Search

Major histocompatibility complex (MHC) class II genes are regulated at the transcriptional level by coordinate action of a limited number of transcription factors that include regulatory factor X (RFX), class II transcriptional activator (CIITA), nuclear factor Y (NF-Y), and cyclic AMP-response element binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB were expressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical and biophysical methods. Analytical ultracentrifugation (AUC) has demonstrated that RFX is a heterotrimer. A heterodimer of RFX5 and RFX-AP was also observed. A high-affinity interaction (K(d) = 25 nM) between RFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction between RFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that the interaction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescence anisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits and complexes binding to duplex DNA. The RFX5 subunit was found to drive recognition of the promoter, while the auxiliary RFX-AP and RFX-ANK subunits were shown to contribute to the specificity of binding for the overall complex. AUC experiments demonstrate that in the absence of additional subunits, monomeric RFX5 binds to X-box DNA with a 1:1 stoichiometry. Interactions between CREB, CIITA, and RFX in the absence of DNA were demonstrated using bead-based immunoprecipitation assays, confirming that preassociation with DNA is not required for forming the macromolecular assemblies that drive MHC class II gene expression.

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.

Herpes simplex virus infections are the cause of significant morbidity, and currently used therapeutics are largely based on modified nucleoside analogs that inhibit viral DNA polymerase function. To target this disease in a new way, we have identified and optimized selective thiazolylphenyl-containing inhibitors of the herpes simplex virus (HSV) helicase-primase enzyme. The most potent compounds inhibited the helicase, the primase and the DNA-dependent ATPase activities of the enzyme with IC50 (50% inhibitory concentration) values less than 100 nM. Inhibition of the enzymatic activities was through stabilization of the interaction between the helicase-primase and DNA substrates, preventing the progression through helicase or primase catalytic cycles. Helicase-primase inhibitors also prevented viral replication as demonstrated in viral growth assays. One compound, BILS 179 BS, displayed an EC50 (effective concentration inhibiting viral growth by 50%) of 27 nM against viral growth with a selectivity index greater than 2,000. Antiviral activity was also demonstrated for multiple strains of HSV, including strains resistant to nucleoside-based therapies. Most importantly, BILS 179 BS was orally active against HSV infections in murine models of HSV-1 and HSV-2 disease and more effective than acyclovir when the treatment frequency per day was reduced or when initiation of treatment was delayed up to 65 hours after infection. These studies validate the use of helicase-primase inhibitors for the treatment of acute herpesvirus infections and provide new lead compounds for optimization and design of superior anti-HSV agents.

One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR ζ ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon ζ ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of ζ chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein–phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein–phosphopeptide complex relative to the protein alone.

The development of a sensitive fluorescence binding assay for evaluating the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides containing the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the acetamido-anilino-naphthyl sulfonic acid (AANS), acrylodan, or dansyl groups as fluorophores. The spectral features of these probes were characterized in the presence and absence of the lck SH2 domain. The binding affinities (Kd) for the fluorescent peptides studied ranged from 40 to 500 nM. The fluorescent peptide containing the sequence FTATEC(AANS)QpYEEIP exhibited the highest binding affinity (Kd = 3.98 x 10(-8) M) and largest change in emission intensity (approximately 8.7-fold) upon binding the SH2 domain. This probe was subsequently used in competitive binding assays to study the interaction of the lck SH2 domain with a series of phosphopeptides related to the pYEEIP and pYQPQP (the pY505 C-terminal) consensus sequences. The effects of peptide length and substitutions of residues within the pYEEIP sequence are discussed in terms of binding affinities. Comparison between the two peptide series revealed that the contributions of individual substitutions to binding affinity are context-dependent. The data also led to the conclusion that the presence of P at +2 results in a functional "truncation" of the binding sequence; i.e., residues at positions higher than +2 do not participate significantly in binding. This implicit truncation may actually be a desired property for the autoregulatory nature of the pYQPQP sequence, since it retains specificity for the SH2 domain while adjusting the Kd to a value appropriate for maintaining the delicate balance of receptor-ligand interactions that are involved in signal transduction events.

Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC 50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC 50 determined for the unlabeled T-22 peptide via competitive binding (59 nM). (Journal of Biomolecular Screening 2008:424-429)

Resonance Raman spectra have been obtained at several uv wavelengths (200–266 nm) for poly(rA)-poly(rU), poly(dA-dU), poly(dA)-poly(dT), and poly(dA-dT), representing nucleic acid duplexes containing A-U and A-T base pairs with different stacking interactions and different backbone conformations. Frequency shifts are seen in the exocyclic modes corresponding to coupled C4O and C5C6 stretching of U and T, although the NH2 scissors frequency of A is unshifted relative to that of the mononucleotide. These frequency patterns are interpreted as the superposition of H bonding and dipole–dipole coupling effects. Strong hypochromism is seen for most of the ring modes, resulting from the absorption hypochromism and from shifts in the electronic transitions due to stacking. The effects are larger for A bands in the homopolymers than in the heteropolymer duplexes, reflecting the larger influence of A/A than A/U(T) stacking. Poly(rA)-poly(rU) stands out among these polymers in showing 10 cm−1 downshifts in one U and several A modes. These shifts may be related to the A form structural parameters of this duplex, but the physical mechanism is not obvious.

