Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.     

Crystal structure of the interleukin-4/receptor alpha chain complex reveals a mosaic binding interface

Interleukin-4 (IL-4) is a principal regulatory cytokine during an immune response and a crucial determinant for allergy and asthma. IL-4 binds with high affinity and specificity to the ectodomain of the IL-4 receptor alpha chain (IL4-BP). Subsequently, this intermediate complex recruits the common gamma chain (gamma c), thereby initiating transmembrane signaling. The crystal structure of the intermediate complex between human IL-4 and IL4-BP was determined at 2.3 A resolution. It reveals a novel spatial orientation of the two proteins, a small but unexpected conformational change in the receptor-bound IL-4, and an interface with three separate clusters of trans-interacting residues. Novel insights on ligand binding in the cytokine receptor family and a paradigm for receptors of IL-2, IL-7, IL-9, and IL-15, which all utilize gamma c, are provided.     

Identification of essential regions in the cytoplasmic tail of interleukin-1 receptor accessory protein critical for interleukin-1 signaling

Interleukin (IL)-1 plays an important role in inflammation and regulation of immune responses. The activated IL-1 receptor complex, which consists of the IL-1 receptor type I and the IL-1 receptor accessory protein (IL-1RAcP), generates multiple cellular responses including NF-kappaB activation, IL-2 secretion, and IL-2 promoter activation. Reconstitution experiments in EL4D6/76 cells lacking IL-1RAcP expression and IL-1 responsiveness were used to analyze structure-function relationships of the IL-1RAcP cytoplasmic tail. Mutating a potential tyrosine kinase phosphorylation motif and various conserved amino acid (aa) residues had no effect on IL-1 responsiveness. Truncation analyses revealed that box 3 of the TIR domain was required for NF-kappaB activation, IL-2 production, and c-Jun N-terminal kinase (JNK) activation, whereas IL-2 promoter activation was only partially inhibited. Surprisingly, deletion of aa 527-534 resulted in almost complete loss of all IL-1 responsiveness. Replacement of these aa with alanyl residues did not reconstitute NF-kappaB activation, IL-2 production, or JNK activation but partly restored IL-2 promoter activation. Immunoprecipitation data revealed a strong correlation between MyD88 binding with NF-kappaB activation and IL-2 production but not with IL-2 promoter activation. Taken together, our data indicate that box 3 of IL-1RAcP is critical for IL-1-dependent NF-kappaB activation and stabilization of IL-2 mRNA via JNK, whereas aa 527-534 largely contribute to IL-2 promoter activation.     

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.     

TspO of rhodobacter sphaeroides. A structural and functional model for the mammalian peripheral benzodiazepine receptor

The function and specific structural aspects of the tryptophan-rich sensory protein (TspO) of Rhodobacter sphaeroides 2.4.1 were studied using site-directed mutagenesis involving some 17 different amino acids. The choice of these amino acids changes was dictated from an analysis of the TspO family of proteins derived from the data bases. These studies demonstrated the importance of several highly conserved tryptophan residues in the sensory transduction pathway involving TspO through the proposed binding of an intermediate(s) in the tetrapyrrole biosynthesis pathway. These studies also revealed that the substitution of one or several of the amino acid residues dramatically affected, either directly or indirectly, the levels of TspO in the membranes of R. sphaeroides. Mounting evidence is presented suggesting that TspO normally forms a dimer within the bacterial outer membrane, and the dimer form of TspO may be the active form for TspO function. Because our earlier studies provided us with a functional framework within which to view these amino acid substitutions, we are able to suggest a preliminary model for TspO structure-function. Not only do these studies tell us more about TspO, but they also shed light on the TspO homologue, the drug-binding component of the mitochondrial peripheral benzodiazepine receptor. Mounting evidence draws numerous parallelism between these proteins and supports the significance of using TspO as a model for the structure and function of the mitochondrial protein.     

