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abstracts
In all organisms, secretion systems mediate the passage of macromolecules across cellular membranes. The bacterial type IV secretion system (T4SS) family can be divided into three functional groups. First, as typified by the Brucella suis system, T4SSs deliver effector macromolecules into eukaryotic cells during the course of infection. Second, in some Gram-negative bacteria, such as in Helicobacter pylori (ComB system), T4SSs mediate DNA uptake from and release into the extracellular environment. Thirdly, as in the IncN plasmid pKM101, T4SSs can mediate the conjugative transfer of plasmid DNA or transposons into a wide range of bacterial species. This conjugation phenomenon contributes to the spread of antibiotic resistance genes among pathogenic bacteria, leading to the emergence of multidrug-resistant pathogenic strains. TraE of the IncN plasmid pKM101 belongs to the VirB8 family of proteins, an essential component of most T4SSs that form functional dimmers in the T4SS core. Here, we present the X-ray crystallographic structure of the periplasmic domain of TraE at 2.4 Å resolution. The structure shows many similarities to the known VirB8-like protein structures from Brucella suis [1] and Agrobacterium tumefaciens [2]. However, the nature and the number of residues implicated in the dimerization interface differ considerably from those in the TraE structure [2]. Similar to other VirB8 homologs we have shown by analytical gel filtration that there is a concentration dependant equilibrium between monomeric and dimeric forms of TraE. Moreover, using a bacterial two-hybrid assay, in vivo dimerization has been demonstrated with full-length TraE and key residues for dimerization were identified by site-directed mutagenesis. Our work adds novel insights into the growing body of knowledge on VirB8-like proteins and it will inform future strategies aimed at developing inhibitors of TraE protein interactions and of plasmid transfer.
abstracts
The similarity of crystal engineering to organic synthesis has been noted by Desiraju and many concepts and strategies have been successfully transferred. We aim to combine the two fields of research into one new concept called "Covalent Assistance to Supramolecular Synthesis". The supramolecular reagent isonicotinic acid hydrazide (isoniazid) is a promising molecule in the supramolecular synthesis of multi-component molecular complexes (Lemmerer at al., 2010). Due to the covalent reaction of the carbohydrazide functional group with simple ketones and aldehydes, the hydrogen bonding functionality of isoniazid can be modified, where two of the hydrogen bond donors are replaced with hydrogen bonding "inert" hydrocarbons (Lemmerer et al., 2011). The "modifiers" bonded to the isoniazid then give a measure of control of the outcome of the supramolecular synthesis with various carboxylic acids depending on the identity and steric size of the modifier used. The steric size itself can be used to shield or to "mask" the remaining hydrogen bonding functionality of isoniazid such that common homomeric and heteromeric interactions are prevented from taking place.
