Yves Bourne

Structures of Aplysia Achbp Complexes with Nicotinic Agonists and Antagonists Reveal Distinctive Binding Interfaces and Conformations.

Upon ligand binding at the subunit interfaces, the extracellular domain of the nicotinic acetylcholine receptor undergoes conformational changes, and agonist binding allosterically triggers opening of the ion channel. The soluble acetylcholine‐binding protein (AChBP) from snail has been shown to be a structural and functional surrogate of the ligand‐binding domain (LBD) of the receptor. Yet, individual AChBP species display disparate affinities for nicotinic ligands. The crystal structure of AChBP from Aplysia californica in the apo form reveals a more open loop C and distinctive positions for other surface loops, compared with previous structures. Analysis of Aplysia AChBP complexes with nicotinic ligands shows that loop C, which does not significantly change conformation upon binding of the antagonist, methyllycaconitine, further opens to accommodate the peptidic antagonist, α‐conotoxin ImI, but wraps around the agonists lobeline and epibatidine. The structures also reveal extended and nonoverlapping interaction surfaces for the two antagonists, outside the binding loci for agonists. This comprehensive set of structures reflects a dynamic template for delineating further conformational changes of the LBD of the nicotinic receptor.

Glycoside hydrolases and glycosyltransferases: families and functional modules.

The past year has witnessed the expected increase in the number of solved structures of glycoside hydrolases and glycosyltransferases, and their constitutive modules. These structures show that, while glycoside hydrolases display an extraordinary variety of folds, glycosyltransferases and carbohydrate-binding modules appear to belong to a much smaller number of folding families.

Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site

The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20–2.35 Å resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non‐catalytic heterologous protein associations.

Acetylcholinesterase inhibition by fasciculin: crystal structure of the complex.

The crystal structure of the snake toxin fasciculin, bound to mouse acetylcholinesterase (mAChE), at 3.2 A resolution reveals a synergistic three-point anchorage consistent with the picomolar dissociation constant of the complex. Loop II of fasciculin contains a cluster of hydrophobic residues that interact with the peripheral anionic site of the enzyme and sterically occlude substrate access to the catalytic site. Loop I fits in a crevice near the lip of the gorge to maximize the surface area of contact of loop II at the gorge entry. The fasciculin core surrounds a protruding loop on the enzyme surface and stabilizes the whole assembly. Upon binding of fasciculin, subtle structural rearrangements of AChE occur that could explain the observed residual catalytic activity of the fasciculin-enzyme complex.

Crystal structures of the bovine beta4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose.

β1,4‐galactosyltransferase T1 (β4Gal‐T1, EC 2.4.1.90/38), a Golgi resident membrane‐bound enzyme, transfers galactose from uridine diphosphogalactose to the terminal β‐N‐acetylglucosamine residues forming the poly‐N‐acetyllactosamine core structures present in glycoproteins and glycosphingolipids. In mammals, β4Gal‐T1 binds to α‐lactalbumin, a protein that is structurally homologous to lyzozyme, to produce lactose. β4Gal‐T1 is a member of a large family of homologous β4galactosyltransferases that use different types of glycoproteins and glycolipids as substrates. Here we solved and refined the crystal structures of recombinant bovine β4Gal‐T1 to 2.4 Å resolution in the presence and absence of the substrate uridine diphosphogalactose. The crystal structure of the bovine substrate‐free β4Gal‐T1 catalytic domain showed a new fold consisting of a single conical domain with a large open pocket at its base. In the substrate‐bound complex, the pocket encompassed residues interacting with uridine diphosphogalactose. The structure of the complex contained clear regions of electron density for the uridine diphosphate portion of the substrate, where its β‐phosphate group was stabilized by hydrogen‐bonding contacts with conserved residues including the Asp252ValAsp254 motif. These results help the interpretation of engineered β4Gal‐T1 point mutations. They suggest a mechanism possibly involved in galactose transfer and enable identification of the critical amino acids involved in α‐lactalbumin interactions.

