Materials and Methods

ATP synthase was isolated from wild type Ilyobacter tartaricus cells. After disrupting the complex with N-lauroylsarcosine, pure c-ring was obtained by precipication of all other subunits of the enzyme with ammonium sulfate [107].

The purified c-ring was dialyzed against 10 mM Tris/HCl buffer (pH 8.0) to precipitate the protein by removal of the detergent. The material was col-lected by centrifugation and dissolved at 10 mg protein/ml in dialysis buffer containing 0.02% (w/v) NaN₃ and 0.78% (w/v) Zwittergent 3-12 (Calbiochem,

Figure 8.5: Motional flexibility within the c-ring. The low frequency normal modes of the c-ring were analyzed according to [112]. The data presented show two different views of the c-ring from I. tartaricus ATP synthase in the lowest-order nontrivial normal mode. (This image was modified due to the fact, that motion is not visible on paper. The arrows indicate the direction of movement; black stands for the cytoplasmic and light grey for the periplasmic endings, the dashed line specifies the hinge region.)

La Jolla, CA, USA). Crystals were grown within 2-3 days at 17^◦C to a size of approximately 400×100×100µm³ using the vapor diffusion method in hang-ing drops. A drop consisted of 1µl protein solution (passed through a 0.22µm sterile filter) and 1 µl crystallization buffer containing 15% (v/v) polyethy-lene glycol 400 in 0.1 M sodium acetate buffer, pH 4.5. The crystals were flash-frozen in liquid nitrogen after an equilibration for 24 h with 30% (v/v) polyethylene glycol 400 as cryo-protectant in the reservoir buffer at 17^◦C. Data used for structure solution were collected at the beamline X06SA of the Swiss Light Source (SLS, Switzerland) and the structure was refined against 2.4-˚A data obtained at beamline ID29 of ESRF (France).

Figure 8.6: Van der Waals surface of the c-ring. The surface is colored light-grey for apolar, yellow for polar, red for acidic and blue for basic surface exposed residues. A: The outer ring surface is shown. A putative DCCD-binding pocket is indicated by an arrow. B: The proximal half of the ring is removed to view the inner surface and some of the helices are shown in ribbon representation for clarity.

Dashed lines near Phe⁵ and Tyr³⁴ mark the putative positions of polar/apolar interfaces of the inner lipid bilayer. The cavity below the binding site is marked by an arrow.

Figure 8.7: Ribbon model of the E. coli c₁₀ oligomer obtained by homology modeling [126] according to the I. tartaricus c₁₁ structure. The residues which form cross-links with subunit a after cysteine sub-stitution and the proton binding Asp⁶¹ are also shown.

Figure 8.8: Schematic model for the interconversion of the binding site in the subunit a/c interface from an alternately locked conformation to an open one. The view is from the cytoplasmic side on the sec-tion of the interface at the level of the binding sites. The side chain of Glu⁶⁵ locks the Na⁺ ion (yellow circle) in the binding site by its orientation approximately horizontal to the membrane plane (Fig. 8.7). We propose that a site entering the interface as indi-cated by the solid arrow encounters electrostatic interactions upon approaching Arg²²⁷ in two ways: the Na⁺ is repelled into the cyto-plasmic outlet path (i.e. in a plane perpendicular to the membrane) and the Glu⁶⁵ is attracted, maintaining approximately its original conformation. Please note that the Glu⁶⁵ is on the distal helix with respect to the Arg²²⁷ of the functional unit if rotation proceeds in the ATP synthesis direction. After rotation of the binding site to the opposite side of Arg²²⁷, Glu⁶⁵ is pulled backwards towards the arginine thus opening the gate to the periplasmic inlet channel.

Na⁺ ions can now pass from subunit a to subunit c (dashed arrow) and the locked conformation of the binding site reforms when the site moves on and the electrostatic attraction between Arg²²⁷ and Glu⁶⁵ is attenuated. The reverse order of events takes place in the ATP hydrolysis direction.

The nucleotide-sugar UTP-α-D-glucose is a key metabolite in procaryotes.

UDP-glucose plays a central role in the synthesis of the cell envelope ofE. coli and is the glycosyl donor during the biosynthesis of lipopolysaccharides [127, 128], membrane-derived oligosaccharides (MDO) [129], and capsular polysac-charides [130]. It is also necessary in galactose and trehalose metabolism.

UDP-α-D-glucose is an essential intermediate for growth on trehalose [131], the synthesis of trehalose [132] and growth on galactose [133]. In addition, it interferes with the expression of σ^s during steady state growth [134].

