Biology subjects

Brandao-Neto, J.

Publications and source records attributed to Brandao-Neto, J..

3 recordsLinked to original sources

bioRxiv2018

The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleotide selectivity of PNPs

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, named as SmPNP2. Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands. The binding of cytosine was confirmed by isothermal titration calorimetry, with a KD of 27 M, and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of 76.3 M. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystallographic structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capability of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site allowing an alternative binding mode for nucleosides, with a 180{degrees} rotation from the canonical binding mode. The remarkable plasticity of this binding site deepens the understanding of the correlation between structure and nucleotide selectivity, offering new ways to analyses PNP activity.\n\nAuthor SummarySchistosoma mansoni is a human parasite dependent on purine salvage for purine bases supply. Purine nucleoside phosphorylase (PNP) is a key enzyme in this pathway. It carries two PNP isoforms, one previously characterized (SmPNP1) and one unknown (SmPNP2). Here we present the crystallographic structure of SmPNP2 and its complex with cytosine, cytidine, ribose-l-phosphate, adenine, hypoxanthine, and tubercidin. Cytidine and cytosine were identified as ligands of SmPNP2 using a thermofluorescence approach. Binding of cytosine was proven by Isothermal Titration Calorimetry (ITC) and cytidine, inosine, and adenosine kinetic parameters were also obtained. Purine bases showed different binding in the active site, rotated 180{degrees} from the canonical binding mode. Its the first report showing a Low Molecular Mass PNP capable of catalyzing both types of nucleotide bases. The SmPNP2 odd behavior sheds a new light on the Schistosoma mansonis life cycle metabolic adaptation.

biochemistry↗

bioRxiv2017

New routes for PTP1B allosteric inhibition by multitemperature crystallography, fragment screening and covalent tethering

Allostery is an inherent feature of proteins and provides alternative routes to regulating function. Small-molecule allosteric inhibitors are often desirable; however, it remains challenging to identify surface sites in proteins which can bind small molecules and modulate function. We identified new allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal \"hidden\" low-occupancy conformational states for protein and ligands. Our results converge on a new allosteric site that is conformationally coupled to the active-site WPD loop, a hotspot for fragment binding, not conserved in the closest homolog, and distinct from other recently reported allosteric sites in PTP1B. Targeting this site with covalently tethered molecules allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure revealed by multitemperature crystallography can be exploited for developing allosteric modulators.

biophysics↗

bioRxiv2016

Gentle, fast and effective crystal soaking by acoustic dispensing

SynopsisA high-throughput method is described for crystal soaking using acoustic droplet ejection, and its effectiveness demonstrated.\n\nAbstractBright light sources, agile robotics, and fast detectors are continually reducing the time it takes to perform an X-ray diffraction experiment, making high throughput experiments more feasible than ever. But this is also pushing the upstream bottleneck towards sample preparation, even for robust and well characterised crystal systems. Crystal soaking is routinely used to generate protein-ligand complex structures, yet protein crystals are often sensitive to changes in solvent composition, and frequently require gentle or careful stepwise soaking techniques, limiting overall throughput. Here, we describe the use of acoustic droplet ejection for soaking of protein crystals with small molecules, and show that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 minutes. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables targeted placement of the compound/solvent away from crystals and towards drop edges, allowing for gradual diffusion of solvent across the drop. This ensures both an improvement in reproducibility of X-ray diffraction and an increased solvent tolerance of the crystals, thus enabling higher effective compound soaking concentrations. We detail the technique here with examples from the protein target JMJD2D, a histone lysine demethylase, having roles in cancer and the focus of active structure based drug design efforts.

biophysics↗