Enrico O Purisima

Enrico O Purisima
National Research Council Canada | NRC

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78
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Publications

Publications (78)
Article
Full-text available
Single-domain antibodies (sdAbs) are a promising class of biotherapeutics with unique structural traits within their paratope region. The distribution of canonical conformations explored by their complementarity determining region (CDR) loops differs to some extent from conventional two-chain Fv fragments of monoclonal antibodies (mAbs). In this st...
Article
Full-text available
Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring func...
Data
Consensus z-scores for double and triple mutants of bH1-VEGF. The z-scores shown are relative to the distribution of scores in the exhaustive scan of single mutants, i.e., the median (or mean) absolute deviations of the single-mutant scores were used in computing the z-scores. In red are the mutants that were produced and validated experimentally....
Data
Consensus z-scores for double and triple mutants of bH1-HER2. The z-scores shown are relative to the distribution of scores in the exhaustive scan of single mutants, i.e., the median (or mean) absolute deviations of the single-mutant scores were used in computing the z-scores. In red are the mutants that were produced and validated experimentally....
Data
Consensus z-scores for double and triple mutants of Herceptin-HER2. The z-scores shown are relative to the distribution of scores in the exhaustive scan of single mutants, i.e., the median (or mean) absolute deviations of the single-mutant scores were used in computing the z-scores. In red are the mutants that were produced and validated experiment...
Data
Top 50 consensus Z-scores for single mutants. The first column in S1 Table lists the mutation sites involved in the top 50 consensus z-scores. The first letter (H or L) refers to the heavy or light chain, respectively. Scores in red correspond to mutants that were selected for production and experimental validation. (PDF)
Data
Binding titration curves to determine dissociation constants, KD. Mean fluorescence intensity (MFI) from FACS data is plotted versus the concentration of different anti-HER2 Fabs on MCF-7 (A) or SKOV-3 (B) cell lines. Data are fit using One-site specific with Hill slope four-parameter nonlinear regression curve fitting model (see Methods in main te...
Data
Fold improvements in binding affinity and relative changes in binding free energy (kcal/mol) relative to the parent Fab during three rounds of mutations (Excel spreadsheet of data in Fig 1). (XLSX)
Data
Apparent KD values of Herceptin Fab and its triple mutants by flow cytometry analysis (n = 3). (PDF)
Data
FR sequences of five non-SpA-binding VHHs and their humanized SpA-binding counterparts. (DOCX)
Data
Binding of wild-type or SpA-engineered AFAI VHH pentamers at either 1 nM (A) or 50 nM (B) to immobilized CEACAM6 N-terminal domain by SPR. VHH pentamers were injected over immobilized CEACAM6 N-terminal domain for 3 min and allowed to dissociate as described in methods. (TIF)
Data
Binding of VHH pentamers at 100 nM to immobilized SpA by SPR. Each pentamer (FR sequences listed in S2 Table) was injected for 2 min and the number of response units bound at the end of the injection was measured. For pentamers bearing the human IGHV3 consensus residue at all 13 SpA contact positions, no residues are plotted on the graph; instead,...
Data
SpA binding by SPR of four llama VHH monomers (ICAM11-4, ICAM34-1, IGF1R-4 and IGF1R-5; dotted lines) and one llama VHH pentamer (AFAI; dotted line) along with their humanized counterparts (solid line). VHH monomers and pentamers (250 nM) were injected over immobilized SpA for 2 min and allowed to dissociate as described in methods. (TIF)
Data
FR sequences of SpA-binding and non-SpA-binding VHH monomers. (DOCX)
Data
FR and CDR sequences of a SpA-binding (Thr57) and non-SpA-binding (Ile57) dromedary VHH and effect of CDR1-CDR3 disulfide bridge on SpA binding. (DOCX)
Data
FR sequences of SpA-binding and non-SpA-binding VHH pentamers. (DOCX)
Data
Metrics for Illumina MiSeq NGS data used in this study. (DOCX)
Article
To determine if the conjugation of a small receptor ligand to a peptidic carrier potentially facilitating the transport across the BBB by "Molecular Trojan Horse" transcytosis is feasible, we synthesized several transport peptide-fallypride-fusion molecules as model systems and determined their binding affinities to the hD2 receptor. Although being...