UDP-α-D-glucose is produced enzymatically by the glucose-1-phosphate

uri-Figure 9.1: Ribbon diagram of the search model G1P-TT in blue. The red part was cut out before molecular replacement attempts.

Figure 9.2: Ribbon diagram of the GalU tetramer.

dylyltransferase (GalU). This enzyme consists of 301 amino acid residues and catalyses the reaction of α-D-glucose-1-phosphate and UTP to UDP-glucose and pyrophosphate, according to the following scheme:

UTP + glucose-1-phosphate ↔ UDP-glucose + pyrophosphate.

The overexpression system was constructed by Reinhold Horlacher (AG Boos, University of Konstanz). Crystallization and preliminary x-ray structure analysis were performed by Joachim Diez (AG Welte/Diederichs, University of Konstanz) [135]. He collected data sets of native crystals and various deriva-tives down to a resolution of 1.7 ˚A but structure solution with experimental phases failed.

Figure 9.3: Superposition of GalU (in red) and G1P-TT (in blue). The bound sugar molecule (in ball-and-stick representation) belongs to the structure of G1P-TT.

A BLAST search¹ through the PDB-database allowed us to recognize en-zymes with known structures and similar sequences. They are all nucleotidyl-transferases, but none of them uses UTP as nucleotidyldonor: glucose-1-phosphate thymidylyltransferase (G1P-TT; PDB-code 1H5R) [136] from E.

coli, glucose-1-phosphate thymidylyltransferase (RmlA) [137] fromPseudomonas aeruginosa, glucose-1-phosphate thymidylyltransferase (Ep) [138] fromSalmonella eneterica L2 the phosphocholine cytidylyltransferase (LicC) [139] from Strep-tococcus Pneumoniae. Like GalU, all these are active as homotetramers.

During this PhD-thesis, the structure of GalU was determined in coopera-tion with Alexei Vagin using his program MOLREP [50]. The search model for molecular replacement was a truncated model of the glucose-1-phosphate thymidylyltransferase from E. coli [136] missing the 44 C-terminal residues following residue 247. This structural fragment was omitted because it con-sists of three helices which protrude out of the globular domain of G1P-TT as can be seen in figure 9.1. After an initial rotation search with MOLREP

1http://www.expasy.org/tools/blast/

Figure 9.4: Superposition of the active sites of GalU (in red) and G1P-TT (in blue). The substrate (in ball-and-stick representation) belongs to the structure of G1P-TT.

[50], 50 peaks were used for a subsequent dyad search [140] which also incorpo-rated informations from a previously performed calculation of the self-rotation function. The dyad search lead to a dimer in which two properly rotated monomers were oriented correctly with respect to each other. Subsequently this dimer was used as a search model in a conventional MOLREP [50] search yealding one tetramer, as expected. Model building was done with several automated, semi-automated and manual methods. Automated model building was performed with the programs ARP/wARP [141] and RESOLVE [142, 74].

Semiautomated and manual model building was done with O [53] and COOT [76]. Refinement was performed with REFMAC5 [51] (Collaborative Compu-tational Project Number 4, 1994 [52]).

The structure of GalU was solved at 1.7 ˚A resolution despite a very high

Figure 9.5: Superposition of the B-site of G1P-TT (in blue and light blue) with bound substrate (in ball-and-stick representation) and the cor-responding area in one monomer of GalU (in red). The putative B-site in GalU is blocked completely by the residues 140-146.

root mean square deviation of 1.8 ˚A (for 222 Cα-atoms in a cutoff range of 4.5 ˚A (calculated with LSQMAN [143])) and a low sequence identity of only 24% compared with the search model G1P-TT. It was refined to a final R-factor of ∼ 19% (R_{f ree} ∼ 24%). Figure 9.2 gives an overview of the GalU tetramer and figure 9.3 shows the superposition of one monomer of GalU with one of G1P-TT. A comparison of the active sites of both structures (figure 9.4) suggests the same catalytic mechanism for GalU as predicted for G1P-TT [136], despite the absence of substrate in the structure of GalU.

Many nucleotidylyltransferases possess a so called secondary binding site (B-site) whose function is not yet understood. This secondary site is missing in GalU due to a different mode of tetramerisation (figure 9.5). The B-site in G1P-TT is formed in the interface of two monomers. Due to the different tetramerization, this interface is missing in GalU. Furthermore, the loop from residue 140 to 146 blocks the putative B-site completely.

