Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these ...receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6-Å resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor- family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Fox gene family comprises a large and functionally diverse group of forkhead-related transcriptional regulators, many of which are essential for metazoan embryogenesis and physiology. Defining ...conserved functional domains that mediate the transcriptional activity of Fox proteins will contribute to a comprehensive understanding of the biological function of Fox family genes.
Systematic analysis of 458 protein sequences of the metazoan Fox family was performed to identify the presence of the engrailed homology-1 motif (eh1), a motif known to mediate physical interaction with transcriptional corepressors of the TLE/Groucho family. Greater than 50% of Fox proteins contain sequences with high similarity to the eh1 motif, including ten of the nineteen Fox subclasses (A, B, C, D, E, G, H, I, L, and Q) and Fox proteins of early divergent species such as marine sponge. The eh1 motif is not detected in Fox proteins of the F, J, K, M, N, O, P, R and S subclasses, or in yeast Fox proteins. The eh1-like motifs are positioned C-terminal to the winged helix DNA-binding domain in all subclasses except for FoxG proteins, which have an N-terminal motif. Two similar eh1-like motifs are found in the zebrafish FoxQ1 and in FoxG proteins of sea urchin and amphioxus. The identification of eh1-like motifs by manual sequence alignment was validated by statistical analyses of the Swiss protein database, confirming a high frequency of occurrence of eh1-like sequences in Fox family proteins. Structural predictions suggest that the majority of identified eh1-like motifs are short alpha-helices, and wheel modeling revealed an amphipathicity that supports this secondary structure prediction.
A search for eh1 Groucho interaction motifs in the Fox gene family has identified eh1-like sequences in greater than 50% of Fox proteins. The results predict a physical and functional interaction of TLE/Groucho corepressors with many members of the Fox family of transcriptional regulators. Given the functional importance of the eh1 motif in transcriptional regulation, our annotation of this motif in the Fox gene family will facilitate further study of the diverse transcriptional and regulatory roles of Fox family proteins.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A closer look into the function of diiron proteins, such as methane monooxygenase and ribonucleotide reductase, is provided by the crystal structure of a designed diiron protein (the picture shows ...the Fe environment). Cofactor rigidity may be a factor in O2 reactivity and a possible role of HisCεH⋅⋅⋅O hydrogen bonds in cofactor stabilization is implicated.
Although the analysis and design of turns that connect the strands in antiparallel β-hairpins has reached an advanced state, much less is known concerning turns between antiparallel helices in ...helical hairpins. We have conducted an analysis of the structures and sequence preferences of two types of interhelical turns, each of which connects the two helices by a two-residue linker in an α
L–β conformation. Based on this analysis, it became apparent that the turn introduced into a designed four-helix bundle protein, DF1, did not occur within an optimal structural context. DF1 is a dimeric model for the diiron class of proteins. A longer loop with a β–α
R–β conformation was inserted between two helices in the protein, and a sequence was chosen to stabilize its conformation. X-ray crystallography and NMR analysis of the protein showed the structure to be in excellent agreement with design.
The crystal structure of human type III 3α-hydroxysteroid dehydrogenase (HSD)/bile acid binding protein (AKR1C2) complexed with NADP+ and 3α,7β-dihydroxy-5β-cholanic acid (ursodeoxycholate) at 3.0 Å ...resolution is presented. Thus, the three-dimensional structure has now been solved for a human HSD member of the aldo-keto reductase superfamily. AKR1C2 is implicated in the prostatic production of the potent androgen 5α-dihydrotestosterone and the hepatic transport of bile acids. It also catalyzes the formation of the neurosteroid 3α-hydroxy-5α-pregnan-20-one in the central nervous system, and its allosteric modulation by fluoxetine has been linked to the use of this drug for premenstrual dsyphoria. Like other members of the superfamily, AKR1C2 folds into an α/β-barrel and binds NADP+ in an extended conformation. The carboxylate of ursodeoxycholate binds to AKR1C2 in the oxyanion hole at the active site. More interestingly, the orientation of ursodeoxycholate is essentially “backwards” and “upside-down” from that observed for testosterone in the related rat 3α-HSD·NADP+·testosterone ternary complex, where testosterone assumes the position of a 3-ketosteroid substrate. The orientation of ursodeoxycholate is thus similar to that expected of a 17β-HSD substrate. The ternary structure explains the ability of AKR1C2 to catalyze 3α-, 17β-, and 20α-HSD reactions. Comparison of the steroid binding pocket of AKR1C2 with that of rat 3α-HSD reveals significant differences in the positions of conserved and nonconserved loop residues, providing insights into the structural basis for the functional flexibility that is observed in all the human 3α-HSD isoforms but not in the rat isoform.
X-ray Structure of a Maquette Scaffold Huang, Steve S.; Gibney, Brian R.; Stayrook, Steven E. ...