Article
Full-text available
Kininogens, the major plasma cystatin-like inhibitors of cysteine cathepsins, are degraded at sites of inflammation, and cathepsin B has been identified as a prominent mediator of this process. Cathepsin B, in contrast to cathepsins L and S, is poorly inhibited by kininogens. This led us to delineate the molecular interactions between this protease...
Article
Full-text available
Mutations or overexpression of signalling genes can result in cancer development and metastasis. In this study, we manually assembled a human cellular signalling network and developed a robust bioinformatics strategy for extracting cancer-associated single nucleotide polymorphisms (SNPs) using expressed sequence tags (ESTs). We then investigated th...
Article
Full-text available
The exchange of residues 67 and 205 of the S2 pocket of human cysteine cathepsins K and L induces a permutation of their substrate specificity toward fluorogenic peptide substrates. While the cathepsin L-like cathepsin K (Tyr67Leu/Leu205Ala) mutant has a marked preference for Phe, the Leu67Tyr/Ala205Leu cathepsin L variant shows an effective cathep...
Article
A facile and sensitive mass spectrometric method has been developed for the dereplication of natural products. The method provides information about the molecular formula and substructure of a precursor molecule and its fragments, which are invaluable aids in dereplication of natural products at their early stages of purification and characterizati...
Article
Full-text available
Over the past few years, microRNAs (miRNAs) have emerged as a new prominent class of gene regulatory factors that negatively regulate expression of approximately one-third of the genes in animal genomes at post-transcriptional level. However, it is still unclear why some genes are regulated by miRNAs but others are not, i.e. what principles govern...
Article
Full-text available
We conducted a comprehensive analysis of a manually curated human signaling network containing 1634 nodes and 5089 signaling regulatory relations by integrating cancer-associated genetically and epigenetically altered genes. We find that cancer mutated genes are enriched in positive signaling regulatory loops, whereas the cancer-associated methylat...
Article
MicroRNAs (miRNAs) modulate expression of their target genes in various tissues and at different developmental stages, but it is unclear whether they drive cross-species variation in gene expression. By comparing data from mammal and fly species we found that the cross-species expression variation of miRNA targets is significantly lower than that o...
Article
Full-text available
MicroRNAs (miRNAs) are non-coding small RNAs of approximately 22 nt that regulate the gene expression by base pairing with target mRNAs, leading to mRNA cleavage or translational repression. It is currently estimated that miRNAs account for approximately 1% of predicted genes in higher eukaryotic genomes and that up to 30% of genes might be regulat...
Article
Full-text available
Structural studies of the ligand-binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand binding, H12 is stabilized in...
Article
AB - Charge optimization as a tool for both analyzing and enhancing binding electrostatics has become an attractive approach over the past few years. An interesting feature of this method for molecular design is that it provides not only the optimal charge magnitudes, but also the selectivity of a particular atomic center for its optimal charge. Th...
Article
MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs, which suppress gene expression by selectively binding to the 3'-noncoding region of specific messenger RNAs through base-pairing. Given the diversity and abundance of miRNA targets, miRNAs appear to functionally interact with various components of many cellular networks. By analyzi...
Article
Protein-ligand binding occurs through interactions at the molecular surface. Hence, a proper description of this surface is essential to our understanding of the process of molecular recognition. Recent studies have noted the inadequacy of using a fixed 1.4 � solvent probe radius to generate the molecular surface. This assumes that water molecules...
Article
Full-text available
A number of missense mutations in the Na,K-ATPase α2 catalytic subunit have been identified in familial hemiplegic migraine with aura. Two alleles (L764P and W887R) showed loss-of-function, whereas a third (T345A) is fully functional but with altered Na,K-ATPase kinetics. This study describes two additional mutants, R689Q and M731T, originally iden...
Article
A mammalian two-hybrid system was developed for high-throughput screening of compounds that disrupt specific protein-protein interactions. The existing mammalian systems are unsatisfactory for drug screening due to nonregulated expression of interacting proteins. To construct a tightly regulated system, the tetracycline repressor was fused with the...
Article
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...
Article
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 a...
Article
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 mo...
