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Hauke Lilie

Publications and source records attributed to Hauke Lilie.

At least 19 recordsLinked to original sources
PubMed2006

Direct kinetic evidence for half-of-the-sites reactivity in the E1 component of the human pyruvate dehydrogenase multienzyme complex through alternating sites cofactor activation.

Recent kinetic and structural studies on various thiamin-dependent enzymes, including the bacterial E1 component of the pyruvate dehydrogenase complex (PDHc), suggested an active center communication between the cofactors in these multimeric enzymes. This regulatory mode has been inferred from the dissymmetry of active sites in proteolytic patterns and X-ray structures and from a complex macroscopic kinetic behavior not being consistent with independently working active sites. Here, direct microscopic kinetic evidence for this hypothesis is presented for the alpha2beta2-type E1 component of the human pyruvate dehydrogenase complex. Only one of the two thiamin molecules bound to the two active sites is in a chemically activated state exhibiting an apparent C2 ionization rate constant of approximately 50 s(-1) at pH 7.6 and 30 degrees C, whereas the thiamin in the "inactive site" ionizes with a rate that is at least 3 orders of magnitude smaller. The chemical nonequivalence is also exhibited in the ability to bind the substrate analogue methyl acetylphosphonate and in the catalytic turnover of the substrate pyruvate in the E1-only reaction. In the activated active site, pyruvate is rapidly bound and decarboxylated with apparent forward rate constants of covalent pyruvate binding of 2 s(-1) and decarboxylation of the formed 2-lactyl-thiamin intermediate of 5 s(-1). In the dormant site, these steps are as slow as 0.03 s(-1). Under the conditions that were used, only the heterotetramer can be detected by analytical ultracentrifugation, thus ruling out the possibility that multiple oligomeric species with different reactivities cause the observed kinetic effects. The results are consistent with the recently suggested model of an active site synchronization in PDHc-E1 via a proton wire that keeps the two active sites in an alternating activation state [Frank, R. A., et al. (2004) Science 306, 872]. Kinetic studies on the related thiamin enzymes transketolase, pyruvate oxidase, and bacterial pyruvate decarboxylase are not consistent with a chemical and/or functional nonequivalence of the active sites as observed in the E1 component of hsPDHc. We hypothesize that the alternating sites reaction in PDHc-E1 aids in the synchronized acyl transfer to the E2 component in the highly organized multienzyme complex.

Amino Acid Sequence↗
PubMed2006

Crystal structure analysis and solution studies of human Lck-SH3; zinc-induced homodimerization competes with the binding of proline-rich motifs.

In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 A. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn(2+) dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.

Amino Acid Motifs↗
PubMed2006

Crystal structure of 12-oxophytodienoate reductase 3 from tomato: self-inhibition by dimerization.

12-Oxophytodienoate reductase (OPR) 3, a homologue of old yellow enzyme (OYE), catalyzes the reduction of 9S,13S-12-oxophytodienoate to the corresponding cyclopentanone, which is subsequently converted to the plant hormone jasmonic acid (JA). JA and JA derivatives, as well as 12-oxophytodienoate and related cyclopentenones, are known to regulate gene expression in plant development and defense. Together with other oxygenated fatty acid derivatives, they form the oxylipin signature in plants, which resembles the pool of prostaglandins in animals. Here, we report the crystal structure of OPR3 from tomato and of two OPR3 mutants. Although the catalytic residues of OPR3 and related OYEs are highly conserved, several characteristic differences can be discerned in the substrate-binding regions, explaining the remarkable substrate stereoselectivity of OPR isozymes. Interestingly, OPR3 crystallized as an extraordinary self-inhibited dimer. Mutagenesis studies and biochemical analysis confirmed a weak dimerization of OPR3 in vitro, which correlated with a loss of enzymatic activity. Based on structural data of OPR3, a putative mechanism for a strong and reversible dimerization of OPR3 in vivo that involves phosphorylation of OPR3 is suggested. This mechanism could contribute to the shaping of the oxylipin signature, which is critical for fine-tuning gene expression in plants.

