Spectroscopic and thermodynamic properties of Debaryomyces hansenii UFV-1 α-galactosidases

Spectroscopic and thermodynamic properties were determined for Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases. α-Galactosidases showed similar secondary structure compositions (α-helix, β-sheet parallel and β-turn). Effects of pH and temperature on the structure of α-galactosidases were investigated using circular dichroism spectroscopy. It was more pronounced at low pH. Microcalorimetry was employed for the determination of thermodynamic parameters. Immediate thermal denaturation reversibility was not observed for α-galactosidases; it occurred as a thermodynamically driven process. Extracellular α-galactosidase, at pH 5.5, showed lower Tm when compared to the intracellular enzyme. The CD and DSC data suggest that D. hansenii α-galactosidases have different behaviors although they possess some similar secondary structures

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

<div><p>The hexameric purine nucleoside phosphorylase from <i>Bacillus subtilis</i> (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2′deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5′ hydroxyl group of adenosine and Arg^43* side chain contributes for the ribosyl radical to adopt an unusual C3’-<i>endo</i> conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl⁶ and Br⁸ substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser⁹⁰ by a threonine in the <i>B. cereus</i> hexameric adenosine phosphorylase (Thr⁹¹) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.</p></div

Biophysical and Structural Characterization of the Recombinant Human eIF3L

The eukaryotic translation initiation factor 3, subunit L (eIF3L) is one of the subunits of the eIF3 complex, an accessory protein of the Polymerase I enzyme and may have an important role in the Flavivirus replication by interaction with a viral non-structural 5 protein. Considering the importance of eIF3L in a diversity of cellular functions, we have produced the recombinant full-length eIF3L protein in Escherichia coli and performed spectroscopic and in silico analyses to gain insights into its hydrodynamic behavior and structure. Dynamic light scattering showed that eIF3L behaves as monomer when it is not interacting with other molecular partners. Circular dichroism experiments showed a typical spectrum of alpha-helical protein for eIF3L, which is supported by sequence-based predictions of secondary structure and the 3D in silico model. The molecular docking with the K subunit of the eIF3 complex revealed a strong interaction. It was also predicted several potential interaction sites in eIF3L, indicating that the protein is likely capable of interacting with other molecules as experimentally shown in other functional studies. Moreover, bioinformatics analyses showed approximately 8 putative phosphorylation sites and one possible N-glycosylation site, suggesting its regulation by post-translational modifications. The production of the eIF3L protein in E. coli and structural information gained in this study can be instrumental for target-based drug design and inhibitors against Flavivirus replication and to shed light on the molecular mechanisms involved in the eukaryotic translation initiation.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Overall structure of BsPNP233.

<p>A. Cartoon representation of the hexamer BsPNP233 with adenine (red spheres) bound in the active site. Solid and dashed grey arrows indicate the inter-dimeric and catalytic interfaces, respectively. B. Cartoon representation of BsPNP233 protomer in complex with adenosine (<i>green</i> stick). Loops, α-helices and β-strands are shown in <i>yellow</i>, <i>blue</i> and <i>pink</i>. The α-helices and β-strands were numbered according to the Mao and colleagues notation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044282#pone.0044282-Mao1" target="_blank">[50]</a>. C. BsPNP233 protomer colored by B-factors from <i>dark blue</i> (lowest) to <i>red</i> (highest). Adenosine is represented by a <i>black</i> stick.</p

The influence of Cl⁶ and Br⁸ modifications in catalysis and nucleoside binding.

<p>A. Structural alignment of Cl-Guo complex (<i>pink</i> carbon atoms) and dGuo complex (<i>cyan</i> carbon atoms). Spheres represent the van der Waals radius of Cl⁶, Gly⁹²C^α, Asp²⁰³O^δ1 and Val²⁰⁵C^γ2 atoms. B. Superposition of Cl-Guo complex and EcPNP-Ado-PO₄ complex (<i>yellow</i> carbon atoms, PDB code 1PK7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044282#pone.0044282-Bennett2" target="_blank">[54]</a>). Spheres represent the van der Waals radius of Cl⁶ and EcPNP Asp²⁰⁴O^δ1 atoms to highlight the steric conflict imposed by the Cl⁶ atom. C. The Br-Guo complex (carbon atoms in <i>green</i>), dGuo complex (carbon atoms in <i>orange</i>) and sulfate complex (carbon atoms in <i>magenta</i>, form IV, chain B) structures are superimposed. The sphere represents the van der Waals radius of Br⁸ and the dashed lines represent hydrogen bonds colored according to the respective structures. D. Structural comparison of Br-Guo complex and the trimeric HsPNP-Guo-SO₄ complex (<i>purple</i> carbon atoms, PDB code 1RFG, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044282#pone.0044282-Canduri1" target="_blank">[63]</a>). The spheres represent the van der Waals radius of Br⁸ and HsPNP Thr²⁴²O^γ1 atoms. The dashed circle has the same radius of Br⁸ and indicates the steric clash that would occur if BrGuo was placed at the Guo position in the HsPNP active site.</p