Article
Although the crystal structure of Vibrio harveyi luciferase has been elucidated, the binding sites for the flavin mononucleotide and fatty aldehyde substrates are still unknown. The determined location of the phosphate-binding site close to Arg 107 on the alpha subunit of luciferase is supported here by point mutagenesis. This information, together...
Article
We show that for a given binding site and a given spatial arrangement of atoms in a ligand, there exists an optimal set of partial charges at the atom centers that will optimize the net electrostatic binding free energy of the ligand. This optimal value can be calculated quite readily from a simple quadratic polynomial with coefficients derivable f...
Article
The bifunctional enzyme formiminotransferase-cyclodeaminase (FTCD) contains two active sites at different positions on the protein structure. The enzyme binds a gamma-linked polyglutamylated form of the tetrahydrofolate substrate and channels the product of the transferase reaction from the transferase active site to the cyclodeaminase active site....
Article
Cathepsin X is a novel cysteine protease which was identified recently from the EST (expressed sequence tags) database. In a homology model of the mature cathepsin X, a unique three residue insertion between the Gln22 of the oxyanion hole and the active site Cys31 was found to be located in the primed region of the binding cleft as part of a surfac...
Article
The free energy of partial dehydration during molecular association has been mapped on an uniform grid-probe basis. 3D-localized quantities are computed as the difference in the BEM-calculated reaction field energies plus non-electrostatic component of solvation between the bound and free states. The resulting desolvation field encodes useful molec...
Article
We present a boundary element method (BEM) for calculating the reaction field energy of a macromolecule embedded in a high-dielectric medium such as water. In a BEM calculation, the key computational task is the calculation of the induced surface charge distribution at the dielectric boundary. This is obtained by solving a system of linear equation...
Article
A set of 25 low-molecular-weight substrates for protein tyrosine phosphatase PTP1 has been investigated by comparative molecular field analysis (CoMFA). The quality of the model was assessed by cross-validations, predictions for a test set of substrates, bootstrapping, randomization of biological data, grid characteristics tests, and comparison wit...
Article
A method is presented to generate and triangulate molecular surfaces rapidly. It is based on the ‘marching tetrahedra’ approach. The method is fast, simple and easy to implement. Our approach is not analytical in nature. Hence no special treatment is required for complications with singularity, degeneracy, or with self-intersecting re-entrant surfa...
Article
The marching cubes algorithm is widely used to generate isosurfaces from a 3D scalar field. A major problem associated with it is the possibility of mismatch between adjacent surface elements, leading to holes on the surface. In this work, we propose using a tetrahedral tesselation of space which would eliminate this problem. Comparing with existin...
Article
We have designed bivalent thrombin inhibitors, consisting of a nonsubstrate type active site blocking segment, a hirudin-based fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The inhibition provided by the bivalent inhibitors with various linker lengths revealed that a minimum of 15 atoms was required for si...
Article
Full-text available
Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase. This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of the active site cleft. Va...
Article
Nonpolar interactions play a major role in the association of the fibrinogen recognition exosite of thrombin with the C-terminal fragment (55-65), Asp-Phe-Glu-IIe-Pro-Glu-Glu-Tyr-Leu-Gln, of hirudin, which is a naturally occurring thrombin inhibitor. The thermodynamic details (free energy, enthalpy, entropy, and heat capacity) of the molecular reco...
Article
Within the papain family of cysteine proteinases few other residues in addition to the catalytic triad, Cys25-His159-Asn175 (papain numbering) are completely conserved [Berti & Storer (1995) J. Mol. Biol. 246, 273-283]. One such residue is tryptophan 177 which participates in a Trp-His-type interaction with the catalytic His159. In all enzymes of t...
Article
The relative helix propensities of Gly, Ala, Val, Ile, and Leu in the center of a polyalanine helix were calculated using a novel free energy simulation method (Wang et al. J. Mol. Biol. 1995, 253, 473) that permits the decomposition of the free energy into its various thermodynamic components. The calculated relative free energy changes agree well...
Article
Free energy calculations were carried out on a series of exosite-binding inhibitors of thrombin. These inhibitors are based on the C-terminal fragment of hirudin and have the sequence Phe-Glu-Glu-IleH59-Pro-Glu-Glu-Tyr- Leu, where the superscript over Ile indicates its relative position in the natural sequence of hirudin. In this study, the effect...
