Publications by authors named "Honnappa Srinivas"

25 Publications

  • Page 1 of 1

Discovery of LYS006, a Potent and Highly Selective Inhibitor of Leukotriene A Hydrolase.

J Med Chem 2021 Feb 16;64(4):1889-1903. Epub 2021 Feb 16.

The cytosolic metalloenzyme leukotriene A hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B (LTB). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB generation at low exposures , LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01955DOI Listing
February 2021

Discovery and Characterization of Clinical Candidate LXE408 as a Kinetoplastid-Selective Proteasome Inhibitor for the Treatment of Leishmaniases.

J Med Chem 2020 10 29;63(19):10773-10781. Epub 2020 Jul 29.

Genomics Institute of the Novartis Research Foundation (GNF), San Diego, California 92121, United States.

Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas disease, and human African trypanosomiasis. Here, we describe the discovery and characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor currently in Phase 1 human clinical trials. Furthermore, we present high-resolution cryo-EM structures of the proteasome in complex with LXE408, which provides a compelling explanation for the noncompetitive mode of binding of this novel class of inhibitors of the kinetoplastid proteasome.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549094PMC
October 2020

Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex.

Nat Chem Biol 2020 01 9;16(1):15-23. Epub 2019 Dec 9.

Novartis Institutes for Biomedical Research, Cambridge, MA, USA.

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.
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http://dx.doi.org/10.1038/s41589-019-0411-6DOI Listing
January 2020

A small-molecule inhibitor of C5 complement protein.

Nat Chem Biol 2019 07 17;15(7):666-668. Epub 2019 Jun 17.

Chemical Biology and Therapeutics, Novartis Institutes of Biomedical Research, Cambridge, MA, USA.

The complement pathway is an important part of the immune system, and uncontrolled activation is implicated in many diseases. The human complement component 5 protein (C5) is a validated drug target within the complement pathway, as an anti-C5 antibody (Soliris) is an approved therapy for paroxysmal nocturnal hemoglobinuria. Here, we report the identification, optimization and mechanism of action for the first small-molecule inhibitor of C5 complement protein.
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http://dx.doi.org/10.1038/s41589-019-0303-9DOI Listing
July 2019

Human Memory B Cells Harbor Diverse Cross-Neutralizing Antibodies against BK and JC Polyomaviruses.

Immunity 2019 03 26;50(3):668-676.e5. Epub 2019 Feb 26.

Novartis Institutes for BioMedical Research, Basel, Switzerland, Cambridge, MA, USA, and Emeryville, CA, USA. Electronic address:

Human polyomaviruses cause a common childhood infection worldwide and typically elicit a neutralizing antibody and cellular immune response, while establishing a dormant infection in the kidney with minimal clinical manifestations. However, viral reactivation can cause severe pathology in immunocompromised individuals. We developed a high-throughput, functional antibody screen to examine the humoral response to BK polyomavirus. This approach enabled the isolation of antibodies from all peripheral B cell subsets and revealed the anti-BK virus antibody repertoire as clonally complex with respect to immunoglobulin sequences and isotypes (both IgM and IgG), including a high frequency of monoclonal antibodies that broadly neutralize BK virus subtypes and the related JC polyomavirus. Cryo-electron microscopy of a broadly neutralizing IgG single-chain variable fragment complexed with BK virus-like particles revealed the quaternary nature of a conserved viral epitope at the junction between capsid pentamers. These features unravel a potent modality for inhibiting polyomavirus infection in kidney transplant recipients and other immunocompromised patients.
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http://dx.doi.org/10.1016/j.immuni.2019.02.003DOI Listing
March 2019

Feasibility and physiological relevance of designing highly potent aminopeptidase-sparing leukotriene A4 hydrolase inhibitors.

Sci Rep 2017 10 19;7(1):13591. Epub 2017 Oct 19.

Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

Leukotriene A4 Hydrolase (LTA4H) is a bifunctional zinc metalloenzyme that comprises both epoxide hydrolase and aminopeptidase activity, exerted by two overlapping catalytic sites. The epoxide hydrolase function of the enzyme catalyzes the biosynthesis of the pro-inflammatory lipid mediator leukotriene (LT) B4. Recent literature suggests that the aminopeptidase function of LTA4H is responsible for degradation of the tripeptide Pro-Gly-Pro (PGP) for which neutrophil chemotactic activity has been postulated. It has been speculated that the design of epoxide hydrolase selective LTA4H inhibitors that spare the aminopeptidase pocket may therefore lead to more efficacious anti-inflammatory drugs. In this study, we conducted a high throughput screen (HTS) for LTA4H inhibitors and attempted to rationally design compounds that would spare the PGP degrading function. While we were able to identify compounds with preference for the epoxide hydrolase function, absolute selectivity was not achievable for highly potent compounds. In order to assess the relevance of designing such aminopeptidase-sparing LTA4H inhibitors, we studied the role of PGP in inducing inflammation in different settings in wild type and LTA4H deficient (LTA4H KO) animals but could not confirm its chemotactic potential.  Attempting to design highly potent epoxide hydrolase selective LTA4H inhibitors, therefore seems to be neither feasible nor relevant.
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http://dx.doi.org/10.1038/s41598-017-13490-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648829PMC
October 2017

Identification of a Novel Allosteric Inhibitory Site on Tryptophan Hydroxylase 1 Enabling Unprecedented Selectivity Over all Related Hydroxylases.

Front Pharmacol 2017 5;8:240. Epub 2017 May 5.

Respiratory Disease Area, Novartis Institutes for BioMedical ResearchHorsham, UK.

Pulmonary arterial hypertension (PAH) has demonstrated multi-serotonin receptor dependent pathologies, characterized by increased tone (5-HT receptor) and complex lesions (SERT, 5-HT, 5-HT receptors) of the pulmonary vasculature together with right ventricular hypertrophy, ischemia and fibrosis (5-HT receptor). Selective inhibitors of individual signaling elements - SERT, 5-HT, 5HT, and combined 5-HT2 receptors, have all been tested clinically and failed. Thus, inhibition of tryptophan hydroxylase 1 (TPH1), the rate limiting step in 5-HT synthesis, has been suggested as a more broad, and thereby more effective, mode of 5-HT inhibition. However, selectivity over non-pathogenic enzyme family members, TPH2, phenylalanine hydroxylase, and tyrosine hydroxylase has hampered therapeutic development. Here we describe the site/sequence, biochemical, and biophysical characterization of a novel allosteric site on TPH1 through which selectivity over TPH2 and related aromatic amino acid hydroxylases is achieved. We demonstrate the mechanism of action by which novel compounds selectively inhibit TPH1 using surface plasma resonance and enzyme competition assays with both tryptophan ligand and BH4 co-factor. We demonstrate 15-fold greater potency within a human carcinoid cell line versus the most potent known TPH1/2 non-specific inhibitor. Lastly, we detail a novel canine system utilized to determine effective biologic inhibition of newly synthesized 5-HT. These findings are the first to demonstrate TPH1-selective inhibition and may pave the way to a truly effective means to reduce pathologic 5-HT and thereby treat complex remodeling diseases such as PAH.
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http://dx.doi.org/10.3389/fphar.2017.00240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418348PMC
May 2017

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency.

J Clin Invest 2017 Mar 6;127(3):912-928. Epub 2017 Feb 6.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.
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http://dx.doi.org/10.1172/JCI89626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330730PMC
March 2017

Orally active 7-substituted (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitriles as active-site inhibitors of sphingosine 1-phosphate lyase for the treatment of multiple sclerosis.

J Med Chem 2014 Jun 21;57(12):5074-84. Epub 2014 May 21.

Novartis Institutes for BioMedical Research , Basel, CH-4002, Switzerland.

Sphingosine 1-phosphate (S1P) lyase has recently been implicated as a therapeutic target for the treatment of multiple sclerosis (MS), based on studies in a genetic mouse model. Potent active site directed inhibitors of the enzyme are not known so far. Here we describe the discovery of (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitrile 5 in a high-throughput screen using a biochemical assay, and its further optimization. This class of compounds was found to inhibit catalytic activity of S1PL by binding to the active site of the enzyme, as seen in the cocrystal structure of derivative 31 with the homodimeric human S1P lyase. 31 induces profound reduction of peripheral T cell numbers after oral dosage and confers pronounced protection in a rat model of multiple sclerosis. In conclusion, this novel class of direct S1P lyase inhibitors provides excellent tools to further explore the therapeutic potential of T cell-targeted therapies in multiple sclerosis and other autoimmune and inflammatory diseases.
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http://dx.doi.org/10.1021/jm500338nDOI Listing
June 2014

Molecular basis of coiled-coil oligomerization-state specificity.

