A light-triggered transmembrane porin.

Porins are ideal model systems for channel engineering. OmpG is a robust, monomeric, transmembrane ß-barrel without ion selectivity. Here, we present a photocaged diethylaminocoumarin (DEACM) hybrid of OmpG. Blockage of the pore by DEACM is confirmed by reduced conductivity. An optimal effect was obtained when two bulky butyl-substituted coumarin cages were attached on the inside of the pore. Irradiation at 385 nm removed the photocages, leading to a restoration of channel conductivity.

Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana.

The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD(ox)), semiquinone form (FADH(.)), and anionic hydroquinone form (FAD(red)H(-)). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD(ox) to the anionic semiquinone form (FAD()(-)) with a quantum efficiency of about 2% and a back recovery time of about 10s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD(ox) to FADH(.), FADH(.) to FAD(red)H(-), and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.

Chapter 13. Nonribosomal peptide synthetases mechanistic and structural aspects of essential domains.

A widespread class of therapeutically important natural products is of peptidic origin. They are produced nonribosomally by large "assembly line"-like multienzyme complexes, the nonribosomal peptide synthetases (NRPS). In contrast to ribosomal peptide synthesis, nonribosomally assembled peptides contain unique structural features such as D-amino acids, N-terminally attached fatty acid chains, N- and C-methylated amino acids, N-formylated residues, heterocyclic elements, glycosylated amino acids, and phosphorylated residues. In recent research using genetic, biochemical, and structural methods, experiments have revealed profound insights into the molecular mechanism of nonribosomal peptide synthesis. Based on this, it was possible to alter existing nonribosomally produced peptides either by changing their biosynthetic templates or by the combined action of chemical peptide synthesis and subsequent enzyme catalysis. An overview of the structural aspects of the NRPS machinery with a focus on mechanistic and structural aspects of essential domains is presented.

Light-driven DNA repair by photolyases.

DNA photolyases are highly efficient light-driven DNA repair enzymes which revert the genome-damaging effects caused by ultraviolet (UV) radiation. These enzymes occur in almost all living organisms exposed to sunlight, the only exception being placental mammals like humans and mice. Their catalytic mechanism employs the light-driven injection of an electron onto the DNA lesion to trigger the cleavage of cyclobutane- pyrimidine dimers or 6-4 photoproducts inside duplex DNA. Spectroscopic and structural analysis has recently yielded a concise view of how photolyases recognize these DNA lesions involving two neighboring bases, catalyze the repair reaction within a nanosecond and still achieve quantum efficiencies of close to one. Apart from these mechanistic aspects, the potential of DNA photolyases for the generation of highly UV-resistant organisms, or for skin cancer prevention by ectopical application is increasingly recognized.

Absorption and fluorescence spectroscopic characterization of cryptochrome 3 from Arabidopsis thaliana.

The blue light photoreceptor cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized at room temperature in vitro in aqueous solution by optical absorption and emission spectroscopic studies. The protein non-covalently binds the chromophores flavin adenine dinucleotide (FAD) and N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). In the dark-adapted state of cry3, the bound FAD is present in the oxidized form (FAD(ox), ca. 38.5%), in the semiquinone form (FADH., ca. 5%), and in the fully reduced neutral form (FAD(red)H2) or fully reduced anionic form (FAD(red)H-, ca. 55%). Some amount of FAD (ca. 1.5%) in the oxidized state remains unbound probably caused by chromophore release and/or denaturation. Förster-type energy transfer from MTHF to FAD(ox) is observed. Photo-excitation reversibly modifies the protein conformation causing a slight rise of the MTHF absorption strength and an increase of the MTHF fluorescence efficiency (efficient protein conformation photo-cycle). Additionally there occurs reversible reduction of bound FAD(ox) to FAD(red)H2 (or FAD(red)H-, FAD(ox) photo-cycle of moderate efficiency), reversible reduction of FADH. to FAD(red)H2 (or FAD(red)H-, FADH. photo-cycle of high efficiency), and modification of re-oxidable FAD(red)H2 (or FAD(red)H-) to permanent FAD(red)H2 (or FAD(red)H-) with low quantum efficiency. Photo-excitation of MTHF causes the reversible formation of a MTHF species (MTHF', MTHF photo-cycle, moderate quantum efficiency) with slow recovery to the initial dark state, and also the formation of an irreversible photoproduct (MTHF'').

Purification, crystallization, and preliminary X-ray diffraction analysis of the Tricorn protease hexamer from Thermoplasma acidophilum.