Construction of protein binding sites in scaffold structures

We have developed a strategy for grafting a protein-protein interface based on the known crystal structure of a native ligand and receptor proteins in a complex. The key interaction residues at the ligand protein binding interface are transferred onto a scaffold protein so that the mutated scaffold protein will bind the receptor protein in the same manner as the ligand protein. First, our method identifies key residues and atoms in the ligand protein, which strongly interact with the receptor protein. Second, this method searches the scaffold protein for combinations of candidate residues, among which the distance between any two candidate residues is similar to that between relevant key interaction residues in the ligand protein. These candidate residues are mutated to key interaction residues in the ligand protein respectively. The scaffold protein is superposed onto the ligand protein based upon the coordinates of corresponding atoms, which are assumed to strongly interact with the receptor protein. Complementarity between scaffold and receptor proteins is evaluated. Scaffold proteins with a low superposing rms difference and high complementary score are accepted for further analysis. Then, the relative position of the scaffold protein is adjusted so that the interfaces between the scaffold and receptor proteins have a reasonable packing density. Other mutations are also considered to reduce the desolvation energy or bad steric contacts. Finally, the scaffold protein is cominimized with the receptor protein and evaluated. To test the method, the binding interface of barstar, the inhibitor of barnase, was grafted onto small proteins. Four scaffold proteins with high complementary scores are accepted.     

The cytoplasmic tyrosine motifs in full-length glycoprotein 130 have different roles in IL-6 signal transduction

The function of the signal-transducing receptor subunit glycoprotein 130 (gp130) in the IL-6-receptor complex has previously been studied using carboxyl-terminal deletion mutants or a truncated molecule of approximately 60 membrane-proximal amino acids (containing box 1 and box 2) linked to the individual gp130 tyrosine motifs. However, the redundancy of the tyrosine motifs within the cytoplasmic part of gp130 has been neglected. Here we describe the analysis of the function of the individual cytoplasmic tyrosine residues of gp130 in the context of the full-length receptor protein in IL-6 signaling as measured by STAT activation, acute phase protein induction, and stimulation of proliferation. Add-back receptor mutants containing only one cytoplasmic tyrosine have been generated and tested for their efficiency in IL-6 signal transduction. Our studies revealed that tyrosine motifs which have been described to recruit STAT proteins are not equivalent with respect to their potential to activate STAT factors and acute phase protein gene promoters: the two distal tyrosines, Tyr905 and Tyr915, of gp130 were more potent than Tyr767 and Tyr814. Surprisingly, Tyr905 and Tyr915 mediate acute phase protein gene promoter activation stronger than the wild-type receptor containing all six cytoplasmic tyrosine residues. In contrast, Ba/F3 cells stably transfected with add-back receptors containing Tyr767 or Tyr905 were more sensitive to IL-6-induced proliferation than cells expressing the other add-back receptor mutants. Thus, the tyrosine residues in the cytoplasmic part of gp130 were found to contribute differentially to IL-6 signal transduction in the full- length gp130 protein.     

Structural Basis of Interleukin-5 Inhibition by the Small Cyclic Peptide AF17121

Interleukin-5 (IL-5) is a T-helper cell of subtype 2 cytokine involved in many aspects of eosinophil life. Eosinophilic granulocytes play a pathogenic role in the progression of atopic diseases, such as allergy, asthma and atopic dermatitis and hypereosinophilic syndromes. Here, eosinophils upon activation degranulate leading to the release of proinflammatory proteins and mediators stored in intracellular vesicles termed granula thereby causing local inflammation, which when persisting leads to tissue damage and organ failure. As a key regulator of eosinophil function, IL-5 therefore presents a major pharmaceutical target and approaches to interfere with IL-5 receptor activation are of great interest. Here we present the structure of the IL-5 inhibiting peptide AF17121 bound to the extracellular domain of the IL-5 receptor IL-5Rα. The small 18mer cyclic peptide snugly fits into the wrench-like cleft of the IL-5 receptor, thereby blocking access of key residues for IL-5 binding. While AF17121 and IL-5 seemingly bind to a similar epitope at IL-5Rα, functional studies show that recognition and binding of both ligands differ. Using the structure data, peptide variants with improved IL-5 inhibition have been generated, which might present valuable starting points for superior peptide-based IL-5 antagonists.     

