. Navarro, A. Koller, R. Nordfelth, H. Wolf-Watz, S. Taylor, J. E. Dixon, Identification of a molecular target for the Yersinia protein kinase A. Mol. Cell 26 , 465-477 (2007). [PubMed]