Expanding the repertoire of sortases applicable for advanced protein engineering

Sortases are transpeptidases which are used to modify, ligate and immobilize peptides or proteins in a site-specific manner 1. To extend the portfolio of sortases used to engineer proteins in vitro, a new type of assay was developed, applicable as high throughput screening tool but also as very sensitive analytical method. With this so-called Reporter Immobilization Assay (REIA) it was possible to perform a biodiversity screening of a variety of wild type sortases found in different species as well as a directed evolution campaign on one of those wild type enzymes 2. The biodiversity screening revealed that the sortase A from Listeria monocytogenes (Lm-SrtA) holds a high reactivity to the unusual LPXTA sort motif. This newly described specificity of Lm-SrtA can be used for efficient orthogonal double-labeling approaches e.g. in combination with sortase A from S. aureus (Sa-SrtA) catalyzing conjugation reactions at the conventional LPXTG sort motif. Furthermore, the Lm-SrtA appears calcium-independent and for this reason is able to catalyze conjugation reactions in vivo. Based on this finding, the Lm-SrtA is suggested as intracellular bioengineering tool. Another conclusion of the biodiversity screening was that Sa-Srt appears as the most efficient wild type enzyme for conjugation at the LPXTG motif, as described also by previous studies performed by others 3. Based on its high starting efficiency, a directed evolution approach was performed with the REIA with the intent to further increase activity and affinity of Sa-SrtA. After four rounds of directed evolution, indeed a five-time mutant of Sa-SrtA was identified possessing a dramatically increased catalytic efficiency and even slightly increased thermo stability. Finally, molecular dynamics simulations gave first insights that two of these five beneficial mutations found in the engineered Sa-SrtA variant are stabilizing the catalytically active Arginine which is in charge of substrate binding and oxyanion stabilization. 1. Schmohl, L.; Schwarzer, D., Curr Opin Chem Biol 2014, 22, 122-8. 2. Schatte, M.; Bocola, M.; Roth, T.; Martinez, R.; Kopetzki, E.; Schwaneberg, U.; Bönitz-Dulat, M., Bioconjugate chemistry 2016, 27, 1484-1492. 3. Navarre, W. W.; Schneewind, O., Mol Microbiol 1994, 14, 115-21

Poly[μ-(1,3-dihy­droxy­propan-2-olato)-potassium]

The asymmetric unit of the title compound, [K(C3H7O3)]n or K[H2gl]n, common name potassium glycerolate, contains half the K+ cation and half of the glycerolate anion. The other half of the anion is generated through a mirror plane passing through the K atom, and a C, an H and an O atom of the glycerolate ligand. The K+ ion is coordinated by the O atoms of the OH groups, leading to a six-membered chelate ring that adopts a very distorted boat conformation. The negatively charged O atom of the glycerolate anion, [H2gl−], is found in the flagpole position and forms an ionic bond with the K+ ion. The O atoms of the hydroxo groups are coordinated to two K+ ions, whereas the negatively charged O atom is bonded to one K+ ion. The K+ ion is coordinated by three other symmetry-related monodentate H2gl− ligands, so that each H2gl− ligand is bonded to two K+ ions, and the potassium has a seven-coordinate environment. The H2gl− ligands are connected via a strong O—H⋯O hydrogen bond and, together with the K⋯O inter­connections, form polymeric sheets which propagate in the directions of the a and b axes

Poly[μ-2,3-dihydroxy­propan-1-olato-sodium]

The Na+ cation in the title compound, [Na(C3H7O3)]n or Na[H2gl], is coordinated by five O atoms leading to a distorted trigonal-bipyramidal geometry. The negatively charged O atom of the glycerolate anion is in an equatorial position, and the O atom of the hydroxo group, attached to the secondary C atom, occupies an axial position completing a five-membered non-planar chelate ring; this defines the asymmetric unit. The Na+ cation is coordinated by three other symmetry-related monodentate H2gl− ligands, so that each H2gl− ligand is bonded to four Na+ ions. The H2gl− ligands are connected via strong O—H⋯O hydrogen bonds and these, together with the Na⋯O inter­connections, are responsible for the formation of polymeric sheets which propagate in the directions of the b and c axes

Cyclo­linopeptide B methanol tris­olvate

The title compound, C56H83N9O9S·3CH3OH, is a methanol tris­olvate of the cyclo­linopeptide cyclo(Met1—Leu2—Ile3—Pro4—Pro5—Phe6—Phe7—Val8—Ile9) (henceforth referred to as CLP-B), which was isolated from flaxseed oil. All the amino acid residues are in an l-configuration based on the CORN rule. The cyclic nona­peptide exhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α-turn and two consecutive β-turns each containing a N—H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) or the third residue (β-turns), repectively. In the crystal, the components of the structure are linked by N—H⋯O and O—H⋯O hydrogen bonds into chains parallel to the a axis