Tricorn protease from Thermoplasma acidophilum is a hexameric enzyme; in vivo the hexamers assemble further to form large icosahedral capsids of 14.6 MDa. Recombinant Tricorn protease was purified as an enzymatically active hexamer of 0.72 MDa that formed crystals of octahedral morphology under low-ionic-strength conditions. These crystals belong to space group C2 with unit cell dimensions a = 307.5 A, b = 163.2 A, c = 220.9 A, beta = 105.5 degrees and diffract to 2.2-A resolution using high-brilliance synchrotron radiation. Based on analysis of the self-rotation function and the presence of a pseudo-origin peak in the native Patterson map, a packing model was derived for the complex, comprising 1.5 hexamers per asymmetric unit with a solvent content of 43%. Due to the ninefold noncrystallographic symmetry the Tricorn crystals represent an interesting case for phasing X-ray crystallographic data by electron microscopic phase information.

Crystal structure of the catalytic core component of the alkylhydroperoxide reductase AhpF from Escherichia coli.

Alkylhydroperoxide reductases (AhpR, EC 1.6.4.*) are essential for the oxygen tolerance of aerobic organisms by converting otherwise toxic hydroperoxides of lipids or nucleic acids to the corresponding alcohols. The AhpF component belongs to the family of pyridine nucleotide-disulphide oxidoreductases and channels electrons from NAD(P)H towards the AhpC component which finally reduces cognate substrates. The structure of the catalytic core of the Escherichia coli AhpF (A212-A521) with a bound FAD cofactor was determined at 1.9 A resolution in its oxidized state. The dimeric arrangement of the AhpF catalytic core and the predicted interaction mode between the N-terminal PDO-like domain and the NADPH domain favours an intramolecular electron transfer between the two redox-active disulphide centres of AhpF.

Structural analysis of adenylate cyclases from Trypanosoma brucei in their monomeric state.

Cyclic AMP is a major trigger of the differentiation process of Trypanosoma brucei, a bloodstream parasite causing sleeping sickness. Its generation in trypanosomes is accomplished by a unique battery of membrane-bound adenylate cyclases (ACs). We have determined the high-resolution X-ray structures of the catalytic domains of two trypanosomal ACs (tACs), GRESAG4.1 and GRESAG4.3. The tAC domains are structurally highly related to the AC domains of higher eukaryotes, but also comprise a highly conserved structural element near the active site, the Delta-subdomain. A cavity below the Delta-subdomain might correspond to an allosteric regulator site as indicated by the stereospecific binding of a single (2S,3S)-1,4- dimercapto-2,3-butanediol molecule. In three different crystal forms, the tAC domains are exclusively observed in a monomeric, catalytically inactive state. Biochemical analysis and the mutagenesis profile of GRESAG4.1 confirmed a common catalytic mechanism of tACs that involves transient dimerization of the AC domain. A low dimerization tendency might play a regulatory role in T. brucei if the activation of tACs is similarly driven by ligand-induced dimerization as in membrane-bound guanylate cyclases.

Crystal structure of the beta-apical domain of the thermosome reveals structural plasticity in the protrusion region.

The crystal structure of the beta-apical domain of the thermosome, an archaeal group II chaperonin from Thermoplasma acidophilum, has been determined at 2.8 A resolution. The structure shows an invariant globular core from which a 25 A long protrusion emanates, composed of an elongated alpha-helix (H10) and a long extended stretch consisting of residues GluB245-ThrB253. A comparison with previous apical domain structures reveals a large segmental displacement of the protruding part of helix H10 via the hinge GluB276-ValB278. The region comprising residues GluB245-ThrB253 adopts an extended beta-like conformation rather than the alpha-helix seen in the alpha-apical domain. Consequently, it appears that the protrusions of the apical domains from group II chaperonins might assume a variety of context-dependent conformations during an open, substrate-accepting state of the chaperonin. Sequence variations in the protrusion regions that are found in the eukaryotic TRiC/CCT subunits may provide different structural propensities and hence serve different roles in substrate recognition.

Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution.

Halorhodopsin, an archaeal rhodopsin ubiquitous in Haloarchaea, uses light energy to pump chloride through biological membranes. Halorhodopsin crystals were grown in a cubic lipidic phase, which allowed the x-ray structure determination of this anion pump at 1.8 angstrom resolution. Halorhodopsin assembles to trimers around a central patch consisting of palmitic acid. Next to the protonated Schiff base between Lys(242) and the isomerizable retinal chromophore, a single chloride ion occupies the transport site. Energetic calculations on chloride binding reveal a combination of ion-ion and ion-dipole interactions for stabilizing the anion 18 angstroms below the membrane surface. Ion dragging across the protonated Schiff base explains why chloride and proton translocation modes are mechanistically equivalent in archaeal rhodopsins.

Crystallization and preliminary X-ray analysis of the catalytic domain of the adenylate cyclase GRESAG4.1 from Trypanosoma brucei.

Adenylate cyclases (ACs) are involved in signal transduction by generating the second messenger, cAMP. In Trypanosoma brucei, 3', 5'-cyclic adenosine monophosphate (cAMP) controls the life cycle of this unicellular parasite. cAMP is generated by a class of adenylate cyclases which are either constitutively (GRESAG4.1-4.3) or transiently expressed (ESAG4) during the life cycle. Unlike mammalian ACs, the trypanosomal ACs have a simple topology comprising of a large extracellular region, a transmembrane helix and a cytosolic catalytic region. Two orthorhombic crystal forms of the catalytic AC domain of GRESAG4.1 (residues Ala884-Thr1132) were generated by the hanging-drop vapour-diffusion method. X-ray diffraction data from GRESAG4.1 crystals were collected at 1.9 A resolution using synchrotron radiation. Furthermore, two heavy-metal derivative data sets were collected from crystal form A; heavy-atom sites were subsequently located in difference Patterson maps.

Crystallization and preliminary X-ray analysis of the catalytic core of the alkylhydroperoxide reductase component AhpF from Escherichia coli.

Alkylhydroperoxide reductases (AhpR, E.C. 1.6.4.x) are essential for the oxygen tolerance of aerobic organisms, converting otherwise toxic hydroperoxides of lipids or nucleic acids to their corresponding alcohols. The AhpF component (521 amino-acid residues, 56.2 kDa) belongs to the family of pyridine nucleotide-disulfide oxidoreductases and channels electrons from NAD(P)H via a series of disulfides towards the AhpC component, which finally reduces the hydro-peroxide substrates. Crystals of the proteolytically truncated AhpF component (residues Asn208-Ala521) of the alkyl hydroperoxide reductase from Escherichia coli were grown under oxidizing conditions. The crystals belong to space group P3(2)21, with unit-cell parameters a = 60.4, c = 171.8 A. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. A molecular-replacement solution was found using the structure of thioredoxin reductase from Arabidopsis thaliana as a search model.

Group II chaperonins: new TRiC(k)s and turns of a protein folding machine.

In the past decade, the eubacterial group I chaperonin GroEL became the paradigm of a protein folding machine. More recently, electron microscopy and X-ray crystallography offered insights into the structure of the thermosome, the archetype of the group II chaperonins which also comprise the chaperonin from the eukaryotic cytosol TRiC. Some structural differences from GroEL were revealed, namely the existence of a built-in lid provided by the helical protrusions of the apical domains instead of a GroES-like co-chaperonin. These structural studies provide a framework for understanding the differences in the mode of action between the group II and the group I chaperonins. In vitro analyses of the folding of non-native substrates coupled to ATP binding and hydrolysis are progressing towards establishing a functional cycle for group II chaperonins. A protein complex called GimC/prefoldin has recently been found to cooperate with TRiC in vivo, and its characterization is under way.

Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C.

Phosphoinositide-specific phospholipases C (PI-PLCs) are ubiquitous enzymes that catalyse the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol (DAG). Whereas the eukaryotic PI-PLCs play a central role in most signal transduction cascades by producing two second messengers, inositol-1,4,5-trisphosphate and DAG, prokaryotic PI-PLCs are of interest because they act as virulence factors in some pathogenic bacteria. Bacterial PI-PLCs consist of a single domain of 30 to 35 kDa, while the much larger eukaryotic enzymes (85 to 150 kDa) are organized in several distinct domains. The catalytic domain of eukaryotic PI-PLCs is assembled from two highly conserved polypeptide stretches, called regions X and Y, that are separated by a divergent linker sequence. There is only marginal sequence similarity between the catalytic domain of eukaryotic and prokaryotic PI-PLCs. Recently the crystal structures of a bacterial and a eukaryotic PI-PLC have been determined, both in complexes with substrate analogues thus enabling a comparison of these enzymes in structural and mechanistic terms. Eukaryotic and prokaryotic PI-PLCs contain a distorted (beta alpha)8-barrel as a structural motif with a surprisingly large structural similarity for the first half of the (beta alpha)8-barrel and a much weaker similarity for the second half. The higher degree of structure conservation in the first half of the barrel correlates with the presence of all catalytic residues, in particular two catalytic histidine residues, in this portion of the enzyme. The second half contributes mainly to the features of the substrate binding pocket that result in the distinct substrate preferences exhibited by the prokaryotic and eukaryotic enzymes. A striking difference between the enzymes is the utilization of a catalytic calcium ion that electrostatically stabilizes the transition state in eukaryotic enzymes, whereas this role is filled by an analogously positioned arginine in bacterial PI-PLCs. The catalytic domains of all PI-PLCs may share not only a common fold but also a similar catalytic mechanism utilizing general base/acid catalysis. The conservation of the topology and parts of the active site suggests a divergent evolution from a common ancestral protein.

