Biomedical subjects

Enrico O Purisima

Publications and source records attributed to Enrico O Purisima.

7 recordsLinked to original sources

PubMed2003

Bridging structural biology and genetics by computational methods: an investigation into how the R774C mutation in the AR gene can result in complete androgen insensitivity syndrome.

Recent structural studies of the ligand-binding domain (LBD) of the androgen receptor (AR) have raised more questions than answers, as most of the known pathogenic mutations of the AR gene causing androgen insensitivity syndrome (AIS) are not in the ligand-binding pocket. In this study, we have investigated one such pathogenic mutation, by examining details of its altered atomic structure using a computational technique of molecular dynamics (MD) simulations extended over 4 ns, effectively creating a 4D structural model. The mutation R774C, which is in the LBD of the AR gene, causes complete AIS (CAIS), producing ARs that have a unique thermolabile profile, being thermostable at 22 degrees C but thermolabile at 37 degrees C. We have therefore investigated this mutation by MD simulations at 293 K (20 degrees C), 300 K (27 degrees C), and 310 K (37 degrees C). The MD simulations indicate that: 1) the mutation causes local structural distortions, which result in changes in the shape of the ligand-binding pocket; 2) the mutation alters the dynamic nature of the protein and results in a more diverse conformational distribution of the ligand-binding pocket; and 3) the effect of the mutation on AR structure could be largely reversed by lowering the temperature at which the MD simulations were conducted. These results therefore strongly support the biochemical data, e.g., the mutants' inability to form AR-ligand complexes at 37 degrees C and its characteristic reversible thermolability, clearly indicating the value of such computational methods.

Androgen-Insensitivity Syndrome↗

PubMed2003

On the transferability of hydration-parametrized continuum electrostatics models to solvated binding calculations.

Using molecular mechanics force field partial atomic charges, we show the nonuniqueness of the parametrization of continuum electrostatics models with respect to solute atomic radii and interior dielectric constant based on hydration (vacuum-to-water transfer) free energy data available for small molecules. Moreover, parameter sets that are optimal and equivalent for hydration free energy calculations lead to large variations of calculated absolute and relative electrostatic binding free energies. Hence, parametrization of solvation effects based on hydration data, although a necessary condition, is not sufficient to guarantee its transferability to the calculation of binding free energies in solution.

Journal Article↗

PubMed2003

Profiling charge complementarity and selectivity for binding at the protein surface.

A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins.

Binding Sites↗

PubMed2002

Design of noncovalent inhibitors of human cathepsin L. From the 96-residue proregion to optimized tripeptides.

A novel series of noncovalent inhibitors of cathepsin L have been designed to mimic the mode of autoinhibition of procathepsin L. Just like the propeptide, these peptide-based inhibitors have a reverse-binding mode relative to a substrate and span both the S' and S subsites of the enzyme active site. In contrast to previous studies in which even moderate truncation of the full-length propeptide led to rapid reduction in potency, these blocked tripeptide-sized inhibitors maintain nanomolar potency. Moreover, these short peptides show higher selectivity (up to 310-fold) for inhibiting cathepsin L over K versus only 2-fold selectivity of the 96-residue propeptide of cathepsin L. A 1.9 A X-ray crystallographic structure of the complex of cathepsin L with one of the inhibitors confirms the designed reverse-binding mode of the inhibitor as well as its noncovalent nature. Enzymatic analysis also shows the inhibitors to be resistant to hydrolysis at elevated concentrations of the enzyme. The mode of inhibition of these molecules provides a general strategy for inhibiting other cathepsins as well as other proteases.

Binding Sites↗

PubMed2002

Implications of the reactive thiol and the proximal non-proline cis-peptide bond in the Structure and function of Vibrio harveyi luciferase.

