The structure of an actin-crosslinking domain from human fimbrin.
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Actins↗
Biomedical subjects
S C Almo
Publications and source records attributed to S C Almo.
Structure of the profilin-poly-L-proline complex involved in morphogenesis and cytoskeletal regulation.
Profilin, a ubiquitous low molecular weight (13,000-15,000 M(r)) actin binding protein, regulates the formation of F-actin structures in vivo, and is localized to specific cellular regions through interaction with proline-rich sequences. Here we report the 2.2 A X-ray structure of the complex between human platelet profilin (HPP) and a decamer of L-proline (L-Pro10). The L-Pro10 peptide adopts a left-handed type II poly-L-proline helix (PPII) and binds to a highly conserved patch of aromatic amino acids on the surface of profilin. The peptide and actin binding sites reside on orthogonal surfaces, and L-Pro10 binding does not result in a conformational rearrangement of HPP. This structure suggests a mechanism for the localization of profilin and its actin-related activities to sites of actin filament assembly in vivo.
Amino Acid Sequence↗
Ionic interactions in crystalline bovine pancreatic ribonuclease A.
Isomorphous crystals (space group P3(2)21) of bovine pancreatic ribonuclease A (RNase A) were prepared at a pH of 5.5 in a series of high salt conditions, where both the nature of the ions and the ionic strength varied: 80% ammonium sulfate (mu = 12.5); 8 M sodium formate (mu = 8.0); 3 M NaCl, 30% ammonium sulfate (mu = 7.0); 3 M CsCl, 30% ammonium sulfate (mu = 7.0); and 2.5 M NaCl, 3.3 M sodium formate (mu = 5.8). These structures were independently refined to a resolution of 2.0 A or better with R-factors that range from 16.1% to 17.5%. A comparison of these six structures and the monoclinic crystal form of RNase A grown from alcohol shows that changes in ionic strength do not alter the secondary or tertiary structure and that there are no significant changes in intramolecular salt bridges. These findings support the notion that structures determined from crystals grown in high salt are representative of the overall structural and electrostatic features present under physiological conditions. While little effect was observed on the main chain conformation, several residues adopted different side chain conformations and altered hydrogen-bonding patterns, either as result of direct anion binding or more subtle indirect effects. Changes in the ionic composition of the mother liquor allowed for the occupancy of the active site with different anions. The direct observation of active site-bound chloride and formate anions supports the proposal that these species act as true competitive inhibitors of RNase A and not through nonspecific electrostatic effects. The identification of bound formate anions allowed for an experimental validation of computational-based functional group mapping techniques and suggests a useful modification to these approaches. Electrostatic surface potential calculations identify a nearly continuous band of positive potential, consistent with an extended binding site for polynucleotide ligands and substrates. The majority of these residues are not involved in salt bridges, which may facilitate binding to extended polynucleotide substrates. Selection of the appropriate solvent conditions results in an unoccupied active site, which will allow this crystal form to be used for the crystallographic study of productive ligand-binding modes.
Amino Acid Sequence↗
Molecular and structural analysis of a continuous birch profilin epitope defined by a monoclonal antibody.
The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 --> Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented.
Allergens↗
Comparison of experimental and computational functional group mapping of an RNase A structure: implications for computer-aided drug design.
One relatively new computational approach to the drug discovery process involves calculating functional group maps of a target structure. Experimental functional group mapping techniques have also recently emerged. In this paper, the structure of RNase A with two bound formates (i.e. carboxylate functionalities) is used as a model system to test the computational methodology. Functional group maps of the RNase A structure were calculated using the Multiple Copy Simultaneous Search (MCSS) method and compared with experimentally determined formate and water positions. The calculations indicate that the protonation state of active-site histidines determines the ability of the enzyme to bind formate. The results also suggest an ordered binding mechanism for the two formates. An improved strategy for using the MCSS method to design new candidate ligands is discussed.
Animals↗
X-ray structures of isoforms of the actin-binding protein profilin that differ in their affinity for phosphatidylinositol phosphates.
