Proteins Do Not Have Strong Spines After All

In this issue of Structure, Berkholz et al. show that the detailed backbone geometry of proteins depends on the local conformation and suggest how this information can be practically used in modeling and refining protein structures

A model of the ternary complex of interleukin-10 with its soluble receptors

BACKGROUND: Interleukin-10 (IL-10) is a cytokine whose main biological function is to suppress the immune response by induction of a signal(s) leading to inhibition of synthesis of a number of cytokines and their cellular receptors. Signal transduction is initiated upon formation of a ternary complex of IL-10 with two of its receptor chains, IL-10R1 and IL-10R2, expressed on the cell membrane. The affinity of IL-10R1 toward IL-10 is very high, which allowed determination of the crystal structure of IL-10 complexed with the extracellular/soluble domain of IL-10R1, while the affinity of IL-10R2 toward either IL-10 or IL-10/sIL-10R1 complex is quite low. This so far has prevented any attempts to obtain structural information about the ternary complex of IL-10 with its receptor chains. RESULTS: Structures of the second soluble receptor chain of interleukin-10 (sIL-10R2) and the ternary complex of IL-10/sIL-10R1/sIL-10R2 have been generated by homology modeling, which allowed us to identify residues involved in ligand-receptor and receptor-receptor interactions. CONCLUSION: The previously experimentally determined structure of the intermediate/binary complex IL-10/sIL-10R1 is the same in the ternary complex. There are two binding sites for the second receptor chain on the surface of the IL-10/sIL-10R1 complex, involving both IL-10 and sIL-10R1. Most of the interactions are hydrophilic in nature, although each interface includes two internal hydrophobic clusters. The distance between C-termini of the receptor chains is 25 Å, which is common for known structures of ternary complexes of other cytokines. The structure is likely to represent the biologically active signaling complex of IL-10 with its receptor on the surface of the cell membrane

Impact of synchrotron radiation on macromolecular crystallography: a personal view

This article, largely based on personal experiences of the authors, reviews the early history of the application of synchrotron radiation to structural biology, and particularly protein crystallography, to show the tremendous impact that this experimental innovation has had on these disciplines

A left-handed crossover involved in amidohydrolase catalysis Crystal structure of Erwinia chrysanthemi l-asparaginase with bound l-aspartate

AbstractThe crystal structure of l-asparaginase from Erwinia chrysanthemi in the presence and absence of l-aspartate was determined at 1.8 Å resolution. Conserved residues in a left-handed crossover (a rare occurrence in protein structures) link pairs of dimers into the catalytically active tetrameric form of the enzyme. The structure of ErA containing bound aspartic acid shows that this unusual strand connectivity is an essential part of the active site architecture, responsible for releasing the product of the enzymatic hydrolysis. The orientation of the bound aspartate indicates for the first time a threonine residue as a catalytic nucleophile

The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from cis-splicing inteins

Abstract Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 Å-resolution crystal structure of a fused variant from the naturally split gp41-1 intein, previously identified from environmental metagenomic sequence data. The structure of the 125-residue gp41-1 intein revealed a compact pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N- and C-terminal intein fragments that are common among naturally occurring split inteins. We successfully created orthogonal split inteins by engineering a similar charge network into the same region of a cis-splicing intein. This strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins.Peer reviewe

Is too ‘creative’ language acceptable in crystallography?

A brief comment is made on the need to use carefully selected, novel terms in crystallographic publications, especially publications addressing non-specialists

A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases

BACKGROUND: Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases). In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted. RESULTS: We have analyzed the presence of orthologs of human CLN2 in the genomic sequences of a number of eukaryotic species. Enzymes with sequences sharing over 80% identity have been found in the genomes of macaque, mouse, rat, dog, and cow. Closely related, although clearly distinct, enzymes are present in fish (fugu and zebra), as well as in frogs (Xenopus tropicalis). A three-dimensional model of human CLN2 was built based mainly on the homology with Pseudomonas sp. 101 sedolisin. CONCLUSION: CLN2 is very highly conserved and widely distributed among higher organisms and may play an important role in their life cycles. The model presented here indicates a very open and accessible active site that is almost completely conserved among all known CLN2 enzymes. This result is somehow surprising for a tripeptidase where the presence of a more constrained binding pocket was anticipated. This structural model should be useful in the search for the physiological substrates of these enzymes and in the design of more specific inhibitors of CLN2

X-ray crystallographic structure of a complex between a synthetic protease of human immunodeficiency virus 1 and a substrate-based hydroxyethylamine inhibitor

The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor JG-365 is Ac-Ser-Leu-Asn-Phe-ψ[CH(OH)CH_2N]-Pro-Ile-Val-OMe; the K_i is 0.24 nM. The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, is believed to mimic a tetrahedral reaction intermediate. The structure of the complex has been refined to an R factor of 0.146 at 2.4-Å resolution by using restrained least squares with rms deviations in bond lengths of 0.02 Å and bond angles of 4. The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is positioned between the active site aspartate carboxyl groups within hydrogen bonding distance. Comparison of this structure with a reduced peptide bond inhibitor-protease complex indicates that these contacts confer the exceptional binding strength of JG-365

Structural analysis and molecular substrate recognition properties of Arabidopsis thaliana ornithine transcarbamylase, the molecular target of phaseolotoxin produced by Pseudomonas syringae

Halo blight is a plant disease that leads to a significant decrease in the yield of common bean crops and kiwi fruits. The infection is caused by Pseudomonas syringae pathovars that produce phaseolotoxin, an antimetabolite which targets arginine metabolism, particularly by inhibition of ornithine transcarbamylase (OTC). OTC is responsible for production of citrulline from ornithine and carbamoyl phosphate. Here we present the first crystal structures of the plant OTC from Arabidopsis thaliana (AtOTC). Structural analysis of AtOTC complexed with ornithine and carbamoyl phosphate reveals that OTC undergoes a significant structural transition when ornithine enters the active site, from the opened to the closed state. In this study we discuss the mode of OTC inhibition by phaseolotoxin, which seems to be able to act only on the fully opened active site. Once the toxin is proteolytically cleaved, it mimics the reaction transition state analogue to fit inside the fully closed active site of OTC. Additionally, we indicate the differences around the gate loop region which rationally explain the resistance of some bacterial OTCs to phaseolotoxin