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PubMed2005

Inhibitors of tripeptidyl peptidase II. 3. Derivation of butabindide by successive structure optimizations leading to a potential general approach to designing exopeptidase inhibitors.

The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase that has been shown to be a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the Met-Gly bond to give Asp-Tyr(SO3H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH2. Starting from Val-Pro-NHBu, a dipeptide of submicromolar affinity that had previously been generated to serve as a lead, successive optimization at P3, P1, and then P2 gave Abu-Pro-NHBu (18, Ki = 80 nM). Further transformation (by making a benzologue) gave the indoline analogue, butabindide (33) as a reversible inhibitor having nanomolar affinity (Ki = 7 nM). Retrospective analysis suggested the possibility of a general approach to designing exopeptidase inhibitors starting from the structure of the first hydrolysis product. Application of this approach to CCK-8 led to Abu-Phe-NHBu (37), but this only had Ki = 9.4 microM. Molecular modeling, to determine the minimum energy conformations and explain the 1000-fold better affinity of butabindide, indicated that 37 cannot access the likely active conformation of butabindide.

Aminopeptidases↗
PubMed2000

Trypsin sheds light on the singular case of seminal RNase, a dimer with two quaternary conformations.

Dimeric seminal RNase presents the singular case of a dimer with access at equilibrium to two conformations: one in which the subunits exchange, or swap, their NH(2)-terminal arms; the other with no exchange. Thus a continuous unfolding/refolding of structural elements into two alternative conformations takes place in the native protein at equilibrium. The phenomenon was investigated by kinetic and mass spectrometric analyses of the effects of trypsin on the native protein, on its isolated quaternary forms, as well as on a monomeric derivative of the protein and on homologous dimeric RNase A. The kinetics of tryptic action on the protein forms and on the protein derivatives, as well as the location of the tryptic cleavage sites, and their chronological sequence, led to the identification of relevant interconversion intermediates, to the description of a model for the interconversion process, and to a hypothesis for the unique phenomenon of the dual quaternary conformation of seminal RNase.

Animals↗
PubMed2005

X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease.

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.

Binding Sites↗
PubMed1975

Single strand conformation of adenylate chains analysed by a specific photoreaction. Determination of structure by 5' residue.

The photoreactivity was analysed in various oligo- and polyadenylates: 1) The quantum yields of the specific photoreaction in poly(dA) and dApdA decrease with increasing temperature, whereas the quantum yield of the photodegradation in poly(rA) increases. 2) The photoreactivities of poly(2'MeA) and poly(2'EtA) closely correspond to that of poly(rA). 3) The photodegradation of rApdA is very similar to that of rAprA, whereas dAprA shows the same specific photoreaction as observed for dApdA. These data support the view, that the specific photoreaction observed in oligo(dA) and poly(dA) is dependent upon a specific conformation, which is not accessible to oligo(rA), poly(rA), poly(2'MeA) and poly(2'EtA). The specific conformation is determined by the nucleotide, which carries the internucleotide bond in the 3'-position.

Adenine Nucleotides↗
PubMed1998

Slow inactivation does not affect movement of the fast inactivation gate in voltage-gated Na+ channels.

Voltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (microl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (hinfinity) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

Animals↗
PubMed2006

Conformational analysis of methyl 5-O-methyl septanosides: effect of glycosylation on conformer populations.

Methyl 5-O-methyl-alpha-d-glycero-d-idoseptanoside (3) and methyl 5-O-methyl-beta-d-glycero-d-guloseptanoside (4) were investigated as (1-->5)-linked di-/oligoseptanoside mimetics. Here we report the synthesis of 3 and 4 and describe their preferred solution conformations through a combination of ab initio/DFT calculations and (1)H (3)J(H,H) NMR coupling constant analysis. The conformations of 3 and 4 observed in this study are discussed in comparison to those of the parent (C5 hydroxy) compounds 1 and 2. The results indicate that methyl 5-O-methyl-alpha-septanoside 3 is relatively rigid and adopts the same (3,4)TC(5,6) conformation as 1. Methyl 5-O-methyl-beta-septanoside 4 is somewhat less rigid than its parent septanoside (2). In addition to the (6,O)TC(4,5) conformation adopted by 2, beta-septanoside 4 also populates the adjacent (3,4)TC(5,6) conformation. Glycosylation at C5 on beta-septanoside 4 therefore increases its overall flexibility and allows access to alternative ring conformations.

