Patrick Van Roey

Study of the Structural Dynamics of the E. coli 70S Ribosome Using Real-Space Refinement

Cryo-EM density maps showing the 70S ribosome of E. coli in two different functional states related by a ratchet-like motion were analyzed using real-space refinement. Comparison of the two resulting atomic models shows that the ribosome changes from a compact structure to a looser one, coupled with the rearrangement of many of the proteins. Furthermore, in contrast to the unchanged inter-subunit bridges formed wholly by RNA, the bridges involving proteins undergo large conformational changes following the ratchet-like motion, suggesting an important role of ribosomal proteins in facilitating the dynamics of translation.

Catalytic domain structure and hypothesis for function of GIY-YIG intron endonuclease I-TevI.

I-TevI, a member of the GIY-YIG family of homing endonucleases, consists of an N-terminal catalytic domain and a C-terminal DNA-binding domain joined by a flexible linker. The GIY-YIG motif is in the N-terminal domain of I-TevI, which corresponds to a phylogenetically widespread catalytic cartridge that is often associated with mobile genetic elements. The crystal structure of the catalytic domain of I-TevI, the first of any GIY-YIG endonuclease, reveals a novel α/β-fold with a central three-stranded antiparallel β-sheet flanked by three helices. The most conserved and putative catalytic residues are located on a shallow, concave surface and include a metal coordination site. Similarities in the three-dimensional arrangement of the catalytically important residues and the cation-binding site with those of the His-Cys box endonuclease I-PpoI suggest the possibility of mechanistic relationships among these different families of homing endonucleases despite completely different folds.

Family 18 chitinase-oligosaccharide substrate interaction: subsite preference and anomer selectivity of Serratia marcescens chitinase A.

The sizes and anomers of the products formed during the hydrolysis of chitin oligosaccharides by the Family 18 chitinase A (ChiA) from Serratia marcescens were analysed by hydrophilic interaction chromatography using a novel approach in which reactions were performed at 0 degrees C to stabilize the anomer conformations of the initial products. Crystallographic studies of the enzyme, having the structure of the complex of the ChiA E315L (Glu315-->Leu) mutant with a hexasaccharide, show that the oligosaccharide occupies subsites -4 to +2 in the substrate-binding cleft, consistent with the processing of beta-chitin by the release of disaccharide at the reducing end. Products of the hydrolysis of hexa- and penta-saccharides by wild-type ChiA, as well as by two mutants of the residues Trp275 and Phe396 important in binding the substrate at the +1 and +2 sites, show that the substrates only occupy sites -2 to +2 and that additional N -acetyl-D-glucosamines extend beyond the substrate-binding cleft at the reducing end. The subsites -3 and -4 are not used in this four-site binding mode. The explanation for these results is found in the high importance of individual binding sites for the processing of short oligosaccharides compared with the cumulative recognition and processive hydrolysis mechanism used to digest natural beta-chitin.

Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate

I‐TevI is a site‐specific, sequence‐tolerant intron endonuclease. The crystal structure of the DNA‐binding domain of I‐TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn finger, an elongated segment containing a minor groove‐binding α‐helix, and a helix–turn–helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove‐binding helix and the helix–turn–helix subdomain make hydrophobic contacts, the few base‐specific hydrogen bonds occur in segments that lack secondary structure and flank the intron insertion site. The multiple base‐specific interactions over a long segment of the substrate are consistent with the observed high site specificity in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases.

Structural Biology of Human Follitropin and Its Receptor

In this review, the current understanding of structure-activity relationships of human follitropin and of the extracellular domain of its receptor is described. Comprehensive mutagenesis of human follitropin combined with the three-dimensional structure of human follitropin has ushered in a new era of understanding of how this complex hormone binds to and activates its receptor. Comparison of human choriogonadotropin and follitropin structures has proved invaluable in understanding how these human glycoprotein hormones have conserved primary sequence that enables high affinity binding while diverging in amino acids that provide specificity. Moreover, by comparison of the structures of deglycosylated and glycosylated human choriogonadotropin and glycosylated human follitropin, there appears to be no influence of oligosaccharides upon backbone conformation of human glycoprotein hormones. Extensive structure-activity relationships of human follitropin receptor have been studied, and new insights gained here as well. These studies indicate that follitropin binds to the central module of the extracellular domain of the follitropin receptor. Biophysical analyses of purified follitropin receptor extracellular domain further revealed conformational changes affected by hormone binding and by the solvent environment. Further, secondary structure analysis of the purified extracellular domain of follitropin receptor favors the leucine-rich repeat motif model of the glycoprotein hormone receptors. Together, the studies indicate that there are only a few residues that contribute to the overall energy of binding. Formation of a weak collisional complex between follitropin and its receptor likely involves complementation of compatible surfaces and steric hindrance by oligosaccharides, followed by conformational change and formation of active site residue salt bridges. In this regard and in light of these new data, current models of the glycoprotein hormone receptors may need to be re-evaluated.

Crystal structure of a deletion mutant of human thymidylate synthase Delta (7-29) and its ternary complex with Tomudex and dUMP.

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 Å and 2.5 Å resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild‐type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 Å away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten‐fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.