The role of a highly reactive cysteine residue, Cys106, in Vibrio harveyi luciferase in modulating the substrate-enzyme interactions and in turn affecting the enzyme activity has been extensively investigated over the past three decades. Replacing Cys106 with valine dramatically hinders the ability of luciferase to stabilize the C4a-hydroperoxyflavin intermediate [Abu-Soud, H. M., Clark, A. C., Francisco, W. A., Baldwin, T. O., and Raushel, F. M. (1993) J. Biol. Chem. 268, 7699-7706] and consume aldehyde substrate [Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203], therefore markedly decreasing enzyme activity. On the basis of the structure-activity relationship of flavin analogues and the location of the phosphate binding site of flavin mononucleotide (FMN) coupled with molecular modeling, the functional part of the isoalloxazine ring of FMN, the thiol side chain of Cys106, the methyl group of Ala75, and the unique non-prolyl cis-peptide bond between Ala74 and Ala75 were found to be closely packed [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. Here, by mutating Ala75 to Gly, we restored key wild-type properties to the C106V mutant, in particular, high enzyme activity and a stable C4a-hydroperoxyflavin intermediate, demonstrating that the primary reason for the dark phenotype of the C106V mutant was the unfavorable steric interaction between Val106 and Ala75 side chains, which could in turn disturb the cis-oriented amide linkage of Ala74 and Ala75. Moreover, significant red shifts in light emission of 3-10 nm were measured for luciferases carrying Val106 with the spectrum of the double mutant C106V/A75G now red shifted to that of Photobacterium phosphoreum luciferase, which also has Val and Gly at positions 106 and 75, respectively. These results strengthen the validity of the binding geometry of the modeled flavin with the re-face of the pyrimidine end of the isoalloxazine ring next to Cys106 and implicate the Ala74-Ala75 cis-peptide as a key component in the bioluminescence reaction.

Enzyme Stability↗

PubMed2002

A fast pairwise evaluation of molecular surface area.

A fast and general analytical approach was developed for the calculation of the approximate van der Waals and solvent-accessible surface areas. The method is based on three basic ideas: the use of the Lorentz transformation formula, a rigid-geometry approximation, and a single fitting parameter that can be refitted on the fly during a simulation. The Lorentz transformation equation is used for the summation of the areas of an atom buried by its neighboring contacting atoms, and implies that a sum of the buried pairwise areas cannot be larger than the surface area of the isolated spherical atom itself. In a rigid-geometry approximation we numerically calculate and keep constant the surface of each atom buried by the atoms involved in 1-2 and 1-3 interactions. Only the contributions from the nonbonded atoms (1-4 and higher interactions) are considered in terms of the pairwise approximation. The accuracy and speed of the method is competitive with other pairwise algorithms. A major strength of the method is the ease of parametrization.

Journal Article↗

PubMedDate not supplied

Effect of limited solid-state glycation on the conformation of lysozyme by ESI-MSMS peptide mapping and molecular modeling.

Although protein glycation has been implicated in the alteration of protein functionality, both in vivo (in biological systems) and in vitro (in food systems), the effect of the protein-bound glycan moiety on the structure/conformation of proteins that result in the modification of functionality is not clear. In this article, we report a study of the conformational changes of glycated lysozyme using LC-ESI-MSMS peptide mapping, and molecular modeling. A comparison of the RP-HPLC of the tryptic digests of unglycated and glycated lysozyme showed markedly different chromatographic profiles. Analysis of the peptide composition of the chromatographic fractions of the tryptic digests revealed that glycation of lysozyme resulted in the modification of its conformation. Glycation-induced changes in the conformation of lysozyme resulted in the exposure of its active site region to increased proteolytic activity of trypsin. Molecular simulation of triglycated lysozyme also showed that limited glycation of lysozyme caused reorientation of the active site residues (Arg 45, Arg 68, Asn 44, and Trp 62) and increased solvent accessibility into the active site region of the protein. The results of the modeling experiment corroborated the results of the RP-HPLC and ESI-MSMS peptide mapping.

Animals↗