Carbohydrate Conformation↗
PubMed1997

Access to phosphorylation in isocitrate dehydrogenase may occur by domain shifting.

To clarify further the mechanism of regulation by phosphorylation of isocitrate dehydrogenase, cocrystallization of isocitrate dehydrogenase and isocitrate dehydrogenase kinase/phosphatase in the presence of an ATP analog was attempted. Although cocrystallization was unsuccessful, a new crystal form of isocitrate dehydrogenase was obtained which provides insight into the phosphorylation mechanism. The new, orthorhombic crystal form of isocitrate dehydrogenase is related to the previously reported tetragonal form largely by an approximately 16 degrees shift of a large domain relative to the small domain and clasp region within each subunit of the dimeric enzyme. The NADP+ cofactor binding surface is significantly disrupted by the shift to the open conformation. The solvent-accessible surface area and surface-enclosed volume increase by 2% relative to the dimeric tetragonal form. Most of the increase results from expansion of the active site cleft such that the distance across its opening increases from approximately 5 to 13 A, significantly increasing accessibility to Ser-113. The conformation of isocitrate dehydrogenase in the orthorhombic crystal form more closely resembles that of the crystal structure of the homologous enzyme 3-isopropylmalate dehydrogenase than does the tetragonal isocitrate dehydrogenase conformation. Since the crystal lattice forces are fairly weak, it appears that isocitrate dehydrogenase is a flexible molecule that can easily undergo domain shifts and possibly other induced fit conformational changes, to accommodate binding to isocitrate dehydrogenase kinase/phosphatase.

Binding Sites↗
PubMed2002

Can we separate active from inactive conformations?

Molecular modeling methodologies such as molecular docking, pharmacophore modeling, and 3D-QSAR, rely on conformational searches of small molecules as a starting point. All of these methodologies seek conformations of the small molecules as they bind to target proteins, i.e., their active conformations. Thus the question as to whether active conformations can be separated from inactive conformations is extremely relevant. In this paper, 3D-descriptors that separate random conformations from active conformations of small molecules are sought. To select appropriate descriptors, 65 protein-ligand complexes were taken from the protein data bank. For each ligand the active conformation was compared to randomly generated low energy conformations. Descriptors such as solvent accessible surface area, number of internal interactions and radius of gyration appear to be useful for separating the active conformations from the random conformations. The results with all these descriptors indicate that active conformations are less compact that random conformations, i.e., they have more solvent accessible surface area, fewer internal interactions and a larger radius of gyration than random conformations. Thus these descriptors could be useful as weights to bias conformational search procedures to conformations more likely to bind to proteins or as filters to eliminate conformations unlikely to bind to any protein.

Computer Simulation↗
PubMed1994

Accessibility of nucleic acid-complexed biomolecules to hydroxyl radicals correlates with their conformation: a fluorescence polarization spectroscopy study.

