Entwicklung von Methoden für das computergestützte Design von Mimotopen

Die wachsende Menge an experimentell aufgeklärten Protein-Protein-Komplexen oder allgemeiner: Protein-Ligand-Komplexen, erlaubt das immer genauere Studium von biomolekularen Wechselwirkungen. Eine Teilmenge der existierenden Wechselwirkungen bilden die für die adaptive Immunantworten wichtigen Antigen-Antikörper-Wechselwirkungen. Die chemischen Gruppen an der Oberfläche der Antigene entscheiden über die spezifischen Wechselwirkungen mit Antikörpern, und werden als antigene Determinanten oder Epitope bezeichnet. Die dazu komplementären Bindestellen auf den Antikörpern werden als Paratope bezeichnet. Häufig verwendet man den Begriff „Epitop“ allgemein für Molekülteile, die spezifisch erkannt werden. Die Spezifität der Epitope wird sowohl durch die geometrische Anordnung als auch durch die chemische Konfiguration der monomeren Gruppen bestimmt. Mimotope sind synthetisch hergestellte Proteine, die die strukturellen Erkennungsmerkmale der Epitope nachahmen und somit z.B. eine definierte Immunantwort auslösen können. Beispielsweise ist es nun möglich, Epitope bis zur atomaren Auflösung zu identifizieren und nach ähnlichen Strukturmotiven auf anderen Proteinstrukturen zu suchen. Diese Art des Strukturvergleichs eröffnet interessante Anwendungen: Epitope lassen sich ggf. auf andere Trägermoleküle transplantieren, oder es könnten Kreuzreaktivitäten vorhergesagt werden. Entscheidend für diese Ansätze ist die Verfügbarkeit einer Methode, mit der sich Strukturmotive schnell und genau vergleichen lassen. Die Entwicklung einer solchen Methode (EpitopeMatch) ist das Ziel dieser Promotionsarbeit. Im Einzelnen soll EpitopeMatch folgende Eigenschaften besitzen: • Einbeziehung geometrischer und chemischer Ähnlichkeit. • Flexible Definition von i. Allg. diskontinuierlichen Epitopen auf der Grundlage bekannter Komplexstrukturen. • Effiziente Suche auf großen Strukturdatenbanken. • Möglichkeit der Transplantation vollständiger Epitope. • Verknüpfung der Fundstellen mit funktionellen biologischen Daten

Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life

Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNAPhe at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m2G6 (N2-methylguanosine) MTase TTCTrmN from Thermus thermophilus and its ortholog PfTrm14 from Pyrococcus furiosus. Structures of PfTrm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. TTCTrmN and PfTrm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNAPhe of T. thermophilus and via site-directed mutagenesis

A Novel Algorithm for Macromolecular Epitope Matching

algorithm

Switch mechanism triggered by Ca2 calcium ion.

<p>(A) ShhN zinc center in states Ca0 (X-ray structure 1vhh, red), Ca1 (X-ray structure 3n1r, blue), Ca2 (X-ray structure 3d1m, black). Putative catalytic water from 1vhh is close to the zinc ion. From Ca0 to Ca1 and Ca2, E127 carboxylate is drawn towards and drags H-bound H135 side chain with it, away from substrate and the active E177. While Ca0 and Ca1 superimpose well, Ca2 is clearly different. (B) Central components of the switch mechanisms in states Ca0 and Ca2. (C) Distances between H-bonded E127 carboxylate-O and H135 imidazole-proton, and between substrate-clamping side chains of H135 and catalytically active E177. Red (Ca0) and black (Ca2) points are sampled by MD simulations. Green triangle (Ca0) and green square (Ca2) are the corresponding values directly taken from X-ray structures 1vhh and 3d1m, respectively.</p

Phylogenetic tree of all 30 reviewed full length Hedgehog proteins from UniProtKB.

<p>The vertebrate Hedgehogs (bottom subtree) are clearly separated from the <i>Drosophila</i> Hedgehogs (top subtree). In all vertebrates the full catalytic motif is absolutely conserved (red), except in rat with one conservative exchange (blue).</p

<i>In vitro</i> tests of ShhN mutant stabilities against proteolysis.

<p>The logarithm of protein content (relative to maximum protein content) is plotted over time. Proteins are (E177A, red) and (E177, blue). Straight red and blue lines are least squares fits to the measurements, shaded areas around these lines are 95% confidence intervals for the corresponding linear models. All data refer to measurements at .</p

Effect of binding on ShhN structure.

<p>Structures taken from MD trajectories for Ca2 (grey; based on PDB entry 3d1m with two ) and Ca0 (red; 1vhh). Shown are sampled structures close to the cluster centers of Ca2 and Ca0 ensembles. (A) Overview and comparison of full ShhN structures in Ca2 and Ca0 states. Position of calcium ions marked by green mesh. Calcium binding pocket formed by loops , breaks up as is removed (transition grey to red). Neighboring loop is also affected although it does not co-ordinate . (B) Close-up of (green spheres) binding site in Ca2 state. Calcium ions are surrounded by a cage of anionic side chains. (C) Same region as in panel B, but now in Ca0 state. Note the large distance differences of anionic groups between B and C.</p

Signaling Domain of Sonic Hedgehog as Cannibalistic Calcium-Regulated Zinc-Peptidase

<div><p>Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double- center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants <i>in vitro</i>: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The <i>in vitro</i> results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on .</p></div

Statistical comparisons of zinc center geometries.

<p>(A) RMSDs between zinc centers from LAS enzyme X-ray structures to ShhN in states Ca0 (red), Ca1 (blue), Ca2 (grey) sampled by molecular dynamics. (B) RMSDs between zinc centers from MD simulations of LAS enzymes and MD simulations of ShhN in states Ca0, Ca1, Ca2.</p