Search

In eukaryotes, many DNA/RNA-binding proteins possess intrinsically disordered regions (IDRs) with large negative charge, some of which involve a consecutive sequence of aspartate (D) or glutamate (E) residues. We refer to them as D/E repeats. The functional role of D/E repeats is not well understood, though some of them are known to cause autoinhibition through intramolecular electrostatic interaction with functional domains. In this work, we investigated the impacts of D/E repeats on the target DNA search kinetics for the high-mobility group box 1 (HMGB1) protein and the artificial protein constructs of the Antp homeodomain fused with D/E repeats of varied lengths. Our experimental data showed that D/E repeats of particular lengths can accelerate the target association in the overwhelming presence of non-functional high-affinity ligands (‘decoys’). Our coarse-grained molecular dynamics (CGMD) simulations showed that the autoinhibited proteins can bind to DNA and transition into the uninhibited complex with DNA through an electrostatically driven induced-fit process. In conjunction with the CGMD simulations, our kinetic model can explain how D/E repeats can accelerate the target association process in the presence of decoys. This study illuminates an unprecedented role of the negatively charged IDRs in the target search process.

DNA mismatch repair (MMR) is an important postreplication process that eliminates mispaired or unpaired nucleotides to ensure genomic replication fidelity. In humans, Msh2-Msh6 and Msh2-Msh3 are the two mismatch repair initiation factors that recognize DNA lesions. While X-ray crystal structures exist for these proteins in complex with DNA lesions, little is known about their structures during the initial search along nonspecific double-stranded DNA, because they are short-lived and difficult to determine experimentally. In this study, various computational approaches were used to sidestep these difficulties. All-atom and coarse-grained simulations based on the crystal structures of Msh2-Msh3 and Msh2-Msh6 showed no translation along the DNA, suggesting that the initial search conformation differs from the lesion-bound crystal structure. We modeled probable search-mode structures of MSH proteins and showed, using coarse-grained molecular dynamics simulations, that they can perform rotation-coupled diffusion on DNA, which is a suitable and efficient search mechanism for their function and one predicted earlier by fluorescence resonance energy transfer and fluorescence microscopy studies. This search mechanism is implemented by electrostatic interactions among the mismatch-binding domain (MBD), the clamp domains, and the DNA backbone. During simulations, their diffusion rate did not change significantly with an increasing salt concentration, which is consistent with observations from experimental studies. When the gap between their DNA-binding clamps was increased, Msh2-Msh3 diffused mostly via the clamp domains while Msh2-Msh6 still diffused using the MBD, reproducing the experimentally measured lower diffusion coefficient of Msh2-Msh6. Interestingly, Msh2-Msh3 was capable of dissociating from the DNA, whereas Msh2-Msh6 always diffused on the DNA duplex. This is consistent with the experimental observation that Msh2-Msh3, unlike Msh2-Msh6, can overcome obstacles such as nucleosomes. Our models provide a molecular picture of the different mismatch search mechanisms undertaken by Msh2-Msh6 and Msh2-Msh3, despite the similarity of their structures.

Lassa virus (LASV) is a notorious human pathogen in West Africa. Its class I trimeric spike complex displays a distinct architecture, and its cell entry mechanism involves unique attributes not shared by other related viruses. We determined the crystal structure of the GP2 fusion glycoprotein from the spike complex of LASV (GP2LASV) in its post-fusion conformation. GP2LASV adopts a canonical helical bundle configuration similarly to other viruses in its family. The core packing of GP2LASV, however, is more organized compared to GP2 from other viruses reducing the formation of internal hydrophobic cavities. We demonstrate a link between the formation of such unfavorable hydrophobic cavities and the efficiencies of membrane fusion and cell entry. Our study suggests that LASV has evolved a more efficient membrane fusogen compared to other viruses from its family by optimizing the post-fusion configuration of its GP2 module.

Highly negatively charged segments containing only aspartate or glutamate residues (“D/E repeats”) are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40–900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.