Rational design of TNFα binding proteins based on the de novo designed protein DS119

De novo protein design offers templates for engineering tailor‐made protein functions and orthogonal protein interaction networks for synthetic biology research. Various computational methods have been developed to introduce functional sites in known protein structures. De novo designed protein scaffolds provide further opportunities for functional protein design. Here we demonstrate the rational design of novel tumor necrosis factor alpha (TNFα) binding proteins using a home‐made grafting program AutoMatch. We grafted three key residues from a virus 2L protein to a de novo designed small protein, DS119, with consideration of backbone flexibility. The designed proteins bind to TNFα with micromolar affinities. We further optimized the interface residues with RosettaDesign and significantly improved the binding capacity of one protein Tbab1‐4. These designed proteins inhibit the activity of TNFα in cellular luciferase assays. Our work illustrates the potential application of the de novo designed protein DS119 in protein engineering, biomedical research, and protein sequence‐structure‐function studies.     

Probing the surface of a sweet protein: NMR study of MNEI with a paramagnetic probe

The design of safe sweeteners is very important for people who are affected by diabetes, hyperlipemia, and caries and other diseases that are linked to the consumption of sugars. Sweet proteins, which are found in several tropical plants, are many times sweeter than sucrose on a molar basis. A good understanding of their structure-function relationship can complement traditional SAR studies on small molecular weight sweeteners and thus help in the design of safe sweeteners. However, there is virtually no sequence homology and very little structural similarity among known sweet proteins. Studies on mutants of monellin, the best characterized of sweet proteins, proved not decisive in the localization of the main interaction points of monellin with its receptor. Accordingly, we resorted to an unbiased approach to restrict the search of likely areas of interaction on the surface of a typical sweet protein. It has been recently shown that an accurate survey of the surface of proteins by appropriate paramagnetic probes may locate interaction points on protein surface. Here we report the survey of the surface of MNEI, a single chain monellin, by means of a paramagnetic probe, and a direct assessment of bound water based on an application of ePHOGSY, an NMR experiment that is ideally suited to detect interactions of small ligands to a protein. Detailed surface mapping reveals the presence, on the surface of MNEI, of interaction points that include residues previously predicted by ELISA tests and by mutagenesis.     

In vitro characterization of the cysteine-rich capping domains in a plant leucine rich repeat protein

Plant leucine rich repeat (LRR) proteins have diverse functions and cellular locations. An important unresolved question involves the role of the cysteine-rich capping domains which flank the LRR domain. Such studies have been hampered by difficulties in producing recombinant LRR proteins in yields sufficient for biochemical analysis. We have used Escherichia coli to overproduce Leucine Rich Protein (LRP), a small model LRR protein from tomato containing approximately five LRRs. The LRP capping domain sequences resemble those from plant disease resistance proteins and receptor-like protein kinases. LRP was purified as a soluble, crystallizable, monomeric protein by renaturation of a GST-fusion protein. The four cysteine residues in LRP were found to form two disulfide bonds, one each in the N- and C-terminal LRR-capping domains, the presence of which is necessary to protect the LRR domain from proteolysis in vitro. Fluorescence and CD spectroscopies together with molecular modelling revealed that structural features of the N-capping domain may be destabilised on reduction. These include a tryptophan stacking interaction and a long alpha-helix of residues 30-44. LRP deletion mutants lacking the capping domains showed a propensity to aggregate and increased proteolytic sensitivity. These results have important implications for future structure-function studies of plant LRR proteins.     