Proc Natl Acad Sci U S A 2010 Nov 2;107(46):19850-5. Epub 2010 Nov 2.

Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

Coiled coils are extensively and successfully used nowadays to rationally design multistranded structures for applications, including basic research, biotechnology, nanotechnology, materials science, and medicine. The wide range of applications as well as the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomerization. Here, we address the important and unresolved question why the presence of particular oligomerization-state determinants within a coiled coil does frequently not correlate with its topology. We found an unexpected, general link between coiled-coil oligomerization-state specificity and trigger sequences, elements that are indispensable for coiled-coil formation. By using the archetype coiled-coil domain of the yeast transcriptional activator GCN4 as a model system, we show that well-established trimer-specific oligomerization-state determinants switch the peptide's topology from a dimer to a trimer only when inserted into the trigger sequence. We successfully confirmed our results in two other, unrelated coiled-coil dimers, ATF1 and cortexillin-1. We furthermore show that multiple topology determinants can coexist in the same trigger sequence, revealing a delicate balance of the resulting oligomerization state by position-dependent forces. Our experimental results should significantly improve the prediction of the oligomerization state of coiled coils. They therefore should have major implications for the rational design of coiled coils and consequently many applications using these popular oligomerization domains.
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http://dx.doi.org/10.1073/pnas.1008502107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2993330PMC
November 2010

An EB1-binding motif acts as a microtubule tip localization signal.

Cell 2009 Jul;138(2):366-76

Biomolecular Research, Structural Biology, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland.

Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.
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http://dx.doi.org/10.1016/j.cell.2009.04.065DOI Listing
July 2009

Regulation of microtubule dynamic instability in vitro by differentially phosphorylated stathmin.

J Biol Chem 2009 Jun 8;284(23):15640-9. Epub 2009 Apr 8.

Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.
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http://dx.doi.org/10.1074/jbc.M900343200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708860PMC
June 2009

Mammalian end binding proteins control persistent microtubule growth.

J Cell Biol 2009 Mar 2;184(5):691-706. Epub 2009 Mar 2.

Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA.

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.
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http://dx.doi.org/10.1083/jcb.200807179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2686402PMC
March 2009

STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER.

Curr Biol 2008 Feb 31;18(3):177-82. Epub 2008 Jan 31.

Department of Cell Biology, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands.

Stromal interaction molecule 1 (STIM1) is a transmembrane protein that is essential for store-operated Ca(2+) entry, a process of extracellular Ca(2+) influx in response to the depletion of Ca(2+) stores in the endoplasmic reticulum (ER) (reviewed in [1-4]). STIM1 localizes predominantly to the ER; upon Ca(2+) release from the ER, STIM1 translocates to the ER-plasma membrane junctions and activates Ca(2+) channels (reviewed in [1-4]). Here, we show that STIM1 directly binds to the microtubule-plus-end-tracking protein EB1 and forms EB1-dependent comet-like accumulations at the sites where polymerizing microtubule ends come in contact with the ER network. Therefore, the previously observed tubulovesicular motility of GFP-STIM1 [5] is not a motor-based movement but a traveling wave of diffusion-dependent STIM1 concentration in the ER membrane. STIM1 overexpression strongly stimulates ER extension occurring through the microtubule "tip attachment complex" (TAC) mechanism [6, 7], a process whereby an ER tubule attaches to and elongates together with the EB1-positive end of a growing microtubule. Depletion of STIM1 and EB1 decreases TAC-dependent ER protrusion, indicating that microtubule growth-dependent concentration of STIM1 in the ER membrane plays a role in ER remodeling.
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http://dx.doi.org/10.1016/j.cub.2007.12.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2600655PMC
February 2008

Suppression of microtubule dynamic instability by the +TIP protein EB1 and its modulation by the CAP-Gly domain of p150glued.

Biochemistry 2008 Jan 15;47(2):779-86. Epub 2007 Dec 15.

Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California 93106, USA.