The glucocorticoid receptor (GR) is involved in the transcriptional regulation of genes associated with inflammation, glucose homeostasis, and bone turnover through the association with ligands, such as corticosteroids. GR-mediated gene transcription is regulated or fine-tuned via the recruitment of co-factors including coactivators and corepressors. Current therapeutic approaches to targeting GR aim to retain the beneficial anti-inflammatory activity of the corticosteroids while eliminating negative side effects. Towards achieving this goal the experiments discussed here reveal a mechanism of co-factor binding in the presence of either bound agonist or antagonist. The GR ligand binding domain (GR-LBD(F602S)), in the presence of agonist or antagonist, utilizes different modes of binding for coactivator versus corepressor. Coactivator binding to the co-effector binding pocket of GR-LBD(F602S) is driven both by favorable enthalpic and entropic interactions whereas corepressor binding to the same pocket is entropically driven. These data support the hypothesis that ligand-induced conformational changes dictate co-factor binding and subsequent trans-activation or trans-repression.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.

The allosteric inhibition of the lymphocyte function associated antigen-1/intercellullar adhesion molecule (LFA-1/ICAM-1) interaction, by a class of small molecules, is characterized by a battery of mass spectrometric techniques. Binding of hydantoins to the I domain of LFA-1 is observed by size exclusion chromatography/mass spectrometry (SEC/MS) and by direct electrospray ionization mass spectrometry (ESI/MS). A photoactive hydantoin analog specifically labels an amino acid residue of LFA-1 I domain. Competition with this photoaffinity labeling by a panel of inhibitors is correlated with their Kd’s for inhibition of the LFA-1/ICAM interaction. Alterations to the tertiary structure of LFA-1 I domain, upon compound binding, are inferred from perturbation in the ESI mass spectrum of the polypeptide’s charge state distribution and by an altered level of nonspecific multimer formation. The results demonstrate specific, stoichiometric, reversible binding of the hydantoins to LFA-1. They further show correlation of this binding with activity and indicate alterations in the polypeptide’s tertiary structure, on hydantoin binding, consistent with the proposed mechanism for inhibition of the protein–protein interaction.

The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.

The specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.

Recent developments in instrumentation for ultraviolet resonance Raman (UVRR) spectroscopy and its application to proteins are reviewed. With excitation near the ∼ 195 nm amide π-π* transition strong enhancement is seen of the amide vibrational modes, particularly amide II, whose intensity is sensitive to secondary structure. With measurements at 200 and 192 nm one can calculate the fractions of α-helix, β sheet and unordered segments from the known cross sections of these structures. The proline imide II band at ∼ 1460 cm −1 is strongly enhanced with 218 nm excitation and its frequency can be used to monitor cis-trans isomerization. Aromatic residues give rise to strong UVRR bands. Phenylalanine (Phe) and tyrosine (Tyr) show scattering patterns typical of substituted benzenes: enhancement of vibronic modes, especially ν 8a and ν 8b in resonance with the quasi-forbidden L a transition (∼ 205 and ∼ 223 nm for Phe and Tyr), and of breathing modes in resonance with the allowed B a,b transitions (∼ 188 and ∼ 193 nm for Phe and Tyr). Tyrosine, and especially tyrosinate, also show strong enhancement of ν 8a and ν 8b in the B a,b -resonant region, behavior attributed to electronic mixing of L a with B a via the OH (or O − ) substituent. In proteins the Tyr ν 8a and ν 8b bands are most readily observed with 229 nm excitation, where the Phe contributions are minimal. The ν 8b frequency is sensitive to Tyr H-bonding and has been calibrated in terms of the H-bond strength. The Tyr 830/850 cm −1 Fermi doublet intensity ratio, which can be monitored at 200 nm is sensitive to H-bonding, but also to other environmental influences. With 218 nm excitation protein spectra are dominated by tryptophan (Trp) contributions. The 1340/1360 cm −1 Trp Fermi doublet is sensitive to solvent exposure, while the ∼ 880 cm −1 band frequency is sensitive to H-bonding. Histidine (His) excitation profiles show π-π* resonances at ∼ 218 and ∼ 204 nm. The UVRR bands are sensitive to protonation, but the enhancement is relatively weak, and the His bands are obscured in protein UVRR spectra by the other aromatic contributions. Applications of the UVRR technique to the filamentous virus fd are described.

