Biomedical subjects

Ethan A Merritt

Publications and source records attributed to Ethan A Merritt.

15 recordsLinked to original sources

PubMed2006

High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design.

Recent efforts to design de novo or redesign the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol RosettaDesign to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal/mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we have designed and characterized a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations; one mutant gains more than 5 kcal/mol of stability over the wild-type protein with only four amino acid changes. We explore the relationship between force-field smoothing and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using modified force-fields, and both fixed and flexible backbone sampling protocols.

Carboxypeptidases A↗

PubMed2006

Using fragment cocktail crystallography to assist inhibitor design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase.

The 1.8 A resolution de novo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.2.6) from Trypanosoma brucei (TbNDRT) has been determined by SADa phasing in an unliganded state and several ligand-bound states. This enzyme is important in the salvage pathway of nucleoside recycling. To identify novel lead compounds, we exploited "fragment cocktail soaks". Out of 304 compounds tried in 31 cocktails, four compounds could be identified crystallographically in the active site. In addition, we demonstrated that very short soaks of approximately 10 s are sufficient even for rather hydrophobic ligands to bind in the active site groove, which is promising for the application of similar soaking experiments to less robust crystals of other proteins.

Amino Acid Sequence↗

PubMed2006

Structure of ribose 5-phosphate isomerase from Plasmodium falciparum.

The structure of ribose 5-phosphate isomerase from Plasmodium falciparum, PFE0730c, has been determined by molecular replacement at 2.09 angstroms resolution. The enzyme, which catalyzes the isomerization reaction that interconverts ribose 5-phosphate and ribulose 5-phosphate, is a member of the pentose phosphate pathway. The P. falciparum enzyme belongs to the ribose 5-phosphate isomerase A family, Pfam family PF06562 (DUF1124), and is structurally similar to other members of the family.

Aldose-Ketose Isomerases↗

PubMed2006

Optimal description of a protein structure in terms of multiple groups undergoing TLS motion.

A single protein crystal structure contains information about dynamic properties of the protein as well as providing a static view of one three-dimensional conformation. This additional information is to be found in the distribution of observed electron density about the mean position of each atom. It is general practice to account for this by refining a separate atomic displacement parameter (ADP) for each atomic center. However, these same displacements are often described well by simpler models based on TLS (translation/libration/screw) rigid-body motion of large groups of atoms, for example interdomain hinge motion. A procedure, TLSMD, has been developed that analyzes the distribution of ADPs in a previously refined protein crystal structure in order to generate optimal multi-group TLS descriptions of the constituent protein chains. TLSMD is applicable to crystal structures at any resolution. The models generated by TLSMD analysis can significantly improve the standard crystallographic residuals R and R(free) and can reveal intrinsic dynamic properties of the protein.

Algorithms↗

PubMed2006

Crystal structure of glyceraldehyde-3-phosphate dehydrogenase from Plasmodium falciparum at 2.25 A resolution reveals intriguing extra electron density in the active site.

The crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) from the major malaria parasite Plasmodium falciparum is solved at 2.25 A resolution. The structure of PfGAPDH is of interest due to the dependence of the malaria parasite in infected human erythrocytes on the glycolytic pathway for its energy generation. Recent evidence suggests that PfGAPDH may also be required for other critical activities such as apical complex formation. The cofactor NAD(+) is bound to all four subunits of the tetrameric enzyme displaying excellent electron densities. In addition, in all four subunits a completely unexpected large island of extra electron density in the active site is observed, approaching closely the nicotinamide ribose of the NAD(+). This density is most likely the protease inhibitor AEBSF, found in maps from two different crystals. This putative AEBSF molecule is positioned in a crucial location and hence our structure, with expected and unexpected ligands bound, can be of assistance in lead development and design of novel antimalarials.

Amino Acid Sequence↗

PubMed2006

Structure of the conserved hypothetical protein MAL13P1.257 from Plasmodium falciparum.

The structure of a conserved hypothetical protein, PlasmoDB sequence MAL13P1.257 from Plasmodium falciparum, Pfam sequence family PF05907, has been determined as part of the structural genomics effort of the Structural Genomics of Pathogenic Protozoa consortium. The structure was determined by multiple-wavelength anomalous dispersion at 2.17 A resolution. The structure is almost entirely beta-sheet; it consists of 15 beta-strands and one short 3(10)-helix and represents a new protein fold. The packing of the two monomers in the asymmetric unit indicates that the biological unit may be a dimer.

Amino Acid Sequence↗

PubMed2006

Structure of Lmaj006129AAA, a hypothetical protein from Leishmania major.

The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD) using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 (Rfree = 0.229) at 1.60 A resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif.

Animals↗

PubMed2006

Cooperative hydrogen bond interactions in the streptavidin-biotin system.

The thermodynamic and structural cooperativity between the Ser45- and D128-biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128-biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128-OD and biotin-N2 is thermodynamically stronger than that between S45-OG and biotin-N1.

Biotin↗

PubMed2005

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily.

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.

Amino Acid Sequence↗

PubMed2005

A molecular viewer for the analysis of TLS rigid-body motion in macromolecules.

