Anastassios C. Papageorgiou

Molecular recognition of human angiogenin by placental ribonuclease inhibitor—an X-ray crystallographic study at 2.0 Å resolution

Human placental RNase inhibitor (hRI), a leucine‐rich repeat protein, binds the blood vessel‐inducing protein human angiogenin (Ang) with extraordinary affinity (Ki <1 fM). Here we report a 2.0 Å resolution crystal structure for the hRI–Ang complex that, together with extensive mutagenesis data from earlier studies, reveals the molecular features of this tight interaction. The hRI–Ang binding interface is large and encompasses 26 residues from hRI and 24 from Ang, recruited from multiple domains of both proteins. However, a substantial fraction of the energetically important contacts involve only a single region of each: the C‐terminal segment 434–460 of hRI and the ribonucleolytic active centre of Ang, most notably the catalytic residue Lys40. Although the overall docking of Ang resembles that observed for RNase A in the crystal structure of its complex with the porcine RNase inhibitor, the vast majority of the interactions in the two complexes are distinctive, indicating that the broad specificity of the inhibitor for pancreatic RNase superfamily proteins is based largely on its capacity to recognize features unique to each of them. The implications of these findings for the development of small, hRI‐based inhibitors of Ang for therapeutic use are discussed.

Microbial superantigens: from structure to function.

Superantigens are highly potent immune stimulators with a unique ability to interact simultaneously with MHC class II molecules and T cell receptors, forming a trimolecular complex that induces profound T-cell proliferation and massive cytokine production. Recent structural studies have provided a wealth of information regarding these complex interactions, and it is now emerging that, despite their common 3-D architecture, superantigens are able to crosslink MHC class II molecules and T cell receptors in a variety of ways.

Structural basis for the recognition of superantigen streptococcal pyrogenic exotoxin A (SpeA1) by MHC class II molecules and T-cell receptors

Streptococcal pyrogenic exotoxin A (SpeA) is a superantigen produced by Streptococcus pyogenes and is associated with severe infections characterized by rash, hypotension, multiorgan failure and a high mortality rate. In this study, an allelic form of this toxin, SpeA1, was crystallized with four molecules in the crystallographic asymmetric unit and its crystal structure was determined at 2.6 Å resolution. The crystallographic R‐factor was 19.4% (33 497 reflections) for 7031 protein atoms and 88 water molecules. The overall structure of SpeA1 is considerably similar to that of other prototype microbial superantigens, either of staphylococcal or streptococcal origin, but has greatest similarity to staphylococcal enterotoxin C (SEC). Based on structural and mutagenesis data, we have mapped several important residues on the toxin molecule, which are involved in the recognition of major histocompatibility complex (MHC) class II molecules and T‐cell receptors. Also, the toxin appears to possess a potential zinc‐binding site which may have implications in binding to particular MHC class II molecules. Finally, we propose models for SpeA1–MHC class II and SpeA1–T‐cell receptor association and the relevance of this phenomenon to the superantigenic action of this toxin is considered.

Dps-like proteins: structural and functional insights into a versatile protein family

Dps-like proteins are key factors involved in the protection of prokaryotic cells from oxidative damage. They act by either oxidizing iron to prevent the formation of oxidative radicals or by forming Dps-DNA complexes to physically protect DNA. All Dps-like proteins are characterized by a common three-dimensional architecture and are found as spherical dodecamers with a hollow central cavity. Despite their structural similarities, recent biochemical and structural data have suggested different functions among members of the family that range from protection inside the cells in response to various stress signals to adhesion and virulence during bacterial infections. Moreover, the Dps-like proteins have lately attracted considerable interest in the field of nanotechnology owing to their ability to act as protein cages for iron and various other metals. A better understanding of their function and mechanism could therefore lead to novel applications in biotechnology and nanotechnology.

