Alexander A. Fedorov

Structural basis for co-stimulation by the human CTLA-4/B7-2 complex

Regulation of T-cell activity is dependent on antigen-independent co-stimulatory signals provided by the disulphide-linked homodimeric T-cell surface receptors, CD28 and CTLA-4 (ref. 1). Engagement of CD28 with B7-1 and B7-2 ligands on antigen-presenting cells (APCs) provides a stimulatory signal for T-cell activation, whereas subsequent engagement of CTLA-4 with these same ligands results in attenuation of the response. Given their central function in immune modulation, CTLA-4- and CD28-associated signalling pathways are primary therapeutic targets for preventing autoimmune disease, graft versus host disease, graft rejection and promoting tumour immunity. However, little is known about the cell-surface organization of these receptor/ligand complexes and the structural basis for signal transduction. Here we report the 3.2-Å resolution structure of the complex between the disulphide-linked homodimer of human CTLA-4 and the receptor-binding domain of human B7-2. The unusual dimerization properties of both CTLA-4 and B7-2 place their respective ligand-binding sites distal to the dimer interface in each molecule and promote the formation of an alternating arrangement of bivalent CTLA-4 and B7-2 dimers that extends throughout the crystal. Direct observation of this CTLA-4/B7-2 network provides a model for the periodic organization of these molecules within the immunological synapse and suggests a distinct mechanism for signalling by dimeric cell-surface receptors.

Essential functions and actin‐binding surfaces of yeast cofilin revealed by systematic mutagenesis

Cofilin stimulates actin filament turnover in vivo. The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined. Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive‐lethal and three were conditional‐lethal and caused severe actin organization defects. Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F‐actin binding and depolymerization are essential cofilin functions. Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn. First, residues required for actin monomer binding are proximal to each other. Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament. Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment‐1. Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro. Finally, mutation of the cofilin N‐terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases.

The molecular basis for allergen cross-reactivity: crystal structure and IgE-epitope mapping of birch pollen profilin.

BACKGROUND The profilins are a group of ubiquitous actin monomer binding proteins that are responsible for regulating the normal distribution of filamentous actin networks in eukaryotic cells. Profilins also bind polyphosphoinositides, which can disrupt the profilin-action complex, and proline-rich ligands which localize profilin to sites requiring extensive actin filament accumulation. Profilins represent cross-reactive allergens for almost 20 % of all pollen allergic patients. RESULTS We report the X-ray crystal structure of birch pollen profilin (BPP) at 2.4 resolution. The major IgE-reactive epitopes have been mapped and were found to cluster on the N- and C-terminal alpha helices and a segment of the protein containing two strands of the beta sheet. The overall fold of this protein is similar to that of the mammalian and amoeba profilins, however, there is a significant change in the orientation of the N-terminal alpha helix in BPP. This change in orientation alters the topography of a hydrophobic patch on the surface of the molecule, which is thought to be involved in the binding of proline-rich ligands. CONCLUSIONS Profilin has been identified as an important cross-reactive allergen for patients suffering from multivalent type I allergy. The prevalent epitopic areas are located in regions with conserved sequence and secondary structure and overlap the binding sites for natural profilin ligands, indicating that the native ligand-free profilin acts as the original cross-sensitizing agent. Structural homology indicates that the basic features of the G actin-profilin interaction are conserved in all eukaryotic organisms, but suggests that mechanistic differences in the binding of proline-rich ligands may exist. The structure of BPP provides a molecular basis for understanding allergen cross-reactivity.

Structure-based activity prediction for an enzyme of unknown function

With many genomes sequenced, a pressing challenge in biology is predicting the function of the proteins that the genes encode. When proteins are unrelated to others of known activity, bioinformatics inference for function becomes problematic. It would thus be useful to interrogate protein structures for function directly. Here, we predict the function of an enzyme of unknown activity, Tm0936 from Thermotoga maritima, by docking high-energy intermediate forms of thousands of candidate metabolites. The docking hit list was dominated by adenine analogues, which appeared to undergo C6-deamination. Four of these, including 5-methylthioadenosine and S-adenosylhomocysteine (SAH), were tested as substrates, and three had substantial catalytic rate constants (105 M-1 s-1). The X-ray crystal structure of the complex between Tm0936 and the product resulting from the deamination of SAH, S-inosylhomocysteine, was determined, and it corresponded closely to the predicted structure. The deaminated products can be further metabolized by T. maritima in a previously uncharacterized SAH degradation pathway. Structure-based docking with high-energy forms of potential substrates may be a useful tool to annotate enzymes for function.