Structure of the substrate binding domain of the thermosome, an archaeal group II chaperonin.

The crystal structure of the substrate binding domain of the thermosome, the archaeal group II chaperonin, has been determined at 2.3 A resolution. The core resembles the apical domain of GroEL but lacks the hydrophobic residues implied in binding of substrates to group I chaperonins. Rather, a large hydrophobic surface patch is found in a novel helix-turn-helix motif, which is characteristic of all group II chaperonins including the eukaryotic TRiC/CCT complex. Models of the holochaperonin, which are consistent with cryo electron microscopy data, suggest a dual role of this helical protrusion in substrate binding and controlling access to the central cavity independent of a GroES-like cochaperonin.

A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-delta1.

We have determined the crystal structures of complexes of phosphoinositide-specific phospholipase C-delta1 from rat with calcium, barium, and lanthanum at 2.5-2.6 A resolution. Binding of these metal ions is observed in the active site of the catalytic TIM barrel and in the calcium binding region (CBR) of the C2 domain. The C2 domain of PLC-delta1 is a circularly permuted topological variant (P-variant) of the synaptotagmin I C2A domain (S-variant). On the basis of sequence analysis, we propose that both the S-variant and P-variant topologies are present among other C2 domains. Multiple adjacent binding sites in the C2 domain were observed for calcium and the other metal/enzyme complexes. The maximum number of binding sites observed was for the calcium analogue lanthanum. This complex shows an array-like binding of three lanthanum ions (sites I-III) in a crevice on one end of the C2 beta-sandwich. Residues involved in metal binding are contained in three loops, CBR1, CBR2, and CBR3. Sites I and II are maintained in the calcium and barium complexes, whereas sites II and III coincide with a binary calcium binding site in the C2A domain of synaptotagmin I. Several conformers for CBR1 are observed. The conformation of CBR1 does not appear to be strictly dependent on metal binding; however, metal binding may stabilize certain conformers. No significant structural changes are observed for CBR2 or CBR3. The surface of this ternary binding site provides a cluster of freely accessible liganding positions for putative phospholipid ligands of the C2 domain. It may be that the ternary metal binding site is also a feature of calcium-dependent phospholipid binding in solution. A ternary metal binding site might be a conserved feature among C2 domains that contain the critical calcium ligands in their CBR's. The high cooperativity of calcium-mediated lipid binding by C2 domains described previously is explained by this novel type of calcium binding site.

Structural mapping of the catalytic mechanism for a mammalian phosphoinositide-specific phospholipase C.

The crystal structures of various ternary complexes of phosphoinositide-specific phospholipase C-delta 1 from rat with calcium and inositol phosphates have been determined at 2.30-2.95 A resolution. The inositol phosphates used in this study mimic the binding of substrates and the reaction intermediate and include D-myo-inositol-1,4,5-trisphosphate, D-myo-inositol-2,4, 5-trisphosphate. D-myo-inositol-4,5-bisphosphate, and D,1-myo-inositol-2-methylene-1,2-cyclicmonophosphonate. The complexes exhibit an almost invariant mode of binding in the active site, each fitting edge-on into the active site and interacting with both the enzyme and the catalytic calcium at the bottom of the active site. Most of the active site residues do not undergo conformational changes upon binding either calcium or inositol phosphates. The structures are consistent with bidentate liganding of the catalytic calcium to the inositol phosphate intermediate and transition state. The complexes suggest explanations for substrate preference, pH optima, and ratio of cyclic to acyclic reaction products. A reaction mechanism is derived that supports general acid/base catalysis in a sequential mechanism involving a cyclic phosphate intermediate and rules out a parallel mechanism where acyclic and cyclic products are simultaneously generated.