A fluorescence methodology has been developed to examine the relationship between the conformational state of specific biomolecules in simple chromatin models and their accessibility to hydroxyl radicals (OH). Polylysine and histone H1 were labelled with SECCA, the succinimidyl ester of coumarin-3-carboxylic acid, which generates the fluorescent derivative 7-OH-SECCA following its interaction with radiation-induced OH in aqueous solution. The fluorescence induced per unit gamma-ray dose reflecting the accessibility of OH to such SECCA-conjugated biomolecules was recorded. The biomolecules were also labelled with the fluorescent derivative 7-OH-SECCA in trace amounts to study their conformation under identical conditions via fluorescence polarization spectroscopy. When these biomolecules were complexed with a polynucleotide or DNA, a major increase in polarization anisotropy was recorded. Upon salt-induced dissociation of these biomolecules from the nucleic acids, the increase in anisotropy was reversed. The histone H1-DNA complexes also exhibited an initial increase in anisotropy with increasing NaCl concentration (maximum at 100 mmol dm-3) indicating the possible formation of a more compact conformation. The fluctuations in anisotropy were inversely proportional to the recorded fluorescence/Gy. The data indicate a direct correlation between the accessibility of OH to polylysine or histone H1 complexed with nucleic acids and the conformation of these biomolecules.

DNA↗
PubMed2000

Local movement in the S2 region of the voltage-gated potassium channel hKv2.1 studied using cysteine mutagenesis.

The positively charged S4 region of voltage-dependent potassium channels moves outward during depolarization, leading to channel opening, but possible movement of the negatively charged S2 region may be more complex. Here we have studied possible movement of the S2 region of the slowly activating human voltage-dependent potassium channel hKv2.1. For this, cysteine mutants in the S2 region were expressed in Xenopus oocytes by injection of cRNA. Whole-cell currents were measured using the two-electrode voltage-clamp technique, and the effect of the membrane-impermeable cysteine-binding reagent parachloromercuribenzenesulfonate (PCMBS) was studied. For mutant S223C (located just outside the membrane in the S2 region), PCMBS inhibited currents and caused faster deactivation of tail currents. The time course of reactivity of PCMBS on tail current amplitudes was faster at more negative holding potentials. There was no effect of PCMBS on potassium channel currents for mutants D225C, N226C, A230C, and V232C. These data suggest that residue S223 is exposed to the extracellular phase at normal resting potentials, making it accessible to PCMBS, but upon depolarization there is a conformational change, making it less accessible, possibly by a local rather than global movement of S2 residues into the membrane. Voltage-dependent movements of nearby residues could also explain the results.

4-Chloromercuribenzenesulfonate↗
PubMed2004

Conformation of gp120 determines the sensitivity of HIV-1 DH012 to the entry inhibitor IC9564.

The HIV-1 envelope glycoprotein gp120 is the key determinant for the anti-HIV-1 entry activity of IC9564. A T198P mutation in the gp120 of the HIV-1 primary isolate, DH012, drastically increases IC9564 sensitivity, which can be reversed by growing the virus in the presence of IC9564. The reversed resistant variants contain a P198S mutation that fully confers the drug-resistant phenotype. Although the amino acid residue at position 198 of gp120 can alter IC9564 sensitivity, results from this study suggest that T198 is not the direct target of the compound. The mutation at position 198 appears to affect the conformation of gp120 and subsequently decreases the accessibility of the drug target. This conformational effect is evidenced by the fact that the T198P mutation significantly increases the neutralizing activity of the conformational antibodies, 1b12 and 48d. On the other hand, the IC9564 escape variant with the P198S mutation is resistant to these conformational antibodies and highly sensitive to the potent neutralizing antiserum, C1206, which recognizes a conformational epitope involving the sequences from V1, V2, and V3 regions in gp120. Thus, results from this study indicate that the conformation of gp120 can be exploited by HIV-1 to escape IC9564.

Animals↗
PubMed1999

Electrostatic and aromatic microdomains within the binding-site crevice of the D2 receptor: contributions of the second membrane-spanning segment.