Article
Hirudin is the most potent and specific thrombin inhibitor from medicinal leech with a Ki value of 2.2 x 10(-14) M. It consists of an active site inhibitor segment, hirudin1-48, a fibrinogen-recognition exosite inhibitor segment, hirudin55-65, and a linker, hirudin49-54, connecting these inhibitor segments. The role of the side chain of the hirudin...
Article
A simple yet accurate method for calculating electrostatic potentials using the boundary element continuum dielectric method is presented. It is shown that the limiting factor in accuracy is not the evaluation of integrals involving the interaction between boundary elements but rather a proper estimation of the self-polarization of a patch upon its...
Article
N alpha-Acetyl[D-Phe45,Arg47]hirudin45-65 (P53) is a bivalent thrombin inhibitor (Ki = 5.6 nM) that consists of an active site inhibitor segment, [N alpha-acetyl-(dF)PRP]; a fibrinogen recognition exo site inhibitor segment, hirudin55-65 (DFEEIPEEYLQ-OH); and a linker, hirudin49-54 (QSHNDG), connecting these inhibitor segments (DiMaio et al., 1990)...
Article
A new type of thrombin exo-site inhibitor has been designed with enhanced inhibitory potency and increased metabolic stability. With the aid of the model of the structure of the thrombin-hirudin fragment complex [Yue, S.-Y., DiMaio, J., Szewczuk, Z., Purisima, E. O., Ni, F., & Konishi, Y. (1992) Protein Eng. 5, 77-85], cyclic analogs of the hirudin...
Article
The COOH-terminal region of hirudin represents an independent functional domain that binds to an anion-binding exosite of thrombin and inhibits the interaction of thrombin with fibrinogen and regulatory proteins in blood coagulation. The thrombin-bound structure of the peptide fragment, hirudin 55-65, has been determined by use of transferred NOE s...
Article
A potent thrombin inhibitor, [D-Phe45, Arg47] hirudin 45-65, that contains an active site-directed sequence D-Phe-Pro-Arg-Pro, an exosite specific fragment hirudin 55-65 (H55-65) and a linker portion hirudin 49-54, was designed based on the hirudin sequence [DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensional model of the co...
Article
Knowledge of three-dimensional protein structures is one of the foundations of protein design and protein engineering. Nuclear magnetic resonance spectroscopy was recently introduced as a second method for protein structure determination, in addition to the well-established diffraction techniques with protein single crystals. This new approach enab...
Article
Proton NMR assignments have been made for 121 of the 124 residues of bovine pancreatic ribonuclease A (RNase A). During the first stage of assignment, COSY and relayed COSY data were used to identify 40 amino acid spin systems belonging to alanine, valine, threonine, isoleucine, and serine residues. Approximately 60 other NH-alpha CH-beta CH system...
Article
An algorithm for locating the region in conformational space containing the global energy minimum of a polypeptide is described. Distances are used as the primary variables in the minimization of an objective function that incorporates both energetic and distance-geometric terms. The latter are obtained from geometry and energy functions, rather th...
Article
A method is presented for starting from a very-low-energy high-dimensional conformation and obtaining a low-energy three-dimensional structure by gradual contraction of the dimensionality. The contraction in dimensionality is achieved by use of Cayley-Menger determinants, of which a simplified form is derived here. Preliminary results are presented...
Article
A method for generating a complete polypeptide backbone structure from a set of Cα coordinates is presented. Initial trial values of ϕ and ψ for a selected residue are chosen (essentially from an identification of the conformational region of the virtual-bond backbone, e.g., and α-helical region), and values of ϕ and ψ for the remaining residues (b...
Article
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Article
Using the MP1-p14 scaffolding complex from the mitogen-activated protein kinase signaling pathway as model system, we explored a structure-based computational protocol to probe and characterize binding affinity hot spots at protein-protein interfaces. Hot spots are located by virtual alanine-scanning consensus predictions over three different energ...
Article
We present a binding free energy function that consists of force field terms supplemented by solvation terms. We used this function to calibrate the solvation model along with the binding interaction terms in a self-consistent manner. The motivation for this approach was that the solute dielectric-constant dependence of calculated hydration gas-to-...

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