Amino Acid Sequence↗
PubMed2006

The galactose switch in Kluyveromyces lactis depends on nuclear competition between Gal4 and Gal1 for Gal80 binding.

The Gal4 protein represents a universally functional transcription activator, which in yeast is regulated by protein-protein interaction of its transcription activation domain with the inhibitor Gal80. Gal80 inhibition is relieved via galactose-mediated Gal80-Gal1-Gal3 interaction. The Gal4-Gal80-Gal1/3 regulatory module is conserved between Saccharomyces cerevisiae and Kluyveromyces lactis. Here we demonstrate that K. lactis Gal80 (KlGal80) is a nuclear protein independent of the Gal4 activity status, whereas KlGal1 is detected throughout the entire cell, which implies that KlGal80 and KlGal1 interact in the nucleus. Consistently KlGal1 accumulates in the nucleus upon KlGAL80 overexpression. Furthermore, we show that the KlGal80-KlGal1 interaction blocks the galactokinase activity of KlGal1 and is incompatible with KlGal80-KlGal4-AD interaction. Thus, we propose that dissociation of KlGal80 from the AD forms the basis of KlGal4 activation in K. lactis. Quantitation of the dissociation constants for the KlGal80 complexes gives a much lower affinity for KlGal1 as compared with Gal4. Mathematical modeling shows that with these affinities a switch based on competition between Gal1 and Gal4 for Gal80 binding is nevertheless efficient provided two monomeric Gal1 molecules interact with dimeric Gal80. Consistent with such a mechanism, analysis of the sedimentation behavior by analytical ultracentrifugation demonstrates the formation of a heterotetrameric KlGal80-KlGal1 complex of 2:2 stoichiometry.

Binding, Competitive↗
PubMed2006

YcdB from Escherichia coli reveals a novel class of Tat-dependently translocated hemoproteins.

The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin Fe(III)-protoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene. The data demonstrate the existence of a class of heme-containing Tat substrates, the first member of which is YcdB.

Amino Acid Sequence↗
PubMed2006

Adaptor protein controlled oligomerization activates the AAA+ protein ClpC.

The AAA+ protein ClpC is not only involved in the removal of misfolded and aggregated proteins but also controls, through regulated proteolysis, key steps of several developmental processes in the Gram-positive bacterium Bacillus subtilis. In contrast to other AAA+ proteins, ClpC is unable to mediate these processes without an adaptor protein like MecA. Here, we demonstrate that the general activation of ClpC is based upon the ability of MecA to participate in the assembly of an active and substrate-recognizing higher oligomer consisting of ClpC and the adaptor protein, which is a prerequisite for all activities of this AAA+ protein. Using hybrid proteins of ClpA and ClpC, we identified the N-terminal and the Linker domain of the first AAA+ domain of ClpC as the essential MecA interaction sites. This new adaptor-mediated mechanism adds another layer of control to the regulation of the biological activity of AAA+ proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Unexpected novel binding mode of pyrrolidine-based aspartyl protease inhibitors: design, synthesis and crystal structure in complex with HIV protease.

At present nine FDA-approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide-like character or the stabilization of a new and unexpected binding-competent conformation of the protein through a novel ligand-binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non-peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp 25 and 25'. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single-digit micromolar inhibition of HIV-1 protease and cathepsin D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross-links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus.

Amino Acid Sequence↗
PubMed2005

Structural insight into poly(A) binding and catalytic mechanism of human PARN.