Anticoagulant proteins from snake venoms: structure, function and mechanism

Over the last several decades, research on snake venom toxins has provided not only new tools to decipher molecular details of various physiological processes, but also inspiration to design and develop a number of therapeutic agents. Blood circulation, particularly thrombosis and haemostasis, is one of the major targets of several snake venom proteins. Among them, anticoagulant proteins have contributed to our understanding of molecular mechanisms of blood coagulation and have provided potential new leads for the development of drugs to treat or to prevent unwanted clot formation. Some of these anticoagulants exhibit various enzymatic activities whereas others do not. They interfere in normal blood coagulation by different mechanisms. Although significant progress has been made in understanding the structure-function relationships and the mechanisms of some of these anticoagulants, there are still a number of questions to be answered as more new anticoagulants are being discovered. Such studies contribute to our fight against unwanted clot formation, which leads to death and debilitation in cardiac arrest and stroke in patients with cardiovascular and cerebrovascular diseases, arteriosclerosis and hypertension. This review describes the details of the structure, mechanism and structure-function relationships of anticoagulant proteins from snake venoms.     

Mapping the Homodimer Interface of an Optimized,Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor

Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation.     

Definition and spatial location of mouse interleukin-2 residues that interact with its heterotrimeric receptor.

The high affinity receptor for interleukin-2 (IL-2) contains three subunits called IL-2R alpha, beta and gamma. A biological and receptor binding analysis based on 1393 different mutant mouse IL-2 (mIL-2) proteins was used to define the function of each of the 149 residues. By this genetic analysis, 44 residues were assigned important functions, 21 of which were structural. The remaining 23 residues consisted of 19 residues, from three separate regions, that were important for IL-2R alpha interaction; three residues, from two separate regions, that were important for IL-2R beta interaction; and a single residue important for IL-2R gamma interaction. We built a model mIL-2 structure based on the homologous human IL-2 (hIL-2) crystal structure. The roles of the 21 residues presumed to be important for structure were consistent with the model. Despite discontinuity in the primary sequence, the residues specific for each IL-2R subunit interaction were clustered and located to three disparate regions of the tertiary mIL-2 structure. The relative spatial locations of these three surfaces are different from the two receptor binding sites known for the structurally related human growth hormone and the significance of this observation is discussed.     

Limited tryptic digestion of recombinant human interleukin-2: Structure-binding relationships with the α chain of the interleukin-2 receptor

The surface topography and structural features of interleukin-2 (IL-2) in relation to its interaction with the α subunit of its receptor (IL-2Rα) have been probed by limited tryptic digestion followed by detailed structural analyses. Four sensitive cleavage sites in IL-2 (Lys⁸, Lys⁹, Lys³⁵, and Arg³⁸) were identified as surface amino acids, suggesting that they are potential binding sites for IL-2Rα. To examine the involvement of these residues in IL-2Rα binding, a truncated IL-2 molecule lacking the amino-terminal residues through Arg³⁸ was generated and it was found to be incapable of binding IL-2Rα in a solid-phase receptor binding sequencing assay. These studies have led to the conclusion that the IL-2Rα contact region of IL-2 includes residues Lys³⁵ and Arg³⁸. This finding is supported by the refined three-dimensional structure of IL-2 in which these residues are located outside of the compact bundle of four helices and thus are readily available for interaction with IL-2Rα.     

Mutational analysis of the Pyrococcus furiosus holliday junction resolvase hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities

The Holliday junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs. The products of these mutant genes were purified to homogeneity. With analysis of the activity of the mutant proteins to bind and cleave synthetic Holliday junctions, one acidic residue, Glu-9, and two basic residues, Arg-10 and Arg-25, were found to play critical roles in enzyme action. This is in addition to the three conserved residues, Asp-33, Glu-46, and Lys-48, which are also conserved in the motif found in the type II restriction endonuclease family proteins. Two aromatic residues, Phe-68 and Phe-72, are important for the formation of the homodimer probably through hydrophobic interactions. The results of these studies have provided insights into the structure-function relationships of the archaeal Holliday junction resolvase as well as the universality and diversity of the Holliday junction cleavage reaction.     

NMR evaluation of adipocyte fatty acid binding protein (aP2) with R- and S-ibuprofen

We have examined global chemical shift perturbations for aP2 ligand complexes and compared these with amide temperature coefficients. Hydrogen bond potential was monitored by amide chemical shift's temperature coefficient. Based on this information, we propose that the binding energy contribution can be spread out to multiple distant residues. For aP2, the ability of the receptor protein to change its hydrogen bond interactions in the beta-strands to accommodate different ligand scaffolds seems to make this receptor difficult for structure based drug design. While stabilization energy differential on hydrogen bonds is likely to be small for individual residues, the accumulative effect on multiple hydrogen bonds may have a dramatic impact on ligand affinity.     