DNA-binding proteins rely on linear diffusion along the longitudinal DNA axis, supported by their nonspecific electrostatic affinity for DNA, to search for their target recognition sites. One may therefore expect that the ability to engage in linear diffusion along DNA is universal to all DNA-binding proteins, with the detailed biophysical characteristics of that diffusion differing between proteins depending on their structures and functions. One key question is whether the linear diffusion mechanism is defined by translation coupled with rotation, a mechanism that is often termed sliding. We conduct coarse-grained and atomistic molecular dynamics simulations to investigate the minimal requirements for protein sliding along DNA. We show that coupling, while widespread, is not universal. DNA-binding proteins that slide along DNA transition to uncoupled translation-rotation (i.e., hopping) at higher salt concentrations. Furthermore, and consistently with experimental reports, we find that the sliding mechanism is the less dominant mechanism for some DNA-binding proteins, even at low salt concentrations. In particular, the toroidal PCNA protein is shown to follow the hopping rather than the sliding mechanism.

Several DNA-binding proteins, such as topoisomerases, helicases and sliding clamps, have a toroidal (i.e. ring) shape that topologically traps DNA, with this quality being essential to their function. Many DNA-binding proteins that function, for example, as transcription factors or enzymes were shown to be able to diffuse linearly (i.e. slide) along DNA during the search for their target binding sites. The protein's sliding properties and ability to search DNA, which often also involves hopping and dissociation, are expected to be different when it encircles the DNA. In this study, we explored the linear diffusion of four ring-shaped proteins of very similar structure: three sliding clamps (PCNA, β-clamp, and the gp45) and the 9-1-1 protein, with a particular focus on PCNA. Coarse-grained molecular dynamics simulations were performed to decipher the sliding mechanism adopted by these ring-shaped proteins and to determine how the molecular properties of the inner and outer ring govern its search speed. We designed in silico variants to dissect the contributions of ring geometry and electrostatics to the sliding speed of ring-shaped proteins along DNA. We found that the toroidal proteins diffuse when they are tilted relative to the DNA axis and able to rotate during translocation, but that coupling between rotation and translocation is quite weak. Their diffusion speed is affected by the shape of the inner ring and, to a lesser extent, by its electrostatic properties. However, breaking the symmetry of the electrostatic potential can result in deviation of the DNA from the center of the ring and cause slower linear diffusion. The findings are discussed in light of earlier computational and experimental studies on the sliding of clamps.

Glycosylation plays not only a functional role but can also modify the biophysical properties of the modified protein. Usually, natural glycosylation results in protein stabilization; however, in vitro and in silico studies showed that sometimes glycosylation results in thermodynamic destabilization. Here, we applied coarse-grained and all-atom molecular dynamics simulations to understand the mechanism underlying the loss of stability of the MM1 protein by glycosylation. We show that the origin of the destabilization is a conformational distortion of the protein caused by the interaction of the monosaccharide with the protein surface. Though glycosylation creates new short-range glycan-protein interactions that stabilize the conjugated protein, it breaks long-range protein-protein interactions. This has a destabilizing effect because the probability of long- and short-range interactions forming differs between the folded and unfolded states. The destabilization originates not from simple loss of interactions but due to a trade-off between the short- and long-range interactions.

The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 has been produced in and isolated from whole Trichoplusia ni larvae. The recombinant protein was crystallized and its structure was solved to 2.2 A resolution. The results indicate that the larvae-produced enzyme is essentially identical to that isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies.

Organophosphate compounds (OP) are potent inhibitors of acetylcholinesterases (AChEs) and can cause lethal poisoning in humans. Inhibition of AChEs by the OP soman involves phosphonylation of the catalytic serine, and subsequent dealkylation produces a form known as the "aged" enzyme. The nonaged form can be reactivated to a certain extent by nucleophiles, such as pralidoxime (2-PAM), whereas aged forms of OP-inhibited AChEs are totally resistant to reactivation. Here, we solved the X-ray crystal structures of AChE from Torpedo californica (TcAChE) conjugated with soman before and after aging. The absolute configuration of the soman stereoisomer adduct in the nonaged conjugate is P(S)C(R). A structural reorientation of the catalytic His440 side chain was observed during the aging process. Furthermore, the crystal structure of the ternary complex of the aged conjugate with 2-PAM revealed that the orientation of the oxime function does not permit nucleophilic attack on the phosphorus atom, thus providing a plausible explanation for its failure to reactivate the aged soman/AChE conjugate. Together, these three crystal structures provide an experimental basis for the design of new reactivators.

