Axel J. Scheidig

Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex

Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota. This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 Å resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis.

Structure of the N6-adenine DNA methyltransferase M.TaqI in complex with DNA and a cofactor analog.

The 2.0 Å crystal structure of the N6-adenine DNA methyltransferase M•TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5′-TCGA-3′, the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand π-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.

The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins.

BACKGROUND In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides. RESULTS The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique. CONCLUSIONS The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.

Towards structural determination of the water-splitting enzyme

A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the QB binding site. The oxygen-evolving activity, 5000–6000 μmol of O2/(h·mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50 °C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 Å. The space group was determined to be P212121 with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization.

Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins.

We present the 1.9 Å resolution crystal structure of the catalytic domain of Gyp1p, a specific GTPase activating protein (GAP) for Ypt proteins, the yeast homologues of Rab proteins, which are involved in vesicular transport. Gyp1p is a member of a large family of eukaryotic proteins with shared sequence motifs. Previously, no structural information was available for any member of this class of proteins. The GAP domain of Gyp1p was found to be fully α‐helical. However, the observed fold does not superimpose with other α‐helical GAPs (e.g. Ras‐ and Cdc42/Rho‐GAP). The conserved and catalytically crucial arginine residue, identified by mutational analysis, is in a comparable position to the arginine finger in the Ras‐ and Cdc42‐GAPs, suggesting that Gyp1p utilizes an arginine finger in the GAP reaction, in analogy to Ras‐ and Cdc42‐GAPs. A model for the interaction between Gyp1p and the Ypt protein satisfying biochemical data is given.

Rab-subfamily-specific regions of Ypt7p are structurally different from other RabGTPases

The GTPase Ypt7p from S. cerevisiae is involved in late endosome-to-vacuole transport and homotypic vacuole fusion. We present crystal structures of the GDP- and GppNHp-bound conformation of Ypt7p solved at 1.35 and 1.6 A resolution, respectively. Despite the similarity of the overall structure to other Ypt/Rab proteins, Ypt7p displays small but significant differences. The Ypt7p-specific residues Tyr33 and Tyr37 cause a difference in the main chain trace of the RabSF2 region and form a characteristic surface epitope. Ypt7p*GppNHp does not display the helix alpha2, characteristic of the Ras-superfamily, but instead possess an extended loop L4/L5. Due to insertions in loops L3 and L7, the neighboring RabSF1 and RabSF4 regions are different in their conformations to those of other Ypt/Rab proteins.

Moderate discrimination of REP-1 between Rab7-GDP and Rab7-GTP arises from a difference of an order of magnitude in dissociation rates

The kinetics of the interaction of Rab7 with REP‐1 have been investigated using the fluorescence of GDP and GTP analogs at the active site of Rab7. The results show that REP‐1 has higher affinity for the GDP bound form of Rab7 (K d=1 nM) than for the GTP bound form (K d=20 nM). Both affinities should still be sufficient for the formation of stable complexes in the cell. The association reaction proceeds in two steps for the GDP bound form. The initial step is fast (k +1=ca. 107 M−1 s−1) and concentration dependent while the second represents a slow equilibration (k +2+k −2=3.5 s−1) which has little effect on the overall equilibrium. The difference in affinity of the two nucleotide bound forms arises from a difference in dissociation rates (0.012 s−1 for Rab7⋅GDP and 0.2 s−1 for Rab7⋅GTP).