Zinc finger as distance determinant in the flexible linker of intron endonuclease I-TevI

I-TevI, the phage T4 td intron-encoded endonuclease, recognizes a lengthy DNA target and initiates intron mobility by introducing a double-strand break in the homing site. The enzyme uses both sequence and distance determinants to cleave the DNA 23–25 bp upstream of the intron insertion site. I-TevI consists of an N-terminal catalytic domain and a C-terminal DNA-binding domain separated by a long, flexible linker. The DNA-binding domain consists of three subdomains: a zinc finger, a minor-groove binding α-helix, and a helix–turn–helix. In this study, a mutational analysis was undertaken to assess the roles of these subdomains in substrate binding and cleavage. Surprisingly, the zinc finger is not required for DNA binding or catalysis. Rather, the zinc finger is a component of the linker and directs the catalytic domain to cleave the homing site at a fixed distance from the intron insertion site. When the cleavage site (CS) is shifted outside a given range, wild-type I-TevI defaults to the fixed distance, whereas zinc-finger mutants have lost the distance determinant and search out the displaced cleavage sequences. Although counterintuitive, a protein containing a 19-aa deletion of the zinc finger can extend further than can wild-type I-TevI to cleave a distant CS sequence, and a Cys-to-Ala mutant of the ligands for zinc, nominally a longer protein, can retract to cleave at a closer CS sequence. Models are presented for the novel function of the zinc finger, as a molecular constraint, whereby intramolecular protein–protein interactions position the catalytic domain by “catalytic clamp” and/or “linker-organizer” mechanisms.

Crystal structure of endo-β-N-acetylglucosaminidase H at 1.9 å resolution: active-site geometry and substrate recognition

BACKGROUND Endo-beta-N-acetylglucosaminidase H (Endo H), an endoglycosidase secreted by Streptomyces plicatus, hydrolyzes the glycosidic bond between the core N-acetyglucosamine residues of asparagine-linked high-mannose oligosaccharides. Endo H is a commonly used reagent in glycobiology research, including the characterization of oligosaccharides in glycoproteins. On-going crystallographic studies of Endo H and related endoglycosidases are aimed at identifying the molecular features that determine the different substrate specificities of these enzymes. RESULTS The three-dimensional structure of Endo H has been determined to 1.9 A resolution. The overall fold of the enzyme is that of an irregular (alpha/beta)8-barrel comprising eight beta-strand/loop/alpha-helix units. Units 5 and 6 have very short loop sections at the top of the molecule and their alpha-helices are replaced by sections of extended geometry. The loop of unit 2 includes a small two-stranded antiparallel beta-sheet. A shallow curved cleft runs across the surface of the molecule from the area of units 5 and 6, over the core of the beta-barrel to the area of the beta-sheet of loop 2. This cleft contains the putative catalytic residues Asp130 and Glu132 above the core of the beta-barrel. These residues are surrounded by several aromatic residues. The loop 2 area of the cleft is formed by neutral polar residues, mostly asparagines. CONCLUSIONS The structure of Endo H is very similar to that of Endo F1, a closely related endoglycosidase secreted by Flavobacterium meningosepticum. Detailed comparison of the structures of Endo H and Endo F1 supports the model previously proposed for substate binding and recognition, in which the area of loop 2 determines the substrate specificity and the alpha-helices of units 5 and 6 are missing to accommodate the protein moiety of the substrate.

Intron-encoded homing endonuclease I-TevI also functions as a transcriptional autorepressor

Customary binding sites of intron-encoded homing endonucleases lie within cognate intronless alleles, at the so-called homing sites. Here, we describe a novel, high-affinity binding site for I-TevI endonuclease, encoded within the group I td intron of phage T4. This site is an operator that overlaps the T4 late promoter, which drives I-TevI expression from within the td intron. I-TevI binds the operator and homing sites with equal affinity, and functions as a transcriptional autorepressor. Distinct sequence and spacing requirements of the catalytic domain result in reduced cleavage activity on operator DNA. Crystallographic studies showed that the overall interactions of the DNA-binding domain with the operator and homing sites are similar, but have some different hydrogen-bonding contacts. We present a model in which the flexibility in protein-DNA interactions allows I-TevI to bind variant intronless alleles to promote intron mobility while facilitating its function in autorepression, and thereby persistence in its host.

A conserved threonine spring-loads precursor for intein splicing.

Protein splicing is an autocatalytic process where an “intein” self‐cleaves from a precursor and ligates the flanking N‐ and C‐“extein” polypeptides. Inteins occur in all domains of life and have myriad uses in biotechnology. Although the reaction steps of protein splicing are known, mechanistic details remain incomplete, particularly the initial peptide rearrangement at the N‐terminal extein/intein junction. Recently, we proposed that this transformation, an N‐S acyl shift, is accelerated by a localized conformational strain, between the inteins catalytic cysteine (Cys1) and the neighboring glycine (Gly‐1) in the N‐extein. That proposal was based on the crystal structure of a catalytically competent trapped precursor. Here, we define the structural origins and mechanistic relevance of the conformational strain using a combination of quantum mechanical simulations, mutational analysis, and X‐ray crystallography. Our results implicate a conserved, but largely unstudied, threonine residue of the Ssp DnaE intein (Thr69) as the mediator of conformational strain through hydrogen bonding. Further, the strain imposed by this residue is shown to position the splice junction in a manner that enhances the rate of the N‐S acyl shift substantially. Taken together, our results not only provide fundamental understanding of the control of the first step of protein splicing but also have important implications in various biotechnological applications that require precursor manipulation.