Poly(A)-specific ribonuclease (PARN) is a processive, poly(A)-specific 3' exoribonuclease. The crystal structure of C-terminal truncated human PARN determined in two states (free and RNA-bound forms) reveals that PARNn is folded into two domains, an R3H domain and a nuclease domain similar to those of Pop2p and epsilon186. The high similarity of the active site structures of PARNn and epsilon186 suggests that they may have a similar catalytic mechanism. PARNn forms a tight homodimer, with the R3H domain of one subunit partially enclosing the active site of the other subunit and poly(A) bound in a deep cavity of its nuclease domain in a sequence-nonspecific manner. The R3H domain and, possibly, the cap-binding domain are involved in poly(A) binding but these domains alone do not appear to contribute to poly(A) specificity. Mutations disrupting dimerization abolish both the enzymatic and RNA-binding activities, suggesting that the PARN dimer is a structural and functional unit. The cap-binding domain may act in concert with the R3H domain to amplify the processivity of PARN.

Adenosine↗
PubMed2005

Hydroxyethylene sulfones as a new scaffold to address aspartic proteases: design, synthesis, and structural characterization.

Hydroxyethylene sulfones were developed as novel scaffolds against aspartyl proteases. A diastereoselective synthesis has been established to introduce the required side chain decoration with desired stereochemistry. Depending on the substitution of the hydroxyethylene sulfone core, micro- to submicromolar inhibition of HIV-1 protease is achieved for the S-configuration at P1 and R-configuration at the hydroxy-group-bearing backbone atom. This stereochemical preference is consistent with the S,R configuration of amprenavir. The racemic mixture of the most potent derivative (K(i) = 80 nM) was separated by chiral HPLC, revealing the S,R,S-enantiomer to be more active (K(i) = 45 nM). Docking studies suggested this isomer as the more active one. The subsequently determined crystal structure with HIV-1 protease, cocrystallized from a racemic mixture, exclusively reveals the S,R,S-enantiomer accommodated to the binding pocket. The transition state mimicking hydroxy group of the inhibitor is centered between both catalytic aspartates, while either its carbonyl or sulfonyl group forms H-bonds to the structurally conserved water mediating interactions between ligand and Ile50NH/Ile50NH' of both flaps. Biological testing of the stereoisomeric hydroxyethylene sulfones against cathepsin D and beta-secretase did not reveal significant inhibition. Most likely, the latter proteases require inverted configuration at the hydroxy group.

Anti-HIV Agents↗
PubMed2005

Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes.

The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, analysis of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30-60 nm in diameter), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5-10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside molecules by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus/receptor complex by cortical actin filaments.

Actins↗
PubMed2005

Oligomerization and assembly of the matrix protein of Borna disease virus.

The matrix protein M of Borna disease virus (BDV) is a constituent of the viral envelope covering the inner leaflet of the lipid bilayer. BDV-M was expressed as recombinant protein in Escherichia coli, purified to homogeneity and structurally analyzed. Recombinant M (i) forms non-covalently bound multimers with a Stoke's radius of 35 Angstroms estimated by size exclusion chromatography, (ii) consists of tetramers detected by analytical ultracentrifugation, and (iii) appears by electron microscopy studies as tetramers with the tendency to assemble into high molecular mass lattice-like complexes. The structural features suggest that BDV-M possesses a dominant driving force for virus particle formation.

Borna disease virus↗
PubMed2005

L-Arginine increases the solubility of unfolded species of hen egg white lysozyme.

L-Arginine (L-Arg) has been widely used as an enhancer of protein renaturation. The mechanism behind its action is still not fully understood. Using hen egg white lysozyme as a model protein, we present data that clearly demonstrate the suppression of the aggregation of denatured protein by L-Arg. By chemical modification of free cysteines, a series of unfolded lysozyme species were obtained that served as models for unfolded and intermediate states during the process of oxidative refolding. An increased equilibrium solubility of unfolded species and intermediates in the presence of L-Arg seems to be its major mechanism of action.

Arginine↗
PubMed2004

Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis.

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.

Amino Acid Sequence↗
PubMed2004

Structure and function of a regulated archaeal triosephosphate isomerase adapted to high temperature.

