Binding studies of a putative C. pseudotuberculosis target protein from Vitamin B12 Metabolism.

Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis's Vitamin B12 metabolism and related pathways to reduce the pathogen's virulence in its hosts.

Improving predictions of protein-protein interfaces by combining amino acid-specific classifiers based on structural and physicochemical descriptors with their weighted neighbor averages.

Protein-protein interactions are involved in nearly all regulatory processes in the cell and are considered one of the most important issues in molecular biology and pharmaceutical sciences but are still not fully understood. Structural and computational biology contributed greatly to the elucidation of the mechanism of protein interactions. In this paper, we present a collection of the physicochemical and structural characteristics that distinguish interface-forming residues (IFR) from free surface residues (FSR). We formulated a linear discriminative analysis (LDA) classifier to assess whether chosen descriptors from the BlueStar STING database (http://www.cbi.cnptia.embrapa.br/SMS/) are suitable for such a task. Receiver operating characteristic (ROC) analysis indicates that the particular physicochemical and structural descriptors used for building the linear classifier perform much better than a random classifier and in fact, successfully outperform some of the previously published procedures, whose performance indicators were recently compared by other research groups. The results presented here show that the selected set of descriptors can be utilized to predict IFRs, even when homologue proteins are missing (particularly important for orphan proteins where no homologue is available for comparative analysis/indication) or, when certain conformational changes accompany interface formation. The development of amino acid type specific classifiers is shown to increase IFR classification performance. Also, we found that the addition of an amino acid conservation attribute did not improve the classification prediction. This result indicates that the increase in predictive power associated with amino acid conservation is exhausted by adequate use of an extensive list of independent physicochemical and structural parameters that, by themselves, fully describe the nano-environment at protein-protein interfaces. The IFR classifier developed in this study is now integrated into the BlueStar STING suite of programs. Consequently, the prediction of protein-protein interfaces for all proteins available in the PDB is possible through STING_interfaces module, accessible at the following website: (http://www.cbi.cnptia.embrapa.br/SMS/predictions/index.html).