TLS (translation/libration/screw) models describe rigid-body vibrational motions of arbitrary objects. A single-group TLS model can be used to approximate the vibration of an entire protein molecule within a crystal lattice. More complex TLS models are broadly applicable to describing inter-domain and other internal vibrational modes of proteins. Such models can be derived and refined from crystallographic data, but they can also be used to describe the vibrational modes observed through other physical techniques or derived from molecular dynamics. The use of TLS models for protein motion has been relatively limited, partly because the physical meaning of the refined TLS parameters is not intuitive. Here, a molecular viewer, TLSView, is introduced using OpenGL and based on the mmLib library for describing and manipulating macromolecular structural models. This visualization tool allows an intuitive understanding of the physical significance of TLS models derived from crystallographic or other data and may be used as an interactive tool to display and interpret inter-domain or other motions in protein structural models. TLSView may also be used to prepare, analyze and validate TLS models for crystallographic refinement.

Algorithms↗

PubMed2004

Structural biology and structure-based inhibitor design of cholera toxin and heat-labile enterotoxin.

Structural biology studies on cholera toxin and the closely related heat-labile enterotoxin from enterotoxigenic Escherichia coli over the past decade have shed light on the mechanism of toxin action at molecular and atomic levels. Also, components of the extracellular protein secretion apparatus that translocate the toxins across the outer membrane are being investigated. At the same time, structure-based design has led to various classes of compounds targeting different toxin sites, including highly potent multivalent inhibitors that block the toxin receptor-binding process.

Bacterial Toxins↗

PubMed2002

Solution and crystallographic studies of branched multivalent ligands that inhibit the receptor-binding of cholera toxin.

The structure-based design of multivalent ligands offers an attractive strategy toward high affinity protein inhibitors. The spatial arrangement of the receptor-binding sites of cholera toxin, the causative agent of the severe diarrheal disease cholera and a member of the AB(5) bacterial toxin family, provides the opportunity of designing branched multivalent ligands with 5-fold symmetry. Our modular synthesis enabled the construction of a family of complex ligands with five flexible arms each ending with a bivalent ligand. The largest of these ligands has a molecular weight of 10.6 kDa. These ligands are capable of simultaneously binding to two toxin B pentamer molecules with high affinity, thus blocking the receptor-binding process of cholera toxin. A more than million-fold improvement over the monovalent ligand in inhibitory power was achieved with the best branched decavalent ligand. This is better than the improvement observed earlier for the corresponding nonbranched pentavalent ligand. Dynamic light scattering studies demonstrate the formation of concentration-dependent unique 1:1 and 1:2 ligand/toxin complexes in solution with no sign of nonspecific aggregation. This is in complete agreement with a crystal structure of the branched multivalent ligand/toxin B pentamer complex solved at 1.45 A resolution that shows the specific 1:2 ligand/toxin complex formation in the solid state. These results reiterate the power of the structure-based design of multivalent protein ligands as a general strategy for achieving high affinity and potent inhibition.

Binding Sites↗

PubMed2002

Characterization and crystal structure of a high-affinity pentavalent receptor-binding inhibitor for cholera toxin and E. coli heat-labile enterotoxin.

Multivalent ligand design constitutes an attractive avenue to the inhibition of receptor recognition and other biological events mediated by oligomeric proteins with multiple binding sites. One example is the design of multivalent receptor blockers targeting members of the AB(5) bacterial toxin family. We report here the synthesis and characterization of a pentavalent inhibitor for cholera toxin and Escherichia coli heat-labile enterotoxin. This inhibitor is an advance over the symmetric pentacyclen-derived inhibitor described in our earlier work in that it presents five copies of m-nitrophenyl-alpha-D-galactoside (MNPG) rather than five copies of beta-D-galactose. The approximately 100-fold higher single-site affinity of MNPG for the toxin receptor binding site relative to galactose is found to yield a proportionate increase in the affinity and IC50 measured for the respective pentavalent constructs. We show by dynamic light scattering that inhibition of receptor binding by the pentavalent inhibitor is due to 1:1 inhibitor:toxin association rather than to inhibitor-mediated aggregation. This 1:1 association is in complete agreement with a 1.46 A resolution crystal structure of the pentavalent inhibitor:toxin complex, which shows that the favorable single-site binding interactions of MNPG are retained by the five arms of the 5256 Da pentavalent MNPG-based inhibitor and that the initial segment of the linking groups interacts with the surface of the toxin B pentamer.

Bacterial Toxins↗

PubMed2002

Anchor-based design of improved cholera toxin and E. coli heat-labile enterotoxin receptor binding antagonists that display multiple binding modes.

The action of cholera toxin and E. coli heat-labile enterotoxin can be inhibited by blocking their binding to the cell-surface receptor GM1. We have used anchor-based design to create 15 receptor binding inhibitors that contain the previously characterized inhibitor MNPG as a substructure. In ELISA assays, all 15 compounds exhibited increased potency relative to MNPG. Binding affinities for two compounds, each containing a morpholine ring linked to MNPG via a hydrophobic tail, were characterized by pulsed ultrafiltration (PUF) and isothermal titration calorimetry (ITC). Crystal structures for these compounds bound to toxin B pentamer revealed a conserved binding mode for the MNPG moiety, with multiple binding modes adopted by the attached morpholine derivatives. The observed binding interactions can be exploited in the design of improved toxin binding inhibitors.

Bacterial Toxins↗

PubMed2002

Combating infectious diseases through multivalent design.

Many biological interactions are multivalent, linking two particles via many copies of the same ligand-receptor binding pair. Examples of multivalent binding range from cell-cell adhesion to the assembly of large protein complexes from constituent multimers to the binding of AB5 bacterial toxins at the cell surface. Multivalent interactions can be effectively mimicked, inhibited, or disrupted through the design of suitable multivalent ligands. We review here recent work on multivalent ligand design based on a number of different chemical scaffolds, with a specific emphasis on the use of structure-based ligand design to target multimeric bacterial toxins.

Anti-Bacterial Agents↗