We determined the structures of Acanthamoeba profilin I and profilin II by x-ray crystallography at resolutions of 2.0 and 2.8 A, respectively. The polypeptide folds and the actin-binding surfaces of the amoeba profilins are very similar to those of bovine and human profilins. The electrostatic potential surfaces of the two Acanthamoeba isoforms differ. Two areas of high positive potential on the surface of profilin II are candidate binding sites for phosphatidylinositol phosphates. The proximity of these sites to the actin binding site provides an explanation for the competition between actin and lipids for binding profilin.
Acanthamoeba↗
Purification, characterization and crystallization of human platelet profilin expressed in Escherichia coli.
Human platelet profilin was expressed in Escherichia coli using a T7 based expression vector. The recombinant material is similar to authentic human platelet profilin based on the measured Kd for rabbit skeletal muscle actin. Crystals of the recombinant material were obtained from both PEG 8000 and (NH4)2SO4. These crystals are isomorphous and belong to the monoclinic space group C2, a = 75.0, b = 32.0, c = 62.5, beta = 123 degrees. These crystals contain one molecule in the asymmetric unit and diffract to at least 2.0 A.
Actins↗
Purification, characterization and crystallization of Acanthamoeba profilin expressed in Escherichia coli.
Profilin (isoform I) from Acanthamoeba castellani was expressed in Escherichia coli using a bacteriophage T7-based expression vector. The recombinant material is similar to authentic profilin from Acanthamoeba-based on fluorescence monitored urea denaturation, circular dichroism, actin-nucleotide exchange rate and the Kd for rabbit skeletal actin. This recombinant material crystallized from 80% saturated sodium potassium tartrate, yielding monoclinic crystals, space group C2, a = 91.4 A, b = 37.4 A, c = 34.7 A, beta = 109.6 degrees. These crystals contain one molecule in the asymmetric unit and diffract to 2.0 A.
Acanthamoeba↗
The structural basis for the altered substrate specificity of the R292D active site mutant of aspartate aminotransferase from E. coli.
Two refined crystal structures of aspartate aminotransferase from E. coli are reported. The wild type enzyme is in the pyridoxal phosphate (PLP) form and its structure has been determined to 2.4 A resolution, refined to an R-factor of 23.2%. The structure of the Arg292Asp mutant has been determined at 2.8 A resolution, refined to an R-factor of 20.3%. The wild type and mutant crystals are isomorphous and the two structures are very similar, with only minor changes in positions of important active site residues. As residue Arg292 is primarily responsible for the substrate charge specificity in the wild type enzyme, the mutant containing a charge reversal at this position might be expected to catalyze transamination of arginine as efficiently as the wild type enzyme effects transamination of aspartate [Cronin, C.N. and Kirsch, J.F. (1988) Biochemistry, 27, 4572-4579]. This mutant does in fact prefer arginine over aspartate as a substrate, however, the rate of catalysis is much slower than that of the wild type enzyme with its physiological substrate, aspartate. A comparison of these two structures indicates that the poorer catalytic efficiency of R292D, when presented with arginine, is not due to a gross conformational difference, but is rather a consequence of both small side chain and main chain reorientations and the pre-existing active site polar environment, which greatly favors the wild type ion pair interaction.
Arginine↗
Structure determination of turkey egg-white lysozyme using Laue diffraction data.
The three-dimensional structure of turkey egg-white lysozyme (TEWL) has been solved and refined at 2.5 A resolution using X-ray data collected by the Laue method. This is the first protein structure determination undertaken using Laue diffraction data. A re-examination of the existing structure of TEWL was necessary when attempts to refine an atomic model based on the C alpha positions in the Protein Data Bank (entry 1LZ2) failed. The correct orientation and position of the turkey lysozyme molecules within the crystallographic unit cell were determined by molecular replacement using a refined model of the homologous hen egg-white lysozyme crystal structure. After modification of the model to reflect the differences in amino-acid sequence between the chicken and turkey enzymes, the structure was subjected to crystallographic refinement using the simulated-annealing refinement technique and conventional least-squares refinement. This yielded a final residual of R = 20.7%. This crystal form is of potential interest for time-resolved crystallographic studies since the amino-acid residues involved in catalysis (Asp52 and Glu35) are accessible to solvent and not blocked by crystal contacts.