Crystal structure of a Cbtx–AChBP complex reveals essential interactions between snake α-neurotoxins and nicotinic receptors

The crystal structure of the snake long α‐neurotoxin, α‐cobratoxin, bound to the pentameric acetylcholine‐binding protein (AChBP) from Lymnaea stagnalis, was solved from good quality density maps despite a 4.2 Å overall resolution. The structure unambiguously reveals the positions and orientations of all five three‐fingered toxin molecules inserted at the AChBP subunit interfaces and the conformational changes associated with toxin binding. AChBP loops C and F that border the ligand‐binding pocket move markedly from their original positions to wrap around the tips of the toxin first and second fingers and part of its C‐terminus, while rearrangements also occur in the toxin fingers. At the interface of the complex, major interactions involve aromatic and aliphatic side chains within the AChBP binding pocket and, at the buried tip of the toxin second finger, conserved Phe and Arg residues that partially mimic a bound agonist molecule. Hence this structure, in revealing a distinctive and unpredicted conformation of the toxin‐bound AChBP molecule, provides a lead template resembling a resting state conformation of the nicotinic receptor and for understanding selectivity of curaremimetic α‐neurotoxins for the various receptor species.

Crystal Structure and Mutational Analysis of the Human CDK2 Kinase Complex with Cell Cycle–Regulatory Protein CksHs1

The 2.6 Angstrom crystal structure for human cyclin-dependent kinase 2(CDK2) in complex with CksHs1, a human homolog of essential yeast cell cycle-regulatory proteins suc1 and Cks1, reveals that CksHs1 binds via all four beta strands to the kinase C-terminal lobe. This interface is biologically critical, based upon mutational analysis, but far from the CDK2 N-terminal lobe, cyclin, and regulatory phosphorylation sites. CDK2 binds the Cks single domain conformation and interacts with conserved hydrophobic residues plus His-60 and Glu-63 in their closed beta-hinge motif conformation. The beta hinge opening to form the Cks beta-interchanged dimer sterically precludes CDK2 binding, providing a possible mechanism regulating CDK2-Cks interactions. One face of the complex exposes the sequence-conserved phosphate-binding region on Cks and the ATP-binding site on CDK2, suggesting that CKs may target CDK2 to other phosphoproteins during the cell cycle.

Bacterial glycosidases for the production of universal red blood cells.

Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the α-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily

N‐acetylglucosamine 1‐phosphate uridyltransferase (GlmU) is a cytoplasmic bifunctional enzyme involved in the biosynthesis of the nucleotide‐activated UDP‐GlcNAc, which is an essential precursor for the biosynthetic pathways of peptidoglycan and other components in bacteria. The crystal structure of a truncated form of GlmU has been solved at 2.25 Å resolution using the multiwavelength anomalous dispersion technique and its function tested with mutagenesis studies. The molecule is composed of two distinct domains connected by a long α‐helical arm: (i) an N‐terminal domain which resembles the dinucleotide‐binding Rossmann fold; and (ii) a C‐terminal domain which adopts a left‐handed parallel β‐helix structure (LβH) as found in homologous bacterial acetyltransferases. Three GlmU molecules assemble into a trimeric arrangement with tightly packed parallel LβH domains, the long α‐helical linkers being seated on top of the arrangement and the N‐terminal domains projected away from the 3‐fold axis. In addition, the 2.3 Å resolution structure of the GlmU–UDP‐GlcNAc complex reveals the structural bases required for the uridyltransferase activity. These structures exemplify a three‐dimensional template for the development of new antibacterial agents and for studying other members of the large family of XDP‐sugar bacterial pyrophosphorylases.

High-throughput automated refolding screening of inclusion bodies

One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no “universal” refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high‐throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96‐well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large‐scale purification tests, and five gave crystals.