Structural proteomics by NMR spectroscopy

Structural proteomics is one of the powerful research areas in the postgenomic era, elucidating structure-function relationships of uncharacterized gene products based on the 3D protein structure. It proposes biochemical and cellular functions of unannotated proteins and thereby identifies potential drug design and protein engineering targets. Recently, a number of pioneering groups in structural proteomics research have achieved proof of structural proteomic theory by predicting the 3D structures of hypothetical proteins that successfully identified the biological functions of those proteins. The pioneering groups made use of a number of techniques, including NMR spectroscopy, which has been applied successfully to structural proteomics studies over the past 10 years. In addition, advances in hardware design, data acquisition methods, sample preparation and automation of data analysis have been developed and successfully applied to high-throughput structure determination techniques. These efforts ensure that NMR spectroscopy will become an important methodology for performing structural proteomics research on a genomic scale. NMR-based structural proteomics together with x-ray crystallography will provide a comprehensive structural database to predict the basic biological functions of hypothetical proteins identified by the genome projects.     

Biochemical consequences of mutations causing the GM2 gangliosidoses

The hydrolysis of GM2-ganglioside is unusual in its requirements for the correct synthesis, processing, and ultimate combination of three gene products. Whereas two of these proteins are the alpha- (HEXA gene) and beta- (HEXB) subunits of beta-hexosaminidase A, the third is a small glycolipid transport protein, the GM2 activator protein (GM2A), which acts as a substrate specific co-factor for the enzyme. A deficiency of any one of these proteins leads to storage of the ganglioside, primarily in the lysosomes of neuronal cells, and one of the three forms of GM2-gangliosidosis, Tay-Sachs disease, Sandhoff disease or the AB-variant form. Studies of the biochemical impact of naturally occurring mutations associated with the GM2 gangliosidoses on mRNA splicing and stability, and on the intracellular transport and stability of the affected protein have provided some general insights into these complex cellular mechanisms. However, such studies have revealed little in the way of structure-function information on the proteins. It appears that the detrimental effect of most mutations is not specifically on functional elements of the protein, but rather on the proteins' overall folding and/or intracellular transport. The few exceptions to this generalization are missense mutations at two codons in HEXA, causing the unique biochemical phenotype known as the B1-variant, and one codon in both the HEXB and GM2A genes. Biochemical characterization of these mutations has led to the localization of functional residues and/or domains within each of the encoded proteins.     

Structural basis of interleukin-5 dimer recognition by its α receptor

Interleukin-5 (IL-5), a major hematopoietin, stimulates eosinophil proliferation, migration, and activation, which have been implicated in the pathogenesis of allergic inflammatory diseases, such as asthma. The specific IL-5 receptor (IL-5R) consists of the IL-5 receptor α subunit (IL-5RA) and the common receptor β subunit (βc). IL-5 binding to IL-5R on target cells induces rapid tyrosine phosphorylation and activation of various cellular proteins, including JAK1/JAK2 and STAT1/STAT5. Here, we report the crystal structure of dimeric IL-5 in complex with the IL-5RA extracellular domains. The structure revealed that IL-5RA sandwiches the IL-5 homodimer by three tandem domains, arranged in a “wrench-like” architecture. This association mode was confirmed for human cells expressing IL-5 and the full-length IL-5RA by applying expanded genetic code technology: protein photo-cross-linking experiments revealed that the two proteins interact with each other in vivo in the same manner as that in the crystal structure. Furthermore, a comparison with the previously reported, partial GM-CSF•GM-CSFRA•βc structure enabled us to propose complete structural models for the IL-5 and GM-CSF receptor complexes, and to identify the residues conferring the cytokine-specificities of IL-5RA and GM-CSFRA.