Structural basis of distinct kinetic models for phosphate binding in hexameric PNPs.

<p>A. Structural superposition of BsPNP233-sulfate open (<i>green</i>) and closed (<i>pink</i>) conformations with the BcAdoP-Ado complex (<i>yellow</i>, PDB code 3UAW, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044282#pone.0044282-Dessanti1" target="_blank">[52]</a>). The cartoon representation highlights the conformational differences observed in the main chain of the β9-α7 loop and the N-terminal portion of helix α7 in the three structures. Dashed lines represent hydrogen bonds and follow the color code of their respective structures. B. The surface representation of BsPNP233 Phe²²⁰ in the closed conformation (<i>pink</i>) and of the BcAdoP Thr⁹¹ evidence the steric hindrance imposed by the Thr⁹¹C^γ2 atom to that Phe²²⁰ rotamer. C. The surface representation of BsPNP233 Phe²²⁰ and Ser⁹⁰ in the closed conformation shows that the Ser⁹⁰ side chain allows the Phe²²⁰ side chain to perform the conformational change needed for the closed conformation takes place.</p

The binding mode of acyclic nucleosides.

<p>A. Stick representation of GCV bound in the BsPNP233 active site. B. Structural comparison of GCV-complex (<i>blue</i> carbon atoms) with dGuo-complex (<i>orange</i> carbon atoms). C and D show the stick representation of the two conformations of ACV (ACV¹ and ACV²) bound to the BsPNP233 active site. E. The structures of GCV-complex (<i>grey</i>) and ACV^1,2-complex (<i>green</i> carbon atoms) are superimposed. F. Structural alignment of ACV^1,2-complex with HsPNP-ACV complex (<i>pink</i> carbon atoms, PDB code 1PWY <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044282#pone.0044282-dosSantos1" target="_blank">[74]</a>). In all panels dashed lines indicate hydrogen bonds and are color coded according to their respective complexes.</p

The different conformations of Ado ribosyl radical.

<p>Structural superposition of BsPNP233-Ado (<i>magenta</i> carbon atoms), <i>B. cereus</i> adenosine phosphorylase (BcAdoP)-Ado-SO₄ (<i>green</i> carbon atoms, PDB code 3UAW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044282#pone.0044282-Dessanti1" target="_blank">[52]</a>) and <i>Entamoeba histolytica</i> PNP-Ado (<i>cyan</i> carbon atoms, PDB code 3U40 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044282#pone.0044282-Hewitt1" target="_blank">[55]</a>) complexes. The different puckers adopted by the ribose moiety of adenosine are labeled and the hydrogen bonds involving the 5′-OH group of Ado in each complex are represented by <i>dashed lines</i>. The sugar puckers were assigned by the pucker.py script of PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044282#pone.0044282-DeLano1" target="_blank">[47]</a>.</p

Comparison of free bases bound to the BsPNP233 active site.

<p>A. Structural comparison of a representative BsPNP233-Ade complex (<i>purple</i> carbon atoms) with the BsPNP233-Hyp complex (<i>grey</i> carbon atoms). B. The structure of the four BsPNP233-Ade complexes solved independently are superimposed. The sulfate-free Ade-complexes are colored in <i>purple</i> (chain A) and <i>pink</i> (chain B) whereas the two independent complexes solved with sulfate bound (dataset I) are colored in <i>orange</i> (chain A) and <i>blue</i> (chain B). C. The structure of the Ade-complex where Ade presents an alternative conformation (carbon atoms in <i>orange</i>) is superimposed in the structure of Hyp-complex (carbon atoms in <i>grey</i>). The surface of the glycerol molecule present at the Hyp-complex is shown to evidence the influence of this molecule in the position and orientation of Hyp in the active site. The hydrogen bonds are shown as <i>dashed lines.</i></p