Construction of a family of Cys2His2 zinc binding sites in the hydrophobic core of thioredoxin by structure-based design

A semi-automated, rational design strategy has been used to introduce a family of seven single, mononuclear Cys2His2 zinc sites at various locations in the hydrophobic core of Escherichia colithioredoxin, a protein that is normally devoid of metal centers. The electronic absorption spectra of the CoII complexes show that five of these designed proteins bind metal with the intended tetrahedral geometry. The designed sites differ in their metal-binding constants and effects on protein stability. Since these designs are constructed within the same host protein framework, comparison of their behavior allows a qualitative evaluation of dominant factors that contribute to metal-binding and metal-mediated protein stabilization. Metal-binding constants are dominated by steric interactions between the buried, designed coordination sphere and the surrounding protein matrix. Metal-mediated stability is the consequence of differential binding to the native and unfolded states. Increased interactions with the unfolded state decrease the stabilizing effect of metal binding. The affinity for the unfolded state is dependent on the placement of the primary coordination sphere residues within the linear protein sequence. These results indicate that a protein fold can have a remarkably broad potential for accommodating metal-mediated cross-links and suggest strategies for engineering protein stability by constructing metal sites that maximize metal binding to the native state and minimize binding to the unfolded state.     

Evidence for common binding sites for ferrichrome compounds and bacteriophage phi 80 in the cell envelope of Escherichia coli

Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.     

Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease

Phage T5 exonuclease is a 5'-->3'exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.     

Information analysis of Fis binding sites

Originally discovered in the bacteriophage Mu DNA inversion system gin, Fis (Factor for Inversion Stimulation) regulates many genetic systems. To determine the base frequency conservation required for Fis to locate its binding sites, we collected a set of 60 experimentally defined wild-type Fis DNA binding sequences. The sequence logo for Fis binding sites showed the significance and likely kinds of base contacts, and these are consistent with available experimental data. Scanning with an information theory based weight matrix within fis, nrd, tgt/sec and gin revealed Fis sites not previously identified, but for which there are published footprinting and biochemical data. DNA mobility shift experiments showed that a site predicted to be 11 bases from the proximal Salmonella typhimurium hin site and a site predicted to be 7 bases from the proximal P1 cin site are bound by Fis in vitro. Two predicted sites separated by 11 bp found within the nrd promoter region, and one in the tgt/sec promoter, were also confirmed by gel shift analysis. A sequence in aldB previously reported to be a Fis site, for which information theory predicts no site, did not shift. These results demonstrate that information analysis is useful for predicting Fis DNA binding.     

Kinetic investigation of cooperativity in coenzyme binding by transketolase active sites

The two-step mechanism of coenzyme (thiamine diphosphate, ThDP) binding with two initially identical active sites of apotransketolase has been examined with a kinetic model. Cooperativity between sites in the primary ThDP binding and in the following conformational transition has been analyzed. The only reliable difference between sites is shown to be the tenfold difference in the backward rate constants of the conformational transition; this means that the cooperative interaction between sites takes place only after termination of both steps of ThDP binding in both sites.     

Modulation of binding sites for corticotropin-releasing hormone by chronic psychosocial stress

This study was conducted to determine whether long lasting psychosocial stress would affect corticotropin-releasing hormone (CRH) binding sites in the brain, the pituitary, and the adrenal gland. As a model for sustained emotional stress we used chronic psychosocial conflict in male tree shrews. In subordinate tree shrews, repeated confrontation with a dominant conspecific results in constant hyperactivity of the HPA-axis and an elevated neurosympathetic tone. After 24 days of psychosocial conflict, CRH binding sites were quantified by in vitro-autoradiography with 125I-ovine CRH in 23 discrete brain regions, the pituitaries, and the adrenal glands of subordinate and control animals. Chronic stress significantly reduced the number of binding sites (Bmax) in the anterior lobe of the pituitary, the dentate gyrus, the CA1-CA3 areas of the hippocampus, and in both the stratum griseum superficiale and the stratum opticum of the superior colliculus. In cortical area 17, the reduction of Bmax was counterbalanced by an increase in the affinity (Kd) of the radioligand for the binding sites. A significant stress-induced enhancement of Bmax was observed in the frontal cortex, cingulate cortex, claustrocortex, the central and lateral nucleus of the amygdala, and in the choroid plexus. This increase was accompanied by a significant decrease of Kd-values in the frontal and cingulate cortex, the lateral nucleus of the amygdala, and the choroid plexus. These findings represent the first in vivo demonstration of a modulation of extrahypothalamic CRH receptors by a naturally occurring form of stress. The different response patterns of the central CRH binding sites reflect distinct neuroendocrine processes which are presumed to coordinate behavioral, autonomic, endocrine, and immune responses to long-lasting psychosocial conflict.     

Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRI, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been reduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA. Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.     

The elusive family of imidazoline binding sites

Endopeptidase 24.11 (EP 24.11), a membrane-bound cell surface enzyme, modulates chemotactic responsiveness of neutrophils to f-Met-Leu-Phe. It is unknown if the enzyme degrades potent formylmethionyl tetrapeptides or if an enzyme with similar activities, endopeptidase 24.15 (EP 24.15), degrades formylated chemotactic peptides. In a study of five formylmethionyl tetrapeptides and f-Met-Leu-Phe, we found that EP 24.11 had high affinity for all peptides evaluated, although it did not effectively degrade f-Met-Ile-Leu-Phe. EP 24.15 had high affinity for three of the tetrapeptides, and for f-Met-Leu-Phe, although, for unclear reasons, it did not degrade f-Met-Ile-Leu-Phe or f-Met-Leu-Phe, the apparent natural products of Staphylococcus aureus and Escherichia coli, respectively.     

Chromosomal binding sites of Ultrabithorax homeotic proteins

Drosophila homeotic genes and their vertebrate cognates, the Hox genes, encode homeodomain proteins that are thought to control segment-specific morphogenesis by regulating subordinate target genes. Although expression of many genes is thought to be influenced by homeotic/Hox function, little is known about the genes they directly regulate in the developing embryo. One of the Drosophila homeotic genes is Ultrabithorax (Ubx) that specifies the identity of specific thoracic and abdominal metameres. Towards identifying genes directly regulated by Ubx we have mapped the binding sites of Ubx proteins (UBX) in polytene chromosomes. We found that the UBX isoforms Ia and IVa accumulate in about 100 discrete chromosomal sites. Most, if not all, the sites are the same for the two UBX isoforms. These sites are all euchromatic, include both bands and interbands and are reproducible from chromosome to chromosome. Some of these sites correspond to the locations of known genes that are good candidates, or are known to be, under direct Ubx control.     

Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on "active pairs" of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.     

Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry

Low-energy (LE) and high-energy (HE) collisionally activated decompositions (CAD) of calcium/peptide complexes of the form [M - H + Ca]+ and [M + Ca]2+ reflect the site of calcium binding in various gas-phase peptides that are models of the calcium binding site III of rabbit skeletal troponin C. The Ca2+ binding sites involve an aspartic acid, glutamic acid, and asparagine, which are in the metal-binding loops of calcium-binding proteins. Both fast atom bombardment (FAB) and electrospray ionization (ESI) were used to generate the metal/peptide complexes. When submitted to LE CAD, ESI-produced Ca2+/peptide complexes undergo fragmentations that are controlled by Ca2+ binding and provide information on the Ca2+ binding site. The LE CAD spectra are simple, indicating that Ca2+ binding involves specific oxygen ligands including acidic side chains and that only a few low-energy fragmentation channels exist. The HE CAD spectra of FAB-produced Ca2+/peptide complexes are more complex, owing to the introduction of high internal energy into the precursor ion. Interactions of the other alkaline-earth metal ions Mg2+ and Ba2+ with these peptides reveal that the ligand preferences of these metal ions are slightly different than those of Ca2+.     