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 2A 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.

The X-ray crystal structures were solved for complexes with Torpedo californica acetylcholinesterase of two bivalent tacrine derivative compounds in which the two tacrine rings were separated by 5- and 7-carbon spacers. The derivative with the 7-carbon spacer spans the length of the active-site gorge, making sandwich interactions with aromatic residues both in the catalytic anionic site (Trp84 and Phe330) at the bottom of the gorge and at the peripheral anionic site near its mouth (Tyr70 and Trp279). The derivative with the 5-carbon spacer interacts in a similar manner at the bottom of the gorge, but the shorter tether precludes a sandwich interaction at the peripheral anionic site. Although the upper tacrine group does interact with Trp279, it displaces the phenyl residue of Phe331, thus causing a major rearrangement in the Trp279-Ser291 loop. The ability of this inhibitor to induce large-scale structural changes in the active-site gorge of acetylcholinesterase has significant implications for structure-based drug design because such conformational changes in the target enzyme are difficult to predict and to model.

Recently, alkylene-linked heterodimers of tacrine (1) and 5-amino-5,6,7,8-tetrahydroquinolinone (2, hupyridone) were shown to exhibit higher acetylcholinesterase (AChE) inhibition than either monomeric 1 or 2. Such inhibitors are potential drug candidates for ameliorating the cognitive decrements in early Alzheimer patients. In an attempt to understand the inhibition mechanism of one such dimer, (RS)-(+/-)-N-9-(1,2,3,4-tetrahydroacridinyl)-N'-5-[5,6,7,8-tetrahydro-2'(1'H)-quinolinonyl]-1,10-diaminodecane [(RS)-(+/-)-3] bisoxalate, the racemate was soaked in trigonal Torpedo californica AChE (TcAChE) crystals, and the X-ray structure of the resulting complex was solved to 2.30 A resolution. Its structure revealed the 1 unit bound to the "anionic" subsite of the active site, near the bottom of the active-site gorge, as seen for the 1/TcAChE complex. Interestingly, only the (R)-enantiomer of the 2 unit was seen in the peripheral "anionic" site (PAS) at the top of the gorge, and was hydrogen-bonded to the side chains of residues belonging to an adjacent, symmetry-related AChE molecule covering the gorge entrance. When the same racemate was soaked in orthorhombic crystals of TcAChE, in which the entrance to the gorge is more exposed, the crystal structure of the corresponding complex revealed no substantial enantiomeric selectivity. This observation suggests that the apparent enantiomeric selectivity of trigonal crystals of TcAChE for (R)-3 is mainly due to crystal packing, resulting in preferential binding of one enantiomeric inhibitor both to its "host" enzyme and to its neighbor in the asymmetric unit, rather than to steric constraints imposed by the geometry of the active-site gorge.

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.

Bifunctional derivatives of the alkaloid galanthamine, designed to interact with both the active site of the enzyme acetylcholinesterase (AChE) and its peripheral cation binding site, have been assayed with Torpedo californica AChE (TcAChE), and the three-dimensional structures of their complexes with the enzyme have been solved by X-ray crystallography. Differences were noted between the IC(50) values obtained for TcAChE and those for Electrophorus electricus AChE. These differences are ascribed to sequence differences in one or two residues lining the active-site gorge of the enzyme. The binding of one of the inhibitors disrupts the native conformation of one wall of the gorge, formed by the loop Trp279-Phe290. It is proposed that flexibility of this loop may permit the binding of inhibitors such as galanthamine, which are too bulky to penetrate the narrow neck of the gorge formed by Tyr121 and Phe330 as seen in the crystal structure.