Journal of molecular biology,
02/2003, Letnik:
326, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Maquettes are
de novo designed mimicries of nature used to test the construction and engineering criteria of oxidoreductases. One type of scaffold used in maquette construction is a four-α-helical ...bundle. The sequence of the four-α-helix bundle maquettes follows a heptad repeat pattern typical of left-handed coiled-coils. Initial designs were molten globular due partly to the minimalist approach taken by the designers. Subsequent iterative redesign generated several structured scaffolds with similar heme binding properties. Variant I
6F
13
2, a structured scaffold, was partially resolved with NMR spectroscopy and found to have a set of mobile inter-helical packing interfaces. Here, the X-ray structure of a similar peptide (I
6F
13M
31
2 i.e. {CGGG EIWKL HEEFLKK FEELLKL HEERLKKM
2}
2 which we call L31M), has been solved using MAD phasing and refined to 2.8
Å resolution. The structure shows that the maquette scaffold is an anti-parallel four-helix bundle with “up–up–down–down” topology. No pre-formed heme-binding pocket exists in the protein scaffold. We report unexpected inter-helical crossing angles, residue positions and translations between the helices. The crossing angles between the parallel helices are −5° rather than the expected +20° for typical left-handed coiled-coils. Deviation of the scaffold from the design is likely due to the distribution and size of hydrophobic residues. The structure of L31M points out that four identical helices may interact differently in a bundle and heptad repeats with an alternating HPPHHPP/HPPHHPH (H: hydrophobic, P: polar) pattern are not a sufficient design criterion to generate left-hand coiled-coils.
Phospholamban (PLB) is a pentameric transmembrane protein that regulates the Ca
2+-dependent ATPase SERCA2a in sarcoplasmic reticulum membranes. We previously described the computational design of a ...water-soluble variant of phospholamban, WSPLB, which reproduced many of the structural and functional properties of the native membrane-soluble protein. While the full-length WSPLB forms a pentamer in solution, a truncated variant forms very stable tetramers. To obtain insight into the tetramer–pentamer cytoplasmic switch, we solved the crystal structure of the truncated construct, WSPLB 21-52. This peptide has a heptad sequence repeat with Leu residues at
a- and Ile at
d-positions from residues 31–52. The crystal structure revealed that WSPLB 21-52 adopted an antiparallel tetrameric coiled coil. This topology contrasts with the parallel topology of an analogue of the coiled-coil of GCN4 with the same Leu
a Ile
d repeat. Analysis of these structures revealed how the nature of the partially exposed residues at
e- and
g-positions influence the topology formed by the bundle. We also constructed a model for the pentameric form of PLB using the coiled-coil parameters derived from a single monomer in the tetrameric structure. This model suggests that both buried and interfacial hydrogen bonds are important for stabilizing the parallel pentamer.
Natural photosynthetic protein complexes capture sunlight to power the energetic catalysis that supports life on Earth. Yet these natural protein structures carry an evolutionary legacy of complexity ...and fragility that encumbers protein reengineering efforts and obfuscates the underlying design rules for light-driven charge separation. De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build new enzymes for efficient solar-to-fuel energy conversion. Here, we report the rational design, X-ray crystal structure, and electron transfer activity of a multi-cofactor protein that incorporates essential elements of photosynthetic reaction centers. This highly stable, modular artificial protein framework can be reconstituted in vitro with interchangeable redox centers for nanometer-scale photochemical charge separation. Transient absorption spectroscopy demonstrates Photosystem II-like tyrosine and metal cluster oxidation, and we measure charge separation lifetimes exceeding 100 ms, ideal for light-activated catalysis. This de novo-designed reaction center builds upon engineering guidelines established for charge separation in earlier synthetic photochemical triads and modified natural proteins, and it shows how synthetic biology may lead to a new generation of genetically encoded, light-powered catalysts for solar fuel production.
Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis by enhancing the delivery of substrate cholesterol from the outer mitochondrial membrane to the cholesterol side ...chain cleavage enzyme system on the inner membrane. A recombinant StAR protein lacking the first N-terminal 62 amino acid residues that includes the mitochondrial targeting sequence was shown to stimulate the transfer of cholesterol and β-sitosterol from liposomes to heat-treated mitochondria in a dose-, time-, and temperature-dependent manner. A recombinant mutant StAR protein that cannot stimulate steroidogenesis by isolated mitochondria did not promote sterol transfer. Unlike the more promiscuous lipid transfer protein, sterol carrier protein 2 (SCP2), StAR did not stimulate phosphatidylcholine transfer in our assay system. The recombinant StAR protein increased cholesterol transfer to heat-treated microsomes as well as to heat- and trypsin-treated mitochondria. These observations demonstrate that StAR has sterol transfer activity, which may reflect an ability to enhance desorption of cholesterol from sterol-rich donor membranes. We suggest that the ability of StAR to promote sterol transfer explains its steroidogenic activity.
Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with ...heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.
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