Harry M. Greenblatt

Structure of acetylcholinesterase complexed with (-)-galanthamine at 2.3 A resolution.

(−)‐Galanthamine (GAL), an alkaloid from the flower, the common snowdrop (Galanthus nivalis), shows anticholinesterase activity. This property has made GAL the target of research as to its effectiveness in the treatment of Alzheimers disease. We have solved the X‐ray crystal structure of GAL bound in the active site of Torpedo californica acetylcholinesterase (TcAChE) to 2.3 Å resolution. The inhibitor binds at the base of the active site gorge of TcAChE, interacting with both the choline‐binding site (Trp‐84) and the acyl‐binding pocket (Phe‐288, Phe‐290). The tertiary amine group of GAL does not interact closely with Trp‐84; rather, the double bond of its cyclohexene ring stacks against the indole ring. The tertiary amine appears to make a non‐conventional hydrogen bond, via its N‐methyl group, to Asp‐72, near the top of the gorge. The hydroxyl group of the inhibitor makes a strong hydrogen bond (2.7 Å) with Glu‐199. The relatively tight binding of GAL to TcAChE appears to arise from a number of moderate to weak interactions with the protein, coupled to a low entropy cost for binding due to the rigid nature of the inhibitor.

Structural insights into substrate traffic and inhibition in acetylcholinesterase

Acetylcholinesterase (AChE) terminates nerve‐impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active‐site gorge.

Crystal Structures of Complexes of N-Butyl- and N-Nonyl-Deoxynojirimycin Bound to Acid β-Glucosidase INSIGHTS INTO THE MECHANISM OF CHEMICAL CHAPERONE ACTION IN GAUCHER DISEASE

Gaucher disease is caused by mutations in the gene encoding acid β-glucosidase (GlcCerase), resulting in glucosylceramide (GlcCer) accumulation. The only currently available orally administered treatment for Gaucher disease is N-butyl-deoxynojirimycin (Zavesca™, NB-DNJ), which partially inhibits GlcCer synthesis, thus reducing levels of GlcCer accumulation. NB-DNJ also acts as a chemical chaperone for GlcCerase, although at a different concentration than that required to completely inhibit GlcCer synthesis. We now report the crystal structures, at 2Å resolution, of complexes of NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) with recombinant human GlcCerase, expressed in cultured plant cells. Both inhibitors bind at the active site of GlcCerase, with the imino sugar moiety making hydrogen bonds to side chains of active site residues. The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site where they undergo hydrophobic interactions. Based on these structures, we make a number of predictions concerning (i) involvement of loops adjacent to the active site in the catalytic process, (ii) the nature of nucleophilic attack by Glu-340, and (iii) the role of a conserved water molecule located in a solvent cavity adjacent to the active site. Together, these results have significance for understanding the mechanism of action of GlcCerase and the mode of GlcCerase chaperoning by imino sugars.

Acetylcholinesterase: a multifaceted target for structure-based drug design of anticholinesterase agents for the treatment of Alzheimer's disease.

The structure of Torpedo californica acetylcholinesterase is examined in complex with several inhibitors that are either in use or under development for treating Alzheimers disease. The noncovalent inhibitors vary greatly in their structures and bind to different sites of the enzyme, offering many different starting points for future drug design.

Flexibility of metal binding sites in proteins on a database scale.

Protein metal binding sites in the pre‐bound (apo) state, and their rearrangements upon metal binding were not analyzed previously at a database scale. Such a study may provide valuable information for metal binding site prediction and design. A high resolution, nonredundant dataset of 210 metal binding sites was created, containing all available representatives of apo–holo pairs for the most populated metals in the PDB. More than 40% of the sites underwent rearrangements upon metal binding. In 30 cases rearrangements involved the backbone. The tendency for side‐chain rearrangement inversely correlates with the number of first‐shell residues. Analysis of side‐chain reorientations as a result of metal binding showed that in 95% of the rigid‐backbone binding sites at most one side chain moved. Thus, in general, part of the first coordination shell is already in place in the pre‐bound form. The frequencies of side‐chain reorientation directly correlated with metal ligand flexibility and solvent accessibility in the apo state. Proteins 2005.

Criteria for Selecting PEGylation Sites on Proteins for Higher Thermodynamic and Proteolytic Stability

PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.

Refined structure of bovine carboxypeptidase A at 1.25 A resolution.

The crystal structure of the bovine zinc metalloproteinase carboxypeptidase A (CPA) has been refined to 1.25 A resolution based on room-temperature X-ray synchrotron data. The significantly improved structure of CPA at this resolution (anisotropic temperature factors, R factor = 10.4%, R(free) = 14.5%) allowed the modelling of conformational disorders of side chains, improved the description of the protein solvent network (375 water molecules) and provided a more accurate picture of the interactions between the active-site zinc and its ligands. The calculation of standard uncertainties in individual atom positions of the refined model of CPA allowed the deduction of the protonation state of some key residues in the active site and confirmed that Glu72 and Glu270 are negatively charged in the resting state of the enzyme at pH 7.5. These results were further validated by theoretical calculations that showed significant reduction of the pK(a) of these side chains relative to solution values. The distance between the zinc-bound solvent molecule and the metal ion is strongly suggestive of a neutral water molecule and not a hydroxide ion in the resting state of the enzyme. These findings could support both the general acid/general base mechanism, as well as the anhydride mechanism suggested for CPA.

Structure of Estradiol Metal Chelate and Estrogen Receptor Complex: The Basis for Designing a New Class of Selective Estrogen Receptor Modulators

Selective estrogen receptor modulators, such as 17β-estradiol derivatives bound to metal complexes, have been synthesized as targeted probes for the diagnosis and treatment of breast cancer. Here, we report the detailed 3D structure of estrogen receptor α ligand-binding domain (ERα-LBD) bound with a novel estradiol-derived metal complex, estradiol-pyridine tetra acetate europium(III), at 2.6 Å resolution. This structure provides important information pertinent to the design of novel functional ERα targeted probes for clinical applications.

Yeast Mpd1p Reveals the Structural Diversity of the Protein Disulfide Isomerase Family

Oxidoreductases belonging to the protein disulfide isomerase (PDI) family promote proper disulfide bond formation in substrate proteins in the endoplasmic reticulum. In plants and metazoans, new family members continue to be identified and assigned to various functional niches. PDI-like proteins typically contain tandem thioredoxin-fold domains. The limited information available suggested that the relative orientations of these domains may be quite uniform across the family, and structural models based on this assumption are appearing. However, the X-ray crystal structure of the yeast PDI family protein Mpd1p, described here, demonstrates the radically different domain orientations and surface properties achievable with multiple copies of the thioredoxin fold. A comparison of Mpd1p with yeast Pdi1p expands our perspective on the contexts in which redox-active motifs are presented in the PDI family.

Control of the rate of evaporation in protein crystallization by the 'microbatch under oil' method.

A procedure is presented for controlling the rate of evaporation during ‘microbatch under oil’ protein crystallization.