The results of this work are yet unpublished. In this short overview my contribution to the project is outlined.

perspectives

During this PhD-thesis, 4 protein structures were solved. Two of them were fragments of TonB (see Chapters 3-6). As described in chapter 3, TonB is anchored in the inner membrane of E. coli and couples the electrochemical potential over the inner membrane to energy dependent transport of various compounds through the outer membrane via so called TonB-dependent recep-tors. By the time of the here presented work, the mechanism of energy transfer was unknown. One open question was whether TonB interacts as a dimer or a monomer with its outer membrane receptors. To shed more light on this sub-ject, several constructs of C-terminal TonB-fragments with different lengths were created and used for various experiments. Two of them were crystallized, TonB77 and TonB92 (consisting of 77 and 92 C-terminal amino acides, respec-tively). The structure of TonB77 was solved with molecular replacement using the model of TonB86 [10] and was refined to an R_{f ree} of 27.1% (see chapter 4).

In the case of TonB92, the best crystals diffracted to a final resolution of 1.08 ˚A (described in chapter 5) but the structure could not be solved with molecular replacement. Therefore, SeMet crystals were grown and used in a MAD ex-periment where 3 datasets corresponding to the peak-, inflection- and remote high-wavelength of Selen were collected. After determinimg the substructure of the Selen atoms with SOLVE [73], RESOLVE [74, 75] was used for solvent flattening and automated model building. Structure refinement of TonB92 was done at a resolution of 1.13 ˚A and yielded a final R_{f ree} of 18.3% using SHELXL [77]. Since the structure of TonB92 shows a completely different fold as the dimeric TonB77 and behaves monomeric in solution, we suggested, that TonB interacts as a monomer with its respective receptors. This finding is now supportetd by structures of complexes of TonB with FhuA and FecA [8, 9]. In

both cases, TonB clearly interacts as a monomer.

The next project in this PhD-thesis was the structure of the rotor ring of F-type Na⁺-ATPase from Ilyobacter tartaricus (see chapters 7 and 8). This c-ring consists of 11 subunits and was solved with molecular replacement using a likelihood-based program called PHASER [93]. Other molecular replacement programs were incapable to overcome the phase problem and numerous at-tempts to solve the structure with anomalous scatterers also failed. Finally, a low resolution electron crystallography model was used as search model in PHASER and gave an interpretable electron density map. The space group of the successfully used dataset was determined to be P21 with 4 complete c-rings in the asymmetric unit. Manual correction of the model was performed with COOT [76] using only one chain of one 11-mer. Subsequently, the remaining 43 monomers were gained by rotation and translation of the modified one.

The necessary transformation matrices could be calculated from the molecu-lar replacement solution. During refinement, a non-crystallographic symmetry restraint was imposed over the 44 monomers in the asymmetric unit with the exclusion of the loop regions that form crystal contacts which made a modifi-cation of the REFMAC5 source code necessary. The structure was refined at 2.4 ˚A resolution to an R_{f ree} of 24.6% and yielded a model with no Ramachan-dran plot outliers.

Since the transport pathway of sodium ions is not clarified so far, co-crystalli-zation of c-ring with its corresponding a-subunit should be the next important step in understanding the mechanism of ATP synthesis with the F0F1-ATPase.

The last mentioned project of this doctoral work was the determination of the structure of GalU described in chapter 9. GalU is a glucose-1-phosphate uridylyltransferase which catalyzes the reaction of glucose-1-phosphate and UTP to UDP-glucose and pyrophosphate. The structure of the GalU tetramer was solved with a so called dyad search in MOLREP [140] using a trun-cated tymidylyltransferase (G1P-TT) as search model. Manual correction was mainly done with COOT [76] and the structure was refined with REFMAC5 [51] (Collaborative Computational Project Number 4, 1994 [52]) to a final R_{f ree} of ∼24% at 1.7 ˚A so far. From the structure one can deduce the same catalytic mechanism for GalU as is predicted for G1P-TT [136] due to the fact, that the catalytic active sites can be superimposed very well (see figure 9.4).

A publication of the work on this project is in progress.

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First of all I would like to thank Prof. Wolfram Welte for giving me the chance to work on a lot of interesting projects. He helped me with good advices and was ready for discussions at all times.

I am also grateful to Prof. Kay Diederichs who taught me most of what I know about x-ray crystallography. He was also very helpful in solving a multitude of computer problems.

I enjoyed very much to work with Dr. Jiri K¨odding on the TonB project. He gave me the opportunity to solve my first protein structure.

I enjoyed very much to work with Dr. Jiri K¨odding on the TonB project. He gave me the opportunity to solve my first protein structure.

Im Dokument X-ray Analysis of Biological Macromolecules (Seite 85-112)