Acetylcholinesterase (AChE) inhibitors improve the cognitive abilities of Alzheimer patients. (-)-Huperzine A [(-)-HupA], an alkaloid isolated from the club moss, Huperzia serrata, is one such inhibitor, but the search for more potent and selective drugs continues. Recently, alkylene-linked dimers of 5-amino-5,6,7,8-tetrahydroquinolinone (hupyridone, 1a), a fragment of HupA, were shown to serve as more potent inhibitors of AChE than (-)-HupA and monomeric 1a. We soaked two such dimers, (S,S)-(-)-bis(10)-hupyridone [(S,S)-(-)-2a] and (S,S)-(-)-bis(12)-hupyridone [(S,S)-(-)-2b] containing, respectively, 10 and 12 methylenes in the spacer, into trigonal TcAChE crystals, and solved the X-ray structures of the resulting complexes using the difference Fourier technique, both to 2.15 A resolution. The structures revealed one HupA-like 1a unit bound to the "anionic" subsite of the active-site, near the bottom of the active-site gorge, adjacent to Trp84, as seen for the TcAChE/(-)-HupA complex, and the second 1a unit near Trp279 in the "peripheral" anionic site at the top of the gorge, both bivalent molecules thus spanning the active-site gorge. The results confirm that the increased affinity of the dimeric HupA analogues for AChE is conferred by binding to the two "anionic" sites of the enzyme. Inhibition data show that (-)-2a binds to TcAChE approximately 6-7- and > 170-fold more tightly than (-)-2b and (-)-HupA, respectively. In contrast, previous data for rat AChE show that (-)-2b binds approximately 3- and approximately 2-fold more tightly than (-)-2a and (-)-HupA, respectively. Structural comparison of TcAChE with rat AChE, as represented by the closely related mouse AChE structure (1maa.pdb), reveals a narrower gorge for rat AChE, a perpendicular alignment of the Tyr337 ring to the gorge axis, and its conformational rigidity, as a result of hydrogen bonding between its hydroxyl group and that of Tyr341, relative to TcAChE Phe330. These structural differences in the active-site gorge explain the switch in inhibitory potency of (-)-2a and 2b and the larger dimer/(-)-HupA potency ratios observed for TcAChE relative to rat AChE. The results offer new insights into factors affecting protein-ligand complementarity within the gorge and should assist the further development of improved AChE inhibitors.

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.

Chimeras of tacrine and m-(N,N,N-Trimethylammonio)trifluoroacetophenone (1) were designed as novel, reversible inhibitors of acetylcholinesterase. On the basis of the X-ray structure of the apoenzyme, a molecular modeling study determined the favored attachment positions on the 4-aminoquinoline ring (position 3 and the 4-amino nitrogen) and the favored lengths of a polymethylene link between the two moieties (respectively 5-6 and 4-5 sp(3) atoms). Seven compounds matching these criteria were synthesized, and their inhibitory potencies were determined to be in the low nanomolar range. Activity data for close analogues lacking some of the postulated key features showed that our predictions were correct. In addition, a subsequent crystal structure of acetylcholinesterase complexed with the most active compound 27 was in good agreement with our model. The design strategy is therefore validated and can now be developed further.

The structure of Torpedo californica acetylcholinesterase (AChE) was examined in com- plex with several inhibitors which are either in use or under development for treatment of Alzheimer's disease. The inhibitors vary greatly in their structures, offering differing starting points for future drug design. The structure of T. californica AChE is also compared to the recently solved structure of Droso- phila melanogaster AChE, the first invertebrate enzyme to have its structure determined. The structure of D. melanogaster AChE complexed with a potential insecticide is described. Drug Dev. Res. 50:573-583, 2000. © 2000 Wiley-Liss, Inc.

(−)-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 Alzheimer’s disease. We have solved the X-ray crystal structure of GAL bound in the active site of Torpedo californica acetylcholinesterase (TcAChE) to 2.3 A 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 A) 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.

Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the “peripheral” anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4′-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.

Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements.

A procedure is presented for controlling the rate of evaporation during ‘microbatch under oil’ protein crystallization., Microbatch crystallization under oil is a powerful procedure for obtaining protein crystals. Using this method, aqueous protein solutions are dispensed under liquid oil, and water evaporates through the layer of oil, with a concomitant increase in the concentrations of both protein and precipitant until the nucleation point is reached. A technique is presented for regulating the rate of water evaporation, which permits fine tuning of the crystallization conditions as well as preventing complete desiccation of the drops in the microbatch crystallization trays.