Quantitative Labeling of Long Plasmid DNA with Nanometer Precision

Sequence-specific labeling of native DNA is of major interest for functional studies of DNA and DNA-modifying enzymes as well as for nanobiotechnology and medical diagnostics. Bearing in mind the size of DNA and the recurrence of only four major building blocks, sequence-specific chemical modification is a very challenging task. DNA sequence recognition can be achieved by using engineered zinc-finger proteins, hairpin polyamides, triplex-forming oligodeoxynucleotides (TFOs), or peptide nucleic acids. TFOs have been used for covalent DNA labeling, but they generally recognize rare homopurine– homopyrimidine tracks, which limits a broad applicability for sequence-specific DNA labeling. Most interestingly, sequence-specific DNA modification is already performed by nature. DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group from the ubiquitous cofactor S-adenosyl-l-methionine (AdoMet or SAM, 1) to adenine or cytosine residues within specific DNA sequences that range between two and eight base pairs (Scheme 1). By replacing the methionine side chain of AdoMet with an aziridine group, DNA MTases can be tricked to couple the whole cofactor N-adenosylaziridine (2a, R=H) sequence-specifically with DNA. This system has been used to deliver functional groups to short duplex oligodeoxynucleotides that were then coupled with suitable functionalized reporter groups in a second step. However, this two-step procedure only gave moderate labeling yields. Quantitative labeling of long plasmid DNA was achieved with the DNA MTase M.TaqI, and direct attachment of the fluorescent dansyl group to the 8-position of the aziridine cofactor via a flexible linker. We have extended this work and synthesized the biotinylated aziridine cofactor 2b (R=NH ACHTUNGTRENNUNG(CH2)4NH-biotin; Scheme 2). The primary amino group of 8-amino ACHTUNGTRENNUNG[1’’-(4’’-aminobutyl)]-2’,3’O-isopropylideneadenosine was protected as trifluoroacetyl amide, the 5’ position was activated as mesyl ester, and the isopropylidene protecting group was removed under acidic conditions. Nucleophilic substitution of the mesylate with aziridine, removal of the trifluoroacetyl protecting group under basic aqueous work up, and coupling of the resulting primary amine with N-hydroxysuccinimidyl biotin (NHS–biotin) furACHTUNGTRENNUNGnished the desired N-adenosylaziridine derivative 2b (for details see the Supporting Information). Compared to the previously employed photocleavable 6-nitroveratryloxocarbonyl protecting group the trifluoroacetyl group proved superior because of its facile removal. The introduction of the reporter group in the last step by its NHS ester offers a versatile approach to aziridine cofactors with various commonly used labels. Aziridine cofactor 2b comprises three functions for DNA MTase-mediated coupling with DNA: the aziridine ring acts as an electrophilic reactive group after protonation, the adenosyl Scheme 1. Reactions catalyzed by the DNA methyltransferase M.TaqI. Nucleophilic attack of the exocyclic amino group of adenine onto the activated methyl group of S-adenosyl-l-methionine (AdoMet, 1) leads to methyl group transfer to adenine in the 5’-TCGA-3’ recognition sequence (left), and nucleophilic ring opening of the aziridine group in cofactor 2 results in sequence-specific coupling with DNA (right).

Structural Basis for the Oxidation of Protein-bound Sulfur by the Sulfur Cycle Molybdohemo-Enzyme Sulfane Dehydrogenase SoxCD

The sulfur cycle enzyme sulfane dehydrogenase SoxCD is an essential component of the sulfur oxidation (Sox) enzyme system of Paracoccus pantotrophus. SoxCD catalyzes a six-electron oxidation reaction within the Sox cycle. SoxCD is an α2β2 heterotetrameric complex of the molybdenum cofactor-containing SoxC protein and the diheme c-type cytochrome SoxD with the heme domains D1 and D2. SoxCD1 misses the heme-2 domain D2 and is catalytically as active as SoxCD. The crystal structure of SoxCD1 was solved at 1.33 Å. The substrate of SoxCD is the outer (sulfane) sulfur of Cys-110-persulfide located at the C-terminal peptide swinging arm of SoxY of the SoxYZ carrier complex. The SoxCD1 substrate funnel toward the molybdopterin is narrow and partially shielded by side-chain residues of SoxD1. For access of the sulfane-sulfur of SoxY-Cys-110 persulfide we propose that (i) the blockage by SoxD-Arg-98 is opened via interaction with the C terminus of SoxY and (ii) the C-terminal peptide VTIGGCGG of SoxY provides interactions with the entrance path such that the cysteine-bound persulfide is optimally positioned near the molybdenum atom. The subsequent oxidation reactions of the sulfane-sulfur are initiated by the nucleophilic attack of the persulfide anion on the molybdenum atom that is, in turn, reduced. The close proximity of heme-1 to the molybdopterin allows easy acceptance of the electrons. Because SoxYZ, SoxXA, and SoxB are already structurally characterized, with SoxCD1 the structures of all key enzymes of the Sox cycle are known with atomic resolution.

Chemo-enzymatic synthesis of fluorescent Rab 7 proteins: Tools to study vesicular trafficking in cells

The N-methylanthraniloylisoprenoid diphosphate derivatives 1 and 2 bind to RabGGTase II and are enzymatically transferred to Rab 7 (the Rab proteins are small G-proteins that control events of docking and fusion of intracellular vesicles). The fluorescent Rab 7 proteins thus obtained may become important tools for further biological studies on vesicular trafficking in cells.