Animals↗
Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis.
Crystals of Ha-Ras p21 with caged GTP at the active site have been used to investigate the conformational changes of p21 on GTP hydrolysis. The structure of the short-lived p21.GTP complex was determined by Laue diffraction methods. After GTP hydrolysis, substantial structural changes occur in the parts of the molecule implicated in the interaction with GTPase-activating protein. The trigger for this process seems to be a change in coordination of the active-site Mg2+ ion as a result of loss of the gamma-phosphate of GTP.
Binding Sites↗
2.8-A-resolution crystal structure of an active-site mutant of aspartate aminotransferase from Escherichia coli.
The three-dimensional structure of a mutant of the aspartate aminotransferase from Escherichia coli, in which the active-site lysine has been substituted by alanine (K258A), has been determined at 2.8-A resolution by X-ray diffraction. The mutant enzyme contains pyridoxamine phosphate as cofactor. The structure is compared to that of the mitochondrial aspartate aminotransferase. The most striking differences, aside from the absence of the lysine side chain, occur in the positions of the pyridoxamine group and of tryptophan 140.
Aspartate Aminotransferases↗
Structure of the amino-terminal domain of phage 434 repressor at 2.0 A resolution.
The crystal structure of the amino-terminal domain of phage 434 repressor has been solved using molecular replacement methods and refined to an R-factor of 19.3% against data to 2.0 A resolution. The protein comprises five short alpha-helices. Two of these form a helix-turn-helix motif, very similar to those found in related proteins. The protein is remarkably similar to the Cro protein from the same phage.
Amino Acid Sequence↗
X-ray Laue diffraction from crystals of xylose isomerase.
The Laue method (stationary crystal, polychromatic x-rays) was used to collect native and heavy-atom-derivative data on crystals of xylose isomerase (EC 5.3.1.5). These data were used to find the heavy-atom positions. The positions found by use of Laue data are the same as those found by use of monochromatic data collected on a diffractometer. These results confirm that Laue diffraction data sets, which can be obtained on a millisecond time scale, can be used to locate small molecules bound to protein active sites. The successful determination of heavy-atom positions also indicates that x-ray crystallographic data collected by the Laue method can be used to solve protein structures.
Aldose-Ketose Isomerases↗
X-ray crystal structures of birch pollen profilin and Phl p 2.
BACKGROUND: Type 1 allergy affects 20% of industrialized populations and thus represents a major health care issue. The symptoms of type 1 allergy, which include rhinitis, conjunctivitis, dermatitis and asthma, are elicited by the cross-linking of IgE receptors through polyvalent allergens. A detailed understanding of the cell surface phenomena and the rational development of new therapies require high-resolution structural information. METHODS: The structures of two widespread allergens, birch pollen profilin (BPP) and Phl p 2 have been solved by multiple isomorphous replacement. Refinements are underway to 2.4 and 2.0 A, respectively. In addition, the IgE-reactive epitopes of BPP where identified by screening an epitope expression library with the serum IgE of an allergic individual. RESULTS: BPP exhibits an alpha/beta-fold which is similar to the mammalian and amoeba profilins. The structure of Phl p 2 is a compact eight-stranded beta-barrel. Screening an epitope library of BPP identified three major epitopic regions involved in IgE binding, including the amino and carboxy-terminal alpha-helices. These regions also interact with the physiologically relevant ligands of profilin, actin and proline-rich peptides. CONCLUSIONS: The distribution of IgE-binding sites on BPP allows for the productive interaction with IgE antibodies of different epitope specificities required for efficient signal transduction. These epitopes correspond to the most highly conserved regions of the profilin molecule and thus provide the molecular basis for allergen cross-sensitivity. Due to steric considerations, the involvement of these epitopic regions in the binding of physiologically relevant profilin ligands indicates that the native profilin is the species responsible for eliciting the allergic response. A comparison of the BPP and Phl p 2 structures shows that there is no preference for secondary structural elements in the allergic response. The detailed chemical and physical description of the major reactive epitopes provides a data base for the design of tight-binding monovalent ligands which can prevent receptor aggregation and thereby reduce the allergic response.
Allergens↗