Rezensionen

STÜBEN, JENS (ed.) (2007): Ostpreußen – Westpreußen – Danzig. Eine historische Literaturlandschaft. München: Oldenbourg Verlag (=Schriften des Bundesinstituts für Kultur und Geschichte der Deutschen im östlichen Europa 30). 762 S. TRABA, ROBERT (2005): ‚Wschodniopruskość‘. Tożsamość regionalna i narodowa w kulturze politycznej Niemiec. [‚Das Ostpreußentum‘. Die regionale und nationale Identität in der politischen Kultur Deutschlands]. Poznań/ Warszawa: Wydawnictwo Poznańskiego Towarzystwa Przyjaciół Nauk. 471 S. DAMPC-JAROSZ, RENATA / SZEWCZYK, GRAŻYNA BARBARA (eds.) (2006): Friedrich Schiller. W dwusetną rocznicę śmierci. [Zum 200. Todesjahr]. Wrocław: Oficyna Wydawnicza ATUT – Wrocławskie Wydawnictwo Oświatowe. 261 S. STRELKA, JOSEPH P. (2005): Ernst Schönwiese. Werk und Leben. Frankfurt (M.) / Berlin / Bern u.a.: Peter Lang Verlag (=New Yorker Beiträge zur Literaturwissenschaft 6). 193 S.  BIALIK, WŁODZIMIERZ (2005): Die gewöhnliche Trivialität. Zu Sekundärbotschaften und zur Ideologie der En-passant-Aussagen in Heinz Günter Konsaliks später Romanproduktion. Frankfurt (M.)/Berlin/Bern u.a.: Peter Lang Verlag (=Posener Beiträge zur Germanistik 5). 290 S. RUCHNIEWICZ, KRZYSZTOF / ZYBURA, MAREK (eds.) (2006): Niemieckojęzyczni laureaci Literackiej Nagrody Nobla. Wrocław: Oficyna Wydawnicza ATUT – Wrocławskie Wydawnictwo Oświatowe. 285 S. RUCHNIEWICZ, KRZYSZTOF / ZYBURA, MAREK (eds.) (2007): Die höchste Ehrung, die einem Schriftsteller zuteil werden kann. Deutschsprachige Nobelpreisträger für Literatur. Dresden: Neisse Verlag. 354 S. GANSEL, CHRISTINA / JÜRGENS, FRANK (22007): Textlinguistik und Textgrammatik. Eine Einführung. Göttingen: Vandenhoeck & Ruprecht / Westdeutscher Verlag Teutsch (=Studienbücher zur Linguistik 6). 270 S. GLÜCK, HELMUT / SCHRÖDER, KONRAD (eds.) (2007): Deutschlernen in den polnischen Ländern vom 15. Jahrhundert bis 1918. Eine teilkommentierte Bibliographie. Bearbeitet von Yvonne Pörzgen und Marcelina Tkocz. Wiesbaden: Harrassowitz Verlag. 319 S. LIPCZUK, RYSZARD (2007): Geschichte und Gegenwart des Fremdwortpurismus in Deutschland und Polen. Frankfurt (M.)/Berlin/Bern u.a.: Peter Lang Verlag (=Danziger Beiträge zur Germanistik 23). 251 S. RICHTER-VAPAATALO, ULRIKE (2007): „Da hatte das Pferd die Nüstern voll“. Gebrauch und Funktion von Phraseologie im Kinderbuch. Untersuchungen zu Erich Kästner und anderen Autoren. Frankfurt (M.)/Berlin/ Bern u.a.: Peter Lang Verlag (=Finnische Beiträge zur Germanistik 20). 333 S. LÜSEBRINK, HANS-JÜRGEN (2005): Interkulturelle Kommunikation. Interaktion, Fremdwahrnehmung, Kulturtransfer. Stuttgart/Weimar: Metzler. 201 S

Cyclo­linopeptide K butanol disolvate monohydrate

The title compound, C56H83N9O11S·2C4H10O·H2O, is a butanol–water solvate of the cyclo­linopeptide cyclo(Metsulfone1-Leu2–Ile3–Pro4–Pro5–Phe6–Phe7–Val8–Ile9) (henceforth referred to as CLP-K) which was isolated from flax oil. All the amino acid residues are in an l configuration based on the CORN rule. The cyclic nona­peptide exhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α- and a β-turn, each containing an N—H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) and the third residue (β-turn), repectively. In the crystal, the components of the structure are linked by inter­molecular N—H⋯O and O—H⋯O hydrogen bonds into a two-dimensional network parallel to (001). The –C(H2)OH group of one of the butanol solvent mol­ecules is disordered over two sets of sites with refined occupancies of 0.863 (4) and 0.137 (4)

Ca2+ binding induced sequential allosteric activation of sortase A: An example for ion-triggered conformational selection

The allosteric activation of the intrinsically disordered enzyme Staphylococcus aureus sortase A is initiated via binding of a Ca2+ ion. Although Ca2+ binding was shown to initiate structural changes inducing disorder-to-order transitions, the details of the allosteric activation mechanism remain elusive. We performed long-term molecular dynamics simulations of sortase A without (3 simulations of 1.6 mu s) and with bound Ca2+ (simulations of 1.6 mu s, 1.8 mu s, and 2.5 mu s). Our results show that Ca2+ binding causes not only ordering of the disordered beta 6/beta 7 loop of the protein, but also modulates hinge motions in the dynamic beta 7/beta 8 loop, which is important for the catalytic activity of the enzyme. Cation binding triggers signal transmission from the Ca2+ binding site to the dynamic beta 7/beta 8 loop via the repetitive folding/unfolding of short helical stretches of the disordered beta 6/beta 7 loop. These correlated structuralrearrangements lead to several distinct conformational states of the binding groove, which show optimal binding features for the sorting signal motif and feature binding energies up to 20 kcal/mol more favorable than observed for the sortase A without Ca2+. The presented results indicate a highly correlated, conformational selection-based activation mechanism of the enzyme triggered by cation binding. They also demonstrate the importance of the dynamics of intrinsically disordered regions for allosteric regulation