Anatomy of lipase binding sites: the scissile fatty acid binding site

Shape and physico-chemical properties of the scissile fatty acid binding sites of six lipases and two serine esterases were analyzed and compared in order to understand the molecular basis of substrate specificity. All eight serine esterases and lipases have similar architecture and catalytic mechanism of ester hydrolysis, but different substrate specificities for the acyl moiety. Lipases and esterases differ in the geometry of their binding sites, lipases have a large, hydrophobic scissile fatty acid binding site, esterases like acetylcholinesterase and bromoperoxidase have a small acyl binding pocket, which fits exactly to their favorite substrates. The lipases were subdivided into three sub-groups: (1) lipases with a hydrophobic, crevice-like binding site located near the protein surface (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). The length of the scissile fatty acid binding site varies considerably among the lipases between 7.8 A in cutinase and 22 A in Candida rugosa and Rhizomucor miehei lipase. Location and properties of the scissile fatty acid binding sites of all lipases of known structure were characterized. Our model also identifies the residues which mediate chain length specificity and thus may guide protein engineering of lipases for changed chain length specificity. The model was supported by published experimental data on the chain length specificity profile of various lipases and on mutants of fungal lipases with changed fatty acid chain length specificity.     

Determination of the binding constant of ligand to protein by thermal inactivation tehnique. Effect of two binding sites

Theoretical considerations on the determining the binding constants (eta) of ligands to proteins were carried out. Whereas for a one-subunit protein the relationship between thermal inactivation rates and ligand concentration there is a simple linear function, for a protein with two subunits, a second-order relationship is derived. If the theory for one-subunit proteins is applied to multi-subunit proteins, the derived values of eta tend to be lower than the real binding constants. A method of determining the ligand binding constant for a two-subunit protein is described.     

Glutamate receptor binding sites in MPTP-treated mice

Changes in excitatory amino acid (EAA) neurotransmission are thought to play an important role in the development of parkinsonian symptoms. We examined EAA receptor binding sites in substantia nigra, striatum, globus pallidus, and cortex at 2 weeks and 2 months after MPTP (1-methyl-4-phenyl-1,2,3,6-tetra-hydroxypyridine) injection in C57bl6 mice. At 2 weeks striatal dopamine content in MPTP-treated mice was reduced to 7% of control and N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate and [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites were decreased in substantia nigra to 57 and 76% of control, respectively. In globus pallidus only [3H]AMPA binding sites were decreased to 80% of control; no significant changes were found in striatum or cortex. [3H]Kainate binding sites remained unchanged. At 2 months striatal dopamine content was reduced to 31% and no changes in EAA binding sites could be detected in any of the structures examined. [3H]Mazindol binding to striatal monoamine-uptake sites was decreased to 17% of control at 2 weeks versus 37% at 2 months. Our data indicate that modulation of NMDA and AMPA binding sites in substantia nigra and globus pallidus, the major projection areas of the subthalamic nucleus, takes place only after severe impairment of the nigrostriatal system.     

Formation of the nicotinic acetylcholine receptor binding sites

Nicotinic acetylcholine receptors (AChRs) are activated by ACh binding to two sites located on different alpha subunits. The two alpha subunits, alpha gamma and alpha delta, are distinguished by their interface with gamma and delta subunits. We have characterized the formation of the ACh binding sites and found, contrary to the current model, that the sites form at different times and in a set order. The first site forms on alpha gamma subunits during the process of subunit assembly. Our data are consistent with the appearance of this site on alpha beta gamma delta subunit tetramers soon after the site for the competitive antagonist alpha-bungarotoxin has formed and delta subunits have assembled with alpha beta gamma trimers. The second site is located on alpha delta subunits and forms after AChR subunits have assembled into alpha2 beta gamma delta pentamers. By determining the order in which the ACh binding sites form, we have also identified the sites in which the delta and second alpha subunits associate during subunit assembly.     

Identification of binding sites for calcium channel antagonists

The binding sites of three typical calcium channel antagonists, 1, 4-dihydropyridines, benzothiazepines and phenylalkylamines, were successfully identified within the primary structures of calcium channels using a photoaffinity labeling technique. The results confirm pharmacological observations of the three antagonists that had been proposed to interact allosterically with each other. We briefly review the results and discuss the future prospects.