The binding-site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted cysteine accessibility method (SCAM), we previously mapped the residues in the third, fifth, sixth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the second membrane-spanning segment (M2) and expressed the mutant receptors in HEK 293 cells. Eleven of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and 9 of these 11 were protected from reaction by a reversible dopamine antagonist, sulpiride. We infer that the side chains of the residues at the 11 reactive loci (D80, L81, V83, V87, P89, W90, V91, V92, L94, E95, V96) are on the water-accessible surface of the binding-site crevice and that 9 of these are occluded by bound antagonist. The pattern of accessibility suggests an alpha-helical conformation. A broadening of the angle of accessibility near the binding site is consistent with the presence of a kink at Pro89. On the basis of the enhanced rates of reaction of positively charged sulfhydryl reagents, we infer the presence of an electrostatic microdomain composed of three acidic residues in M2 and the adjacent M3 that could attract and orient cationic ligands. Furthermore, based on the enhanced reactivity of the hydrophobic cation-containing reagent, we infer the presence of an aromatic microdomain formed between M2, M3, and M7.

Amino Acid Substitution↗
PubMed2004

Release factors eRF1 and RF2: a universal mechanism controls the large conformational changes.

Class I release factors 1 and 2 (RF1 and RF2) terminate protein synthesis by recognizing stop codons on the mRNA via their conserved amino acid motifs (NIKS in eRF1 and SPF in RF2) and by the conserved tripeptide (GGQ) interactions with the ribosomal peptidyltransferase center. Crystal structures of eRF1 and RF2 do not fit their ribosomal binding pocket (approximately 73 angstroms). Cryoelectron microscopy indicates large conformational changes in the ribosome-bound RF2. Here, we investigate the conformational dynamics of the eRF1 and RF2 using molecular dynamics simulation, structural alignment, and electrostatic analysis of domain interactions. We show that relaxed eRF1 has a shape remarkably similar to the ribosome-bound RF2 observed by cryoelectron microscopy. The similarity between the two release factors is as good as between elongation factor G and elongation factor Tu-guanosine-5'(beta,gamma-imido)triphosphate-tRNA. Further, the conformational transitions and dynamics of eRF1 and RF2 between the free and ribosome-bound states are most likely controlled by protonation of conserved histidines. For eRF1, the distance between the NIKS and GGQ motifs shrinks from 97.5 angstroms in the crystal to 70-80 angstroms. For RF2, the separation between SPF and GGQ elongates from 32 angstroms in the crystal to 50 angstroms. Coulombic interaction strongly favors the open conformation of eRF1; however, solvation and histidine protonation modulate the domain interactions, making the closed conformation of eRF1 more accessible. Thus, RF1 and RF2 function like molecular machines, most likely fueled by histidine protonation. The unified conformational control and the shapes of eRF1 and RF2 support the proposition that the termination of protein synthesis involves similar mechanisms across species.

Amino Acid Motifs↗
PubMed1989

Molecular aspects of heavy-chain class switching.

Ig heavy chain class switching results from recombination that specifically joins together tandemly repeated sequences called S-regions. S-regions are located 5' of every CH gene except C delta. Recombination between S mu and a downstream S-region results in the juxtaposition of the rearranged variable region genes with C gamma, C epsilon, or C alpha genes and expression of the new isotype. Switch recombination is probably mediated by specific recombinase(s) that may be involved in other types of Ig-gene recombination. The efficiency and regulation of class-switch recombination is influenced by a number of factors, both structural and functional. Structurally, S-regions are all composed of tandemly repeated sequences that contain common pentamer sequences, but they differ in their length and their degree of sequence similarity with S mu. The mechanism of switching appears to be influenced by the accessibility of the S-region to recombinase(s). The conformational changes that result in accessibility are unknown but are correlated with transcription and hypomethylation of DNA near S-regions. Torsional stress resulting from progression of RNA polymerase through a GC-rich S-region could serve to further distort the DNA conformation and open the duplex, thus making it available for DNA strand invasion and recombinase activity. Although the mechanistic events of S-S recombination remain to be elucidated, switching is clearly influenced by protein mediators (mitogens and interleukins) which induce differential transcription of S regions.

Animals↗
PubMedDate not supplied

Dehydration: a new alcohol theory.