The EB1+TIP protein family and its binding partners track growing plus ends of microtubules in cells and are thought to regulate their dynamics. Here we determined the effects of EB1 and the N-terminal CAP-Gly domain (p150n) of one of its major binding partners, p150Glued, both separately and together, on the dynamic instability parameters at plus ends of purified steady-state microtubules. With EB1 alone, the shortening rate, the extent of shortening, and the catastrophe frequency were suppressed in the absence of significant effects on the growth rate or rescue frequency. The effects of EB1 on dynamics were significantly different when p150n was added together with EB1. The rate and extent of shortening and the catastrophe frequency were suppressed 3-4 times more strongly than with EB1 alone. In addition, the EB1-p150n complex increased the rescue frequency and the mean length the microtubules grew, parameters that were not significantly affected by EB1 alone. Similarly, deletion of EB1's C-terminal tail, which is a crucial binding region for p150n, significantly increased the ability of EB1 to suppress shortening dynamics. EB1 by itself bound along the length of the microtubules with 1 mol of EB1 dimer bound per approximately 12 mol of tubulin dimer. Approximately twice the amount of EB1 was recruited to the microtubules in the presence of p150n. Our results indicate that inactivation of EB1's flexible C-terminal tail significantly changes EB1's ability to modulate microtubule dynamics. They further suggest that p150Glued may activate and thereby facilitate the recruitment of EB1 to the tips of microtubules to regulate their dynamics.
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http://dx.doi.org/10.1021/bi701912gDOI Listing
January 2008

Structure-function relationship of CAP-Gly domains.

Nat Struct Mol Biol 2007 Oct 9;14(10):959-67. Epub 2007 Sep 9.

Biomolecular Research, Structural Biology, Paul Scherrer Insititut, CH-5232 Villigen PSI, Switzerland.

In all eukaryotes, CAP-Gly proteins control important cellular processes. The molecular mechanisms underlying the functions of CAP-Gly domains, however, are still poorly understood. Here we use the complex formed between the CAP-Gly domain of p150(glued) and the C-terminal zinc knuckle of CLIP170 as a model system to explore the structure-function relationship of CAP-Gly-mediated protein interactions. We demonstrate that the conserved GKNDG motif of CAP-Gly domains is responsible for targeting to the C-terminal EEY/F sequence motifs of CLIP170, EB proteins and microtubules. The CAP-Gly-EEY/F interaction is essential for the recruitment of the dynactin complex by CLIP170 and for activation of CLIP170. Our findings define the molecular basis of CAP-Gly domain function, including the tubulin detyrosination-tyrosination cycle. They further establish fundamental roles for the interaction between CAP-Gly proteins and C-terminal EEY/F sequence motifs in regulating complex and dynamic cellular processes.
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http://dx.doi.org/10.1038/nsmb1291DOI Listing
October 2007

Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.

Proc Natl Acad Sci U S A 2007 Jul 6;104(29):12151-6. Epub 2007 Jul 6.

Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG with the highly selective low-molecular-weight inhibitor AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. Here, we report the 2.4 A x-ray crystal structure of PknG in complex with AX20017. The unique multidomain topology of PknG reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Directed mutagenesis suggests that the rubredoxin domain functions as a regulator of PknG kinase activity. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine-binding site, targeting an active conformation of the kinase domain. Remarkably, although the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017-binding pocket is shaped by a unique set of amino acid side chains that are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compound's inhibitory potency. Our results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of M. tuberculosis.
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http://dx.doi.org/10.1073/pnas.0702842104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1924570PMC
July 2007

Molecular basis of coiled-coil formation.

Proc Natl Acad Sci U S A 2007 Apr 16;104(17):7062-7. Epub 2007 Apr 16.

Biomolecular Research, Structural Biology, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.

Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed "trigger sequence," that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation.
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http://dx.doi.org/10.1073/pnas.0700321104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855353PMC
April 2007

Key interaction modes of dynamic +TIP networks.

Mol Cell 2006 Sep;23(5):663-71

Biomolecular Research, Structural Biology, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.

Dynamic microtubule plus-end tracking protein (+TIP) networks are implicated in all functions of microtubules, but the molecular determinants of their interactions are largely unknown. Here, we have explored key binding modes of +TIPs by analyzing the interactions between selected CAP-Gly, EB-like, and carboxy-terminal EEY/F-COO(-) sequence motifs. X-ray crystallography and biophysical binding studies demonstrate that the beta2-beta3 loop of CAP-Gly domains determines EB-like motif binding specificity. They further show how CAP-Gly domains serve as recognition domains for EEY/F-COO(-) motifs, which represent characteristic and functionally important sequence elements in EB, CLIP-170, and alpha-tubulin. Our findings provide a molecular basis for understanding the modular interaction modes between alpha-tubulin, CLIPs, EB proteins, and the dynactin-dynein motor complex and suggest that multiple low-affinity binding sites in different combinations control dynamic +TIP networks at microtubule ends. They further offer insights into the structural consequences of genetic CAP-Gly domain defects found in severe human disorders.
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http://dx.doi.org/10.1016/j.molcel.2006.07.013DOI Listing
September 2006

Control of intrinsically disordered stathmin by multisite phosphorylation.

