Edward J. Meehan

The solubility of hen egg-white lysozyme

Abstract The equilibrium solubility of chicken egg-white lysozyme in the presence of crystalline solid state was determined as a function of NaCl concentration, pH, and temperature. The solubility curves obtained represent a region of the lysozyme phase diagram. This diagram makes it possible to determine the supersaturation of a given set of conditions or to achieve identical supersaturations by different combinations of parameters. The temperature dependence of the solubility permits the evaluation of Δ H of crystallization and we present a method for its calculation. The data indicates a negative heat of crystallization for the tetragonal crystal form but a positive heat of crystallization for the high temperature orthorhombic form.

Structure of an insect delta-class glutathione S-transferase from a DDT-resistant strain of the malaria vector Anopheles gambiae

Glutathione S-transferases (GSTs) are a major family of detoxification enzymes which possess a wide range of substrate specificities. Most organisms possess many GSTs belonging to multiple classes. Interest in GSTs in insects is focused on their role in insecticide resistance; many resistant insects have elevated levels of GST activity. In the malaria vector Anopheles gambiae, elevated GST levels are associated with resistance to the organochlorine insecticide DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane]. This mosquito is the source of an insect GST, agGSTd1-6, which metabolizes DDT and is inhibited by a number of pyrethroid insecticides. The crystal structure of agGSTd1-6 in complex with its inhibitor S-hexyl glutathione has been determined and refined at 2.0 A resolution. The structure adopts a classical GST fold and is similar to those of other insect delta-class GSTs, implying a common conjugation mechanism. A structure-based model for the binding of DDT to agGSTd1-6 reveals two subpockets in the hydrophobic binding site (H-site), each accommodating one planar p-chlorophenyl ring.

Control of nucleation and growth in protein crystal growth

The potential advantages of nucleation and growth control through temperature, rather than the addition of precipitants or removal of solvent, are discussed. A simple light scattering arrangement for the characterization of nucleation and growth conditions in solutions is described. The temperature dependence of the solubility of low ionic strength lysozyme solutions is applied in preliminary nucleation and growth experiments.

Experimental and theoretical analysis of the rate of solvent equilibration in the hanging drop method of protein crystal growth

Abstract In the hanging drop method of protein crystal growth, a water droplet containing protein, buffer and a precipitating agent, such as ammonium sulfate, is suspended from a glass coverslip above a well containing an aqueous solution of the precipitating agent at a concentration double that in the drop. We present a comprehensive theoretical study of the rate of water evaporation in the hanging drop method. We find that in earths gravity the rate controlling step in the evaporation is the rate of diffusion of water vapor across the air space separating the drop from the well. Using ammonium sulfate as the precipitating agent, we have made careful measurements at both 4°C and 25°C of the evaporation times for some 25 μL droplets at various concentrations. These results are in good agreement with our theory. As determined by the theory, the parameters affecting the rate of evaporation include the temperature, the vapor pressure of water, the ionization constant of the salt, the volume of the droplet, the contact angle between the droplet and the coverslip, the number of moles of salt in the droplet, the number of moles of water and salt in the well, the molar volumes of water and salt, the distance from the droplet to the well, and the coefficient of diffusion of water vapor through air. These parameters do not act independently; rather, they combine to form three dimensionless groups upon which the rate of evaporation depends. We evaluate numerically 18 different drop and well arrangements commonly encountered in the laboratory. In all cases considered at 25°C, the number of moles of water in the droplet achieves 95% of its final value in 3—30 h, after which further evaporation is quite slow. Our experiments confirm this. We consider qualitatively the effect of weightlessness (spaceflight) on the rate of evaporation and find it to be most likely controlled by the rate of the interdiffusion of salt and water in the droplet.

Mutation of a Conserved Tryptophan in the Chitin-Binding Cleft of Serratia marcescens Chitinase A Enhances Transglycosylation

Family 18 chitinases have the signature peptide DGXDXDXE forming the fourth β-strand in the (β⁄α)8-barrel of their catalytic domain. The carboxyl-end glutamic acid, E315 in Serratia marcescens chitinase A, serves as the acid/base during chitin hydrolysis, and the side-chain of the preceding aspartic acid, D313, helps to position correctly the N-acetyl moiety of the glycosyl sugar undergoing hydrolysis. Chitin substrates are bound within a long cleft across the top of the barrel, whose floor consists of aromatic residues that hydrophobically stack with every other GlcNAc. Alanine substitution of the conserved Trp167 at the −3 subsite in Serratia marcescens chitinase A enhanced transglycosylation. Higher oligosaccharides were formed from both chitin tetra- and pentasaccharide, and the only hydrolytic product from chitin trisaccharide was the disaccharide. Greater retention of the glycosyl fragment at the active site of the −3 mutant of Serratia marcescens chitinase A might favor transglycosylation due to a stabilized conformation of its D313.