Crystal Structures of Oligomeric Forms of the Ip-10/Cxcl10 Chemokine

We have determined the structure of wild-type IP-10 from three crystal forms. The crystals provide eight separate models of the IP-10 chain, all differing substantially from a monomeric IP-10 variant examined previously by NMR spectroscopy. In each crystal form, IP-10 chains form conventional beta sheet dimers, which, in turn, form a distinct tetrameric assembly. The M form tetramer is reminiscent of platelet factor 4, whereas the T and H forms feature a novel twelve-stranded beta sheet. Analytical ultracentrifugation indicates that, in free solution, IP-10 exists in a monomer-dimer equilibrium with a dissociation constant of 9 microM. We propose that the tetrameric structures may represent species promoted by the binding of glycosaminoglycans. The binding sites for several IP-10-neutralizing mAbs have also been mapped.

Role of conserved residues in structure and stability: Tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up‐and‐down β‐barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo‐RBP. Crystal structures of recombinant human apo‐RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 Å and 2.0 Å resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the β‐barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by ideying spectroscopic signals for monitoring the formation of different substructures.

Crystallographic and functional characterization of the fluorodifen-inducible glutathione transferase from Glycine max reveals an active site topography suited for diphenylether herbicides and a novel L-site.

Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 A resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrophobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.

Plant GSTome: structure and functional role in xenome network and plant stress response

Glutathione transferases (GSTs) represent a major group of detoxification enzymes. All plants possess multiple cytosolic GSTs, each of which displays distinct catalytic as well as non-catalytic binding properties. The progress made in recent years in the fields of genomics, proteomics and protein crystallography of GSTs, coupled with studies on their molecular evolution, diversity and substrate specificity has provided new insights into the function of these enzymes. In plants, GSTs appear to be implicated in an array of different functions, including detoxification of xenobiotics and endobiotics, primary and secondary metabolism, stress tolerance, and cell signalling. This review focuses on plant GSTome and attempts to give an overview of its catalytic and functional role in xenome and plant stress regulatory networks.

Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme.

Angiotensin I-converting enzyme (ACE) is a highly glycosylated type I integral membrane protein. A series of underglycosylated testicular ACE (tACE) glycoforms, lacking between one and five N-linked glycosylation sites, were used to assess the role of glycosylation in tACE processing, crystallization and enzyme activity. Whereas underglycosylated glycoforms showed differences in expression and processing, their kinetic parameters were similar to that of native tACE. N-glycosylation of Asn-72 or Asn-109 was necessary and sufficient for the production of enzymically active tACE but glycosylation of Asn-90 alone resulted in rapid intracellular degradation. All mutants showed similar levels of phorbol ester stimulation and were solubilized at the same juxtamembrane cleavage site as the native enzyme. Two mutants, tACEDelta36-g1234 and -g13, were successfully crystallized, diffracting to 2.8 and 3.0 A resolution respectively. Furthermore, a truncated, soluble tACE (tACEDelta36NJ), expressed in the presence of the glucosidase-I inhibitor N -butyldeoxynojirimycin, retained the activity of the native enzyme and yielded crystals belonging to the orthorhombic P2(1)2(1)2(1) space group (cell dimensions, a=56.47 A, b=84.90 A, c=133.99 A, alpha=90 degrees, beta=90 degrees and gamma=90 degrees ). These crystals diffracted to 2.0 A resolution. Thus underglycosylated human tACE mutants, lacking O-linked oligosaccharides and most N-linked oligosaccharides or with only simple N-linked oligosaccharides attached throughout the molecule, are suitable for X-ray diffraction studies.

Strain Relief at the Active Site of Phosphoserine Aminotransferase Induced by Radiation Damage.

The X‐ray susceptibility of the lysine‐pyridoxal‐5′‐phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 Å resolution, complemented by on‐line spectroscopic studies. X‐rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal‐5′‐phosphate and the Lys residue. Analysis of the “undamaged” structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X‐rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 × 106 Gγ. Our data provide new insights into the enzymatic activation of pyridoxal‐5′‐phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites.