Biochemical characterization of the relaxase TraI, the coupling protein TraD and the hypothetical protein Yaf of the novel Type IV Secretion System from the human pathogen Neisseria gonorrhoeae

Type IV secretion systems (T4SSs) are able to transport DNA and effector proteins across the inner and outer membranes of prokaryotes. T4SSs consist of a transport complex which forms the channel across the inner and outer membrane, the so called mating pair formation complex (MPF), and DNA processing and transfer proteins (Dtr proteins). A unique T4SS is encoded in the gonococcal genomic island which is found in most Neisseria gonorrhoeae strains. Remarkably, this secretion system is the first T4SSs that was shown to secrete ssDNA during all phases of growth directly into the environment. This property might facilitate functional studies on the transport mechanisms used by T4SSs. Remarkably, the T4SS encoded within the GGI consists of a MPF complex that is homolgous to the MPF complex of the E. coli F-plasmid (MPFF family) and Dtr proteins that belong to the MOBH family of targeting factors. Although many different members of the MOBH family have already been identified, almost no information is currently available on this novel group of targeting factors. To study the conservation of GGI-like T4SSs, similar T4SS were identified in a computational analysis of all currently completely sequenced chromosomes and plasmids. Next to N. gonorrhoeae very similar conserved T4SSs have been found in several other bacteria. These T4SSs are either encoded within the chromosome or on plasmids. Most of the genes encoded in the GGI between the ltgX and exp1 genes, the region which encodes mainly the MPFF proteins, were highly conserved in allmost all of the identified systems, even though, several of theses genes have been shown previously not to be important for ssDNA transport. Remarkably, all identified chromosomal GGI-like T4SSs encode mpf genes of the MPFF family and dtr genes of the MOBH family; whereas, the plasmid encoded GGI-like T4SSs always contain dtr genes of the MOBF family and mpf genes of the MPFF family. This suggests that relaxases of the MOBH family are preferentially found in chromosomally encoded T4SSs. To elucidate the functional mechanism of the DNA processing reaction of this unique DNA secretion system a biochemical approach was set up to functionally characterize the coupling protein TraD, the relaxase TraI and the hypothetical protein Yaf which are different key enzymes of the Dtr proteins of N. gonorrhoeae. The overexpression and purification of coupling proteins and relaxases of other T4SSs has previously been shown to be inherently difficult. Although many different approaches using E. coli as host for a recombinant overexpression have been tested, it was not possible to obtain soluble TraD and large amounts of inclusion bodies were produced. Small amounts of TraD have been detected in isolated inner membranes of E. coli, but all performed solubilization attempts failed. Finally, TraD was isolated and purified from inclusion bodies under denaturing conditions, but all further refolding approaches with the purified protein resulted either in the loss or the aggregation of TraD. Therefore, it was not possible to characterize the enzymatic activity of the neisserial coupling protein TraD. The hypothetical protein Yaf, which is encoded in same operon with traI and traD was sucessfully overexpressed and purified. Interestingly, Yaf forms different oligomeric states in solution. The most abundant oligomer of native Yaf was the dimeric state. The dynamic equilibrium of the different oligomeric states was strongly dependent on the concentration of the protein and the buffer conditions. The interconversion between the different oligomeric states is a slow process. The presence of an N-terminal His-tag strongly stabilized the tetrameric form. Limited proteolysis experiments showed that Yaf is highly resistant against trypsin and most likely has a very compact and stable structure. Tetrameric Yaf showed a higher resistance against proteolytic digestion than dimeric Yaf. Remarkably, the introduction of different point mutations into the sequence of Yaf resulted in a destabilization of the protein and a strong decrease in the amount of soluble protein. Different activity assays have been performed with the purified protein to study the function of Yaf. DNA binding assays showed binding of dimeric and tetrameric Yaf to dsDNA with low affinity (>5 μM). Furthermore, a metal dependent sequence unspecific nuclease activity was observed associated with elution fractions containing dimeric and tetrameric Yaf. This nuclease activity was specific for dsDNA fragments and oligonucleotides that contained secondary structures. In the presence of different divalent metals, a relaxation activity on supercoiled plasmid DNA has been observed for fractions containing dimeric and tetrameric Yaf, and was strongly enhanced in the presence of Mn2+ and Co2+, but was also observed in the presence of Mg2+. To obtain further information on the structure and function of Yaf, crystallization attempts have been started in close collaboration with Dr. S. Smits in the group of Prof. Dr. L. Schmitt (Heinrich Heine University in Düsseldorf). Small crystals of N-terminally His-tagged Yaf diffracted up to 3 Å. Remarkably, no crystals have been obtained for native Yaf in any of the tested conditions. Recentyl, crystals of N-terminal His-tagged Yaf with incorporated selenomethionine have been obtained. To characterize the MOBH relaxase TraI, many different overexpression and purification approaches have been tested. Finally, an overexpression and purification method could be established to purify soluble TraI. Isolated TraI formed stable dimers in solution, and limited proteolysis revealed the presence of at least three trypsin resistant domains in TraI. DNA binding assays showed that TraI binds to ssDNA and dsDNA with high affinity in a sequence unspecific manner. The minimal binding frame of TraI for ssDNA substrates has been determined with 9 nucleotides. Furthermore, a metal dependent sequence unspecific relaxation activity of TraI on supercoiled plasmids has been observed, with Mn2+ as preferred metal cofactor. The relaxation activity of TraI is similar to the activity of E. coli Topoisomerase I, but differs within the preferred metal cofactor and the lack of detectable intermediates during the relaxation reaction of TraI. DNA cleavage assays performed with different ssDNA substrates demonstrated that TraI cleaves oligonucleotides containing the putative oritT sequence of the GGI or the oriT region of the E. coli F plasmid strand specific in a Mn2+ dependent manner. The complementary strands and a polyT oligonucleotide were not cleaved by TraI. Analysis of the putative cleavage sites did not show any sequence similarity between both cleaved oligonucleotides. To obtain structural information about TraI, crystallization attempts in close collaboration with Dr. S. Smits in the group of Prof. L. Schmitt (Heinrich Heine University in Düsseldorf) were started. Besides the characterization of the enzymatic activity of TraI and Yaf, pull down assays were performed to test whether these proteins interact with each other or with the T4SS ATPase TraC. Unfortunately, no protein-protein interactions were detected between these three proteins. The experimental results from the current study help to understand the DNA processing mechanism of the unique T4SS of N. gonorrhoeae. Important steps have been made in the functional overexpression and the purification of Yaf and TraI. The hereby obtained results from the biochemical characterization of TraI represent the first reported biochemical data which is available on the novel MOBH relaxase family. Furthermore, this study revealed first insights into a possible enzymatic activity of the previously uncharacterized hypothetical protein Yaf, even though the function of this protein still remains unknown