J Biol Chem 2006 Jun 22;281(23):16078-83. Epub 2006 Mar 22.

Biomolecular Research, Structural Biology, Paul Scherrer Insititut, CH-5232 Villigen PSI, Switzerland.

Stathmin is an intrinsically disordered protein implicated in the regulation of microtubule dynamics and in the development of cancer. The microtubule destabilizing activity of stathmin is down-regulated by phosphorylation of four serine residues, Ser16, Ser25, Ser38, and Ser63. Here we have used calorimetric and spectroscopic methods, including nuclear magnetic resonance to analyze the properties of seven stathmin phosphoisoforms to bind tubulin and inhibit microtubule formation. We found that stathmin phosphorylation results in a substantial loss in hydration entropy upon tubulin-stathmin complex formation. Remarkably, a linear correlation between the free energy change of complex formation and the microtubule inhibition activities of stathmin phosphoisoforms was observed. This finding provides a biophysical basis for understanding the mechanism by which local stathmin activity gradients important for promoting localized microtubule growth are established. We further found that phosphorylation of Ser16 and Ser63 disrupts the formation of a tubulin-interacting beta-hairpin and a helical segment, respectively, explaining the dominant role of these residues in regulating cell cycle progression. The insight into the tubulin-stathmin interaction offers a molecular basis for understanding the nature and the factors that control intrinsically disordered protein systems in general.
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http://dx.doi.org/10.1074/jbc.M513524200DOI Listing
June 2006

A conserved trimerization motif controls the topology of short coiled coils.

Proc Natl Acad Sci U S A 2005 Sep 19;102(39):13891-6. Epub 2005 Sep 19.

Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 PT, United Kingdom.

In recent years, short coiled coils have been used for applications ranging from biomaterial to medical sciences. For many of these applications knowledge of the factors that control the topology of the engineered protein systems is essential. Here, we demonstrate that trimerization of short coiled coils is determined by a distinct structural motif that encompasses specific networks of surface salt bridges and optimal hydrophobic packing interactions. The motif is conserved among intracellular, extracellular, viral, and synthetic proteins and defines a universal molecular determinant for trimer formation of short coiled coils. In addition to being of particular interest for the biotechnological production of candidate therapeutic proteins, these findings may be of interest for viral drug development strategies.
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http://dx.doi.org/10.1073/pnas.0502390102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1236532PMC
September 2005

Structural insights into the EB1-APC interaction.

EMBO J 2005 Jan 23;24(2):261-9. Epub 2004 Dec 23.

Biomolecular Research, Structural Biology, Paul Scherrer Institut, Villigen PSI, Switzerland.

EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. We find that EB1 is a stable dimer with a parallel coiled coil and show that dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 microM affinity. The conserved APC Ile2805-Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789-Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC-EB1 is post-translationally regulated in cells. Our findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms.
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http://dx.doi.org/10.1038/sj.emboj.7600529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC545816PMC
January 2005

Thermodynamics of the Op18/stathmin-tubulin interaction.

J Biol Chem 2003 Oct 14;278(40):38926-34. Epub 2003 Jul 14.

Structural Biology, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.

Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10 degrees C and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K0 = 6.0 x 10(6) m(-1). The obtained large negative molar heat capacity change of deltaCp0 = -860 cal mol(-1) K(-1) (referring to tubulin) for the tubulin-stathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on beta-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.
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http://dx.doi.org/10.1074/jbc.M305546200DOI Listing
October 2003

Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone.

J Biol Chem 2003 Feb 2;278(8):6194-200. Epub 2002 Dec 2.

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc(1)Man(7-9)GlcNAc(2) present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc(1)Man(7-9)GlcNAc(2), determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K(b)) and changes in enthalpy of binding (Delta H(b) degrees ) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 x 10(4) m(-1) at 279 K is obtained when glucose occurs in alpha-1,3 linkage to Man alpha Me as in Glc alpha 1-3Man alpha Me. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed.
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http://dx.doi.org/10.1074/jbc.M209132200DOI Listing
February 2003