A central belief about ethanol is that it acts mainly by partitioning into the lipid bilayer of membranes. Newer ideas focus on the neuronal synapse and suggest that ethanol can allosterically change protein conformation, as is suggested by studies on GABA-receptor-mediated chloride uptake and on (Na(+)-K+)-ATPase. Several studies from my laboratory suggest that ethanol enhances enzymatic cleavage of sialic acid (SA) from gangliosides, and perhaps also glycoproteins, but does so without stimulating enzyme activity, suggesting conformational changes that affect accessibility. I propose a new model for the cell membrane in the synaptic region, which features gangliosides surrounding membrane proteins, with an interspersed film of water creating hydrogen bonds that anchor SA moieties to membrane protein. I believe that we should consider the possibility that an important action of ethanol, and polar anesthetics, is due to hydrophilic, not hydrophobic, properties and the ability to dehydrate the cell-surface microdomain. Our laboratory has recently advanced the theory that ethanol dehydrates a "solvent regulatory site" of membrane (Na(+)-K+)-ATPase. This principle might be extended to other enzymes and receptor proteins, as well as to the accessibility of sialoglycoconjugates to sialidase (neuraminidase). Hydrogen bonding between SA and polar regions of receptor protein, and the conformation on both imposed by it, would surely be changed by minor degrees of dehydration and substitution of alcohol molecules for water. Ethanol, unlike water, can only hydrogen bond "at one end." Displacement of water by ethanol would not only "free" the SA groups and make them more vulnerable to enzymatic cleavage but also could simultaneously change the conformation of receptor protein. Similarly, ethanol may displace water that links the polar heads of phospholipids to polar portions of receptors proteins. Ethanol may have an even more important and direct effect of substituting for hydrogen-bonded water within protein itself.

Body Water↗
PubMed1993

Molecular mechanism of two noncompetitive inhibitors of Na(+)-glucose cotransporter: comparison of DCCD and PCMB.

The effects of noncompetitive inhibitors of Na(+)-dependent glucose uptake, p-chloromercuribenzoate N,N'-dicyclohexylcarbodiimide (DCCD), on substrate-induced cotransporter conformational changes were examined using fluorescein isothiocyanate (FITC) and tryptophan fluorescence. p-chloromercuribenzoate (PCMB) inhibited both substrate-induced conformational changes with similar concentration required for 50% quenching/enhancement of tryptophan or FITC fluorescence. In contrast, DCCD inhibited the Na(+)-induced conformational change with an apparent concentration resulting in 50% inhibition (K0.5) of 18 microM and the glucose-induced conformational change with an apparent K0.5 of 5 microM. DCCD slightly increased the apparent K0.5 for the Na+ concentration required for Na(+)-induced conformational change. DCCD and PCMB altered tryptophan accessibility to quench reagents in all three conformations. Tryptophan residues on the PCMB-treated cotransporter were more Cs+ than I- sensitive in contrast to the unlabeled cotransporter. The PCMB-treated cotransporter had a reduced response to Na+, suggesting that the mode of PCMB inactivation of cotransporter activity resulted from conformational changes in the substrate-free cotransporter. DCCD had a smaller effect on cotransporter tryptophan quench reagent susceptibility. However, DCCD-labeled cotransporter was equally accessible to I- and Cs+, and the DCCD-labeled cotransporter did not respond to substrates. Loss of charge distribution around cotransporter tryptophans correlated with loss of substrate-induced conformational changes.

Chloromercuribenzoates↗
PubMed2000

Millisecond time scale conformational flexibility in a hyperthermophile protein at ambient temperature.

Rubredoxin from the hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10(-6) s(-1) at 100 degrees C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the approximately millisecond time frame or faster at 28 degrees C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 10(4) and 10(6), for which EX(2) exchange kinetics were directly verified, have exchange activation energy values within 2-3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of hyperthermophile proteins is not supported by these data.

Archaeal Proteins↗