Untersuchung der Wechselwirkung von PCP Domänen mit Interaktionspartnern in Nicht-ribosomalen Peptidsynthetasen
Final Report Abstract
Non-ribosomal peptide synthetases (NRPS) and the related polyketide synthetases constitute two classes of modular multi-domain enzymes found in the cytoplasm of bacteria and fungi that synthesize a large variety of highly diverse peptides and bioorganic compounds with biological functions ranging from cell wall components to iron sequestration and chemical defense against other microorganisms. Many of these products are used as antibiotics, anti-inflammatory, antitumor or immunosuppressive drugs, making the investigation of the structure and function of NRPS and PKS systems of high medical relevance. In particular their modular organization makes them in principle highly suited for the creation of new molecules with potential medical applications. We have investigated the interaction between the central PCP and ACP domains and the enzymatic reaction centers. These studies have revealed that the linker region between the different domains is important for functionality. By using the Curacin A ACP/Halogenase system we could show that the enzyme recognizes an interface consisting of the ACP and the substrate. In addition we could demonstrate that there is no detectable interaction within a unique triple ACP cluster of the Curacin A PKS. By solving the crystal structure of the Phosphopantetheine transferases Sfp in complex with a PCP we could show that the interaction site of Sfp widens and can accommodate the A/H conformation of the PCP. By mutational analysis we could demonstrate that only few and interactions between both proteins are relevant to orient the PCP in the required orientation.
Publications
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2012. Characterization of molecular interactions between ACP and halogenase domains in the Curacin A polyketide synthase. ACS Chem Biol. 7:378-86
Busche A, Gottstein D, Hein C, Ripin N, Pader I, Tufar P, Eisman EB, Gu L, Walsh CT, Sherman DH, Löhr F, Güntert P, Dötsch V
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2013. Molecular crowding drives active Pin1 into nonspecific complexes with endogenous proteins prior to substrate recognition. J Am Chem Soc. 135:13796-803
Luh LM, Hänsel R, Löhr F, Kirchner DK, Krauskopf K, Pitzius S, Schäfer B, Tufar P, Corbeski I, Güntert P, Dötsch V
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2013. SPLICEFINDER - a fast and easy screening method for active protein trans-splicing positions. PLoS One. 8:e72925
Zettler J, Eppmann S, Busche A, Dikovskaya D, Dötsch V, Mootz HD, Sonntag T
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2014. Crystal structure of a PCP/Sfp complex reveals the structural basis for carrier protein posttranslational modification. Chem Biol. 21:552-62
Tufar P, Rahighi S, Kraas FI, Kirchner DK, Löhr F, Henrich E, Köpke J, Dikic I, Güntert P, Marahiel MA, Dötsch V
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2015. Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing. J Biol Chem. 290:27712-22
Mehler M, Eckert CE, Busche A, Kulhei J, Michaelis J, Becker-Baldus J, Wachtveitl J, Dötsch V, Glaubitz C