Trp2313-His2315 of factor VIII C2 domain is involved in membrane binding: structure of a complex between the C2 domain and an inhibitor of membrane binding.

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 Å) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed β-strand of the C2 domain, Trp2313-His2315. This result indicates that the Trp2313-His2315 segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

Structural basis of transport of lysophospholipids by human serum albumin

Lysophospholipids play important roles in cellular signal transduction and are implicated in many biological processes, including tumorigenesis, angiogenesis, immunity, atherosclerosis, arteriosclerosis, cancer and neuronal survival. The intracellular transport of lysophospholipids is through FA (fatty acid)-binding protein. Lysophospholipids are also found in the extracellular space. However, the transport mechanism of lysophospholipids in the extracellular space is unknown. HSA (human serum albumin) is the most abundant carrier protein in blood plasma and plays an important role in determining the absorption, distribution, metabolism and excretion of drugs. In the present study, LPE (lysophosphatidylethanolamine) was used as the ligand to analyse the interaction of lysophospholipids with HSA by fluorescence quenching and crystallography. Fluorescence measurement showed that LPE binds to HSA with a Kd (dissociation constant) of 5.6 microM. The presence of FA (myristate) decreases this binding affinity (Kd of 12.9 microM). Moreover, we determined the crystal structure of HSA in complex with both myristate and LPE and showed that LPE binds at Sudlow site I located in subdomain IIA. LPE occupies two of the three subsites in Sudlow site I, with the LPE acyl chain occupying the hydrophobic bottom of Sudlow site I and the polar head group located at Sudlow site I entrance region pointing to the solvent. This orientation of LPE in HSA suggests that HSA is capable of accommodating other lysophospholipids and phospholipids. The study provides structural information on HSA-lysophospholipid interaction and may facilitate our understanding of the transport and distribution of lysophospholipids.

Structure of catalytic domain of Matriptase in complex with Sunflower trypsin inhibitor-1.

BackgroundMatriptase is a type II transmembrane serine protease that is found on the surfaces of epithelial cells and certain cancer cells. Matriptase has been implicated in the degradation of certain extracellular matrix components as well as the activation of various cellular proteins and proteases, including hepatocyte growth factor and urokinase. Sunflower trypsin inhibitor-1 (SFTI-1), a cyclic peptide inhibitor originally isolated from sunflower seeds, exhibits potent inhibitory activity toward matriptase.ResultsWe have engineered and produced recombinant proteins of the matriptase protease domain, and have determined the crystal structures of the protease:SFTI-1 complex at 2.0 Å as well as the protease:benzamidine complex at 1.2 Å. These structures elaborate the structural basis of substrate selectivity of matriptase, and show that the matriptase S1 substrate specificity pocket is larger enough to allow movement of benzamidine inside the S1 pocket. Our study also reveals that SFTI-1 binds to matriptase in a way similar to its binding to trypsin despite the significantly different isoelectric points of the two proteins (5.6 vs. 8.2).ConclusionsThis work helps to define the structural basis of substrate specificity of matriptase and the interactions between the inhibitor and protease. The complex structure also provides a structural template for designing new SFTI-1 derivatives with better potency and selectivity against matriptase and other proteases.

Convective diffusion in protein crystal growth

We considered a protein crystal in the form of a flat plate suspended in its parent solution so that the normal to the largest face was perpendicular to the acceleration due to gravity. For simplicity, the protein concentration in the solution adjacent to the plate was taken to be the equilibrium solubility. The bulk of the solution was supersaturated, however, which gave rise to a horizontal concentration gradient driving fluid toward the plate. We also took into account the diffusion of the dissolved protein with respect to the moving fluid. In the boundary layer next to the plate, we solved the Navier-Stokes equation and the equation for convective diffusion to determine the flow velocity and the protein mass flux. We found that, because of the convection, the local rate of growth of the plate varied strongly with depth. The variation was diminished by a factor of 1/30 when the local gravity was reduced from g to 10-6 g as occurs aboard the Space Shuttle in earth orbit. For an aqueous solution of lysozyme at a concentration of 40 mg/ml, the boundary layer at the top of a 1 mm high crystal has a thickness of 80 μm in earths gravity and 2570 μm in 10-6 g. We examined the optical transmission of the boundary layer and compared it with the “haloes” observed by Feher et al. about growing hemispherical crystals of lysozyme.

Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268–275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.