β-Secretase (β-site amyloid precursor protein-cleaving enzyme 1; BACE1) is a transmembrane aspartic protease that cleaves the β-amyloid precursor protein en route to generation of the amyloid β-peptide (Aβ) that is believed to be responsible for the Alzheimer's disease amyloid cascade. It is thus a prime target for the development of inhibitors which may serve as drugs in the treatment and/or prevention of Alzheimer's disease. In the following determination of the crystal structures of both apo and complexed BACE1, structural analysis of all crystal structures of BACE1 deposited in the PDB and molecular dynamics (MD) simulations of monomeric and `dimeric' BACE1 were used to study conformational changes in the active-site region of the enzyme. It was observed that a flap able to cover the active site is the most flexible region, adopting multiple conformational states in the various crystal structures. Both the presence or absence of an inhibitor within the active site and the crystal packing are shown to influence the flap's conformation. An open conformation of the flap is mostly observed in the apo structures, while direct hydrogen-bonding interaction between main-chain atoms of the flap and the inhibitor is a prerequisite for the flap to adopt a closed conformation in the crystal structures of complexes. Thus, a systematic study of the conformational flexibility of the enzyme may not only contribute to structure-based drug design of BACE1 inhibitors and of other targets with flexible conformations, but may also help to better understand the mechanistic events associated with the binding of substrates and inhibitors to the enzyme.

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 A resolution. This structure provides important information pertinent to the design of novel functional ERα targeted probes for clinical applications.

A bis-(-)-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and Abeta peptide aggregation. The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 A resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the "anionic" subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440N(epsilon2), which is hydrogen-bonded to Ser200O(gamma) in the native enzyme. The second nor-meptazinol binds at the peripheral "anionic" site at the gorge entrance. A number of GOLD models of the complex, using both native TcAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure. These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors.

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.

Gaucher disease is caused by mutations in the gene encoding acid beta-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 2A 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 (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.

An extracellular alpha-type carbonic anhydrase (dCAII) from the salt-tolerant alga Dunaliella salina differs from its mesophilic counterparts in remaining active from zero to multimolar salt concentrations. To gain insight into the outstanding salt tolerance of dCAII, the enzyme was functionally overexpressed in Escherichia coli, purified by affinity chromatography and crystallized by the hanging-drop method. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 119.9, c = 58.5 A, beta = 94.2 degrees. Data from a single crystal were collected to 2.4 A resolution under cryogenic conditions (120 K) using an R-AXIS IV(++) detector mounted on a Rigaku RU-H3R rotating-anode generator. The asymmetric unit contains two molecules of the protein, which corresponds to V(M) = 2.65 A(3) Da(-1) and a solvent content of 52.7%.

The structure of Torpedo californica acetylcholinesterase is examined in complex with several inhibitors that are either in use or under development for treating Alzheimer's 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.

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.

E2020 is a potent inhibitor of acetylcholinesterase (AChE), displaying high selectivity for AChE relative to butyrylcholinesterase. It was recently licensed under the trade name of Aricept by the FDA for use in the management of Alzheimer’s disease. A series of compounds related to E2020 were synthesized and shown to serve as good inhibitors of Torpedo californica acetylcholinesterase (TcAChE). Several of these inhibitors were soaked into TcAChE crystals, and the three-dimensional structures of the complexes obtained were solved by X-ray crystallography. As might be expected from their elongated structures the ligands bind along the axis of the active-site gorge, interacting with the peripheral anionic site (Trp279), as well as with various other aromatic groups along the length of the gorge. The benzyl-piperidine group binds in the area of Trp84, the residue which binds the quaternary ammonium cation of acetylcholine. Inhibitors in which a trifluoroketo group had been added to the phenyl ring formed a covalent bond with the Oγ of the active site serine, Ser200. It is hoped that these studies will clarify which interactions are most important for tight inhibitor binding, and thus permit the design of more effective and selective insecticides which will simultaneously be acutely toxic for their insect targets and relatively harmless to other animals.