Structure of EVH1, a novel proline-rich ligand-binding module involved in cytoskeletal dynamics and neural function

The Ena-VASP homology (EVH1) domain is a protein interaction module found in several proteins that are involved in transducing migratory and morphological signals into cytoskeletal reorganization. EVH1 specifically recognizes proline-rich sequences in its binding partners and directs the localization and formation of multicomponent assemblies involved in actin-based motile processes and neural development. The structure of the complex between an EVH1 domain and the target peptide sequence EFPPPPT identifies the interactions responsible for recognition and distinguishes it from other proline-rich binding modules, including SH3 and WW domains. Surprisingly, the EVH1 domain has structural similarity to pleckstrin homology (PH), phosphotyrosine-binding (PTB) and ran-binding (RanBD) domains.

Crystal structure of PTP1B complexed with a potent and selective bidentate inhibitor

Protein-tyrosine phosphatase 1B (PTP1B) has been implicated as an important regulator in several signaling pathways including those initiated by insulin and leptin. Potent and specific PTP1B inhibitors could serve as useful tools in elucidating the physiological functions of PTP1B and may constitute valuable therapeutics in the treatment of several human diseases. We have determined the crystal structure of PTP1B in complex with compound 2, the most potent and selective PTP1B inhibitor reported to date. The structure at 2.15-Å resolution reveals that compound 2 simultaneously binds to the active site and a unique proximal noncatalytic site formed by Lys-41, Arg-47, and Asp-48. The structural data are further corroborated by results from kinetic analyses of the interactions of PTP1B and its site-directed mutants with compound 2 and several of its variants. Although many of the residues important for interactions between PTP1B and compound 2 are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which provide a structural basis for potent and selective PTP1B inhibition. Our data further suggest that potent, yet highly selective, PTP1B inhibitory agents can be acquired by targeting the area defined by residues Lys-41, Arg-47, and Asp-48, in addition to the previously identified second aryl phosphate-binding pocket.

Cholesterol-rich Fluid Membranes Solubilize Ceramide Domains IMPLICATIONS FOR THE STRUCTURE AND DYNAMICS OF MAMMALIAN INTRACELLULAR AND PLASMA MEMBRANES

A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.

Profilin binds proline-rich ligands in two distinct amide backbone orientations.

The actin regulatory protein profilin is targeted to specific cellular regions through interactions with highly proline-rich motifs embedded within its binding partners. New X-ray crystallographic results demonstrate that profilin, like SH3 domains, can bind proline-rich ligands in two distinct amide backbone orientations. By further analogy with SH3 domains, these data suggest that non-proline residues in profilin ligands may dictate the polarity and register of binding, and the detailed organization of the assemblies involving profilin. This degeneracy may be a general feature of modules that bind proline-rich ligands, including WW and EVH1 domains, and has implications for the assembly and activity of macromolecular complexes involved in signaling and the regulation of the actin cytoskeleton.

Ceramide-platform formation and -induced biophysical changes in a fluid phospholipid membrane

To understand the formation and properties of ceramide-enriched domains in cell membranes, the biophysical properties of the binary system palmitoyloleoylphosphatidylcholine (POPC)/palmitoylceramide were thoroughly characterized. Diverse fluorescent probes and parameters were necessary to unravel the complexity of this apparently simple system. For the first time, a complete phase diagram is reported, characterizing the lamellar phases of these mixtures, and providing a quantitative framework integrating biophysical and biological studies. The diagram suggests that in resting cells no ceramide domains exist, but upon apoptotic stimuli, platforms may form. Moreover, our data show that 2 mol% of Cer strongly affects the POPC fluid matrix, suggesting that a small increase in Cer levels can significantly affect cell membrane properties. In this work, we also show that Cer domains, formed in conditions similar to physiological, are extremely ordered and rigid. The domains composition is estimated from the phase diagram and their large size was concluded from fluorescence resonance energy transfer. Dynamic light scattering and electron microscopy were used to characterize the system morphology, which is highly dependent on ceramide content and includes vesiculation and tubular structure formation.

Functional Annotation and Three-Dimensional Structure of Dr0930 from Deinococcus radiodurans: A Close Relative of Phosphotriesterase in the Amidohydrolase Superfamily

Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed, and purified to homogeneity. The enzyme crystallized in the space group P3121, and the structure was determined to a resolution of 2.1 A. The protein folds as a (beta/alpha)7beta-barrel, and a binuclear metal center is found at the C-terminal end of the beta-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the beta-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of delta- and gamma-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was delta-nonanoic lactone with a kcat/Km of 1.6 x 10(6) M-1 s-1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of kcat and kcat/Km of 0.07 min-1 and 0.8 M-1 s-1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is 30%. The eight substrate specificity loops were transplanted from PTE to Dr0930, but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a kcat of 1.14 min-1, an increase of 16-fold over the wild-type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.