Triosephophate isomerase (TIM) is a dimeric enzyme in eucarya, bacteria and mesophilic archaea. In hyperthermophilic archaea, however, TIM exists as a tetramer composed of monomers that are about 10% shorter than other eucaryal and bacterial TIM monomers. We report here the crystal structure of TIM from Thermoproteus tenax, a hyperthermophilic archaeon that has an optimum growth temperature of 86 degrees C. The structure was determined from both a hexagonal and an orthorhombic crystal form to resolutions of 2.5A and 2.3A, and refined to R-factors of 19.7% and 21.5%, respectively. In both crystal forms, T.tenax TIM exists as a tetramer of the familiar (betaalpha)(8)-barrel. In solution, however, and unlike other hyperthermophilic TIMs, the T.tenax enzyme exhibits an equilibrium between inactive dimers and active tetramers, which is shifted to the tetramer state through a specific interaction with glycerol-1-phosphate dehydrogenase of T.tenax. This observation is interpreted in physiological terms as a need to reduce the build-up of thermolabile metabolic intermediates that would be susceptible to destruction by heat. A detailed structural comparison with TIMs from organisms with growth optima ranging from 15 degrees C to 100 degrees C emphasizes the importance in hyperthermophilic proteins of the specific location of ionic interactions for thermal stability rather than their numbers, and shows a clear correlation between the reduction of heat-labile, surface-exposed Asn and Gln residues with thermoadaptation. The comparison confirms the increase in charged surface-exposed residues at the expense of polar residues.

Adaptation, Physiological↗
PubMed2004

The mature part of proNGF induces the structure of its pro-peptide.

Human nerve growth factor (NGF) belongs to the structural family of cystine knot proteins, characterized by a disulfide pattern in which one disulfide bond threads through a ring formed by a pair of two other disulfides connecting two adjacent beta-strands. Oxidative folding of NGF revealed that the pro-peptide of NGF stimulates in vitro structure formation. In order to learn more about this folding assisting protein fragment, a biophysical analysis of the pro-peptide structure has been performed. While proNGF is a non-covalent homodimer, the isolated pro-peptide is monomeric. No tertiary contacts stabilize the pro-peptide in its isolated form. In contrast, the pro-peptide appears to be structured when bound to the mature part. The results presented here demonstrate that the mature part stabilizes the structure in the pro-peptide region. This is the first report that provides a biophysical analysis of a pro-peptide of the cystine knot protein family.

Circular Dichroism↗
PubMed2004

Killing of target cells by redirected granzyme B in the absence of perforin.

Granzyme B (GzmB) is a potent apoptosis-inducing serine protease of cytotoxic lymphocytes. Following receptor-mediated endocytosis, GzmB is supposed to enter the cytosol through perforin-mediated membrane disruption. We investigated whether retargeting of GzmB to Lewis Y positive surface receptors could lead to perforin-independent target cell death. We coupled recombinant GzmB to the Lewis Y-binding antibody dsFv-B3. Targeting of GzmB to Lewis Y positive cells triggered cell death with similar efficacy as dsFv-B3 targeted Pseudomonas exotoxin fragment 38 (PE38). Since GzmB was only weakly inhibited by plasma proteins, GzmB-based immunoconjugates should be useful as a new class of immunotoxins with low immunogenicity utilizing programmed cell death for therapeutic purposes.

Animals↗
PubMed2004

Activation of the redox-regulated chaperone Hsp33 by domain unfolding.

The molecular chaperone Hsp33 in Escherichia coli responds to oxidative stress conditions with the rapid activation of its chaperone function. On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer. While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown. To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33. These proteins were studied by a combination of biochemical and NMR spectroscopic techniques. We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, alpha-helical structure. This appears to conceal both the substrate-binding site as well as the dimerization interface. Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33. This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric. Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers. This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress.

Anilino Naphthalenesulfonates↗