Proteins often form crystals of different shapes and sizes, depending on crystallisation conditions such as pH, temperature, concentration and nature of precipitant, and the presence or absence of additives. In addition to the macroscopic variety and the quality of diffraction of the crystals, different crystal forms show important differences in the packing of protein molecules. It was noted by Axelsen et al. [1] that, due to crystal contacts, the entrance to the active-site gorge of every monomer in the trigonal crystal form of Torpedo californica acetyl-cholinesterase (TcAChE) is tightly blocked by a symmetry-related molecule.

Organophosphates (OP) are potent transition state (TS) inhibitors which react rapidly with acetylcholinesterase (AChE), and then may undergo an internal dealkylation to produce an irreversibly inhibited, “aged” OP-enzyme conjugate. To understand the structural basis for the stability of aged enzyme, we crystallized and solved the X-ray structures of conjugates obtained by reaction of Torpedo californica (Tc) AChE with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman). After reaction with OP, unbound inhibitor was removed by gel filtration and aging was allowed to proceed to >90% completion. Aged OP-TcAChE was crystallized using PEG-200 and MES buffer at pH 5.8. X-ray data were collected using trigonal crystals, and refined using difference Fourier techniques at 2.2A (DFP), 2.5A (sarin), and 2.2A (soman) resolution. In each structure, the highest positive difference density peak, corresponding to the OP, was observed to be within covalent bonding distance of Ser200. All three structures suggest that the stability of aged AChE derives from interaction of the two resonance oxygen atoms attached to the phosphorus atom with catalytic subsites of the enzyme. Based upon the geometry of the refined structures, we infer that backbone amides of the oxyanion hole (Gly118, Gly119 and Ala201) stabilize one oxygen by hydrogen bonding, while the His440 imidazolium holds the other oxygen in a salt bridge. The conformations of the active sites of aged sarin- and soman-TcAChE are essentially identical and provide structural models for the rate-limiting deacylation TS that occurs during enzyme hydrolysis of the natural substrate, acetylcholine.

A low molecular weight protein inhibitor of serine proteinases from Russet Burbank potato tubers, polypeptide chymotrypsin inhibitor-1 (PCI-1), has been crystallized in complex with Streptomyces griseus proteinase B (SGPB). The three-dimensional structure of the complex has been solved at 2.1 A resolution by the molecular replacement method and has been refined to a final R-factor (= Σ∥F o ¦−¦F c ∥ Σ¦F o ¦ ) of 0.142 (8.0 to 2.1 A resolution data). The reactive site bond of PCI-1 (Leu38I to Asn39I) is intact in the complex, and there is no significant distortion of the peptide from planarity. The distance between the active site serine Oγ of SGPB and the carbonyl carbon of the scissile bond of PCI-1 is 2.8 A (1 A = 0.1 nm). The inhibitor has little secondary structure, having a three-stranded anti-parallel β-sheet on the side opposite the reactive site and four β-turns. PCI-1 has four disulphide bridges; these presumably take the place of extensive secondary structure in keeping the reactive site conformationally constrained. The pairing of the cystine residues, which had not been characterized chemically, is as follows: Cys3I to Cys40I, Cys6I to Cys24I, Cys7I to Cys36I, and Cys13I to Cys49I. The molecular structure of SGPB in the PCI-1 complex agrees closely with the structure of SGPB complexed with the third domain of the turkey ovomucoid inhibitor (OMTKY3). A least-squares overlap of all atoms in SGPB gives a root-mean-square difference of 0.37 A. One of the loops of SGPB (Ser35 to Gly40) differs in conformation in the two complexes by more than 2.0 A root-mean-square for the main-chain atoms. Thr39 displays the largest differences with the carbonyl carbon atom deviating by 3.6 A. This conformational alternative is a result of the differences in the molecular structures of the P′4 residues following the reactive site bonds of the two inhibitors. This displacement avoids a close contact (1.3 A) between the carbonyl oxygen of Ser38 of SGPB and Pro42I Cβ of PCI-1. The solvent structure of the PCI-1-SGPB complex includes 179 waters, two sulphate or phosphate ions, and one calcium or potassium ion, which appears to play a role in crystal formation. The molecular structure of PCI-1 determined here has allowed the proposal of a model for the structure of a two-domain inhibitor from potatoes and tomatoes, inhibitor II. The two domains of the model are related by a pseudo-2-fold axis between the three-stranded β-sheet of each domain.