Unlike its thiocyanate analogues, which involve the formation of the geminite [Pd{(C 6H 5) 2P(CH 2) 2-P(C 6H 5) 2}(SCN)(NCS)] and the di-N-bound complex [Pd{(C 6H 5) 2P(CH 2) 3P(C 6H 5) 2}(NCS) 2], the corresponding selenocyanate complexes contain only Sebound groups, as determined by the results of single crystal X-ray diffraction studies. Se-bonding is also maintained in the [Pd{(C 6H 5) 2PCH 2 o C 6H 4-CH 2P-(C 6H 5) 2}(SeCN) 2] complex, based on the results of IR studies. Only in the case of the (C 6H 5) 2PCH 2P(C 6H 5) 2 palladium(II) complexes, do both pseudohalides exhibit analogous bonding via their chalcogen atoms. The selenocyanate's bonding pattern is thus dominated by the soft/soft interaction afforded by Pd-SeCN bonding, remaining impervious to increasing steric demands of the diphosphine ligands.

The reaction, at 25 °C in methanol, between [Pd(SeCN)4]2− and bis(diphenylphosphino)methane (dpm) has been found to produce cyano(selenocyanato) [diphenyl(diphenylphosphinomethyl) phosphine selenide]palladium(II), [Pd(dpmSe)(CN)(SeCN)], wherein the cyanide group is trans to an Se atom which has been inserted into one PdP bond, and the selenocyanate group is trans to the unchanged diphenylphosphino group. The structure has been confirmed by the results of a single crystal X-ray diffraction study. The structural isomer, [Pd(dpm)- (SeCN)2], of the foregoing complex has also been prepared by the reaction of Pd(C2H3O2)2 with dpm, followed by reaction with KSeCN. Heating the [Pd(dpm)(SeCN)2] isomer converts it into [Pd- (dpmSe)(CN)(SeCN)]. A mechanism is proposed for the isomerization which involves an intramolecular selenium atom insertion.

Carboxylate and guanidinium groups of amino acids have been studied by ultraviolet resonance Raman (UVRR) spectroscopy. On near-resonant excitation (192 nm) into the allowed π–π* transition of the carboxylate chromophore, the vibrational spectrum of aspartate displays only one band originating from the COO− symmetric stretching mode, consistent with an A-term enhancement mechanism. The UVRR spectra of the guanidinium ion reveals a complex vibrational pattern, and the bands have been assigned with the aid of a normal coordinate analysis. UV enhancement is observed for both asymmetric (E′) and totally symmetric (A1′) modes, and also for the overtone of the out-of-plane CN3 bending mode, Γ(A2″). The E′ enhancement is suggested to reflect Jahn—Teller splitting in the first allowed electronic transition A2″ E″, whereas the 2Γ(A2″) enhancement requires a lowering of the force constant for out-of-plane bending in the excited state, consistent with its expected tendency toward pyramidalization. Similar enhancement patterns are seen in the UVRR spectra of the guanidinium chromophore in methylguanidinium and arginine, which show, in addition, splittings and intensity redistribution due to the substituents. The excitation profiles of guanidinium and arginine reveal a complex structure that may arise from an excited-state Jahn—Teller effect. The Raman enhancements are too low to permit detection of guanidinium or carboxylate in the UVRR spectra of proteins, even at wavelengths as low as 192 nm.

Ultraviolet resonance Raman spectra are reported for cytochrome c (cyt c) in FeII and FeIII oxidation states at low (0.005 M) and high (0.9-1.5 M) ionic strength. With 200-nm excitation the amide band intensities are shown to remain constant, establishing that redox state and ionic strength have no influence on the alpha-helical content. The tyrosine 830/850-cm-1 doublet, however, shows a loss in 830-cm-1 intensity at I = 0.005 M for the FeIII protein, suggesting a weakening or a loss of H-bonding from an internal tyrosine, probably Tyr-48, which is H-bonded to a heme propionate group in cyt c crystals. Excitation profiles of tryptophan peak at approximately 229 nm for both FeII and FeIII forms of cyt c, but at approximately 218 nm for aqueous tryptophan. The approximately 2200-cm-1 red shift of the resonant electronic transition is attributed to the Trp-59 residue being buried and H-bonded. Consistent with this Trp environment, the H-bond-sensitive 877-cm-1 Trp band is strong and sharp, and the 1357/1341-cm-1 doublet has a large intensity ratio, approximately 1.5, for both FeII and FeIII cyt c. The 877-cm-1-band frequency shifts to 860 cm-1 when the Trp indole proton is replaced by a deuteron. This band was used to show that Trp H/D exchange in D2O is much faster for FeIII than FeII cyt c. The half-time for exchange at room temperature is estimated to be approximately 30 and approximately 5 h, respectively, for FeII and FeIII when examined at I = 0.005.(ABSTRACT TRUNCATED AT 250 WORDS)

Raman spectra are reported for distamycin, excited at 320 nm, in resonance with the first strong absorption band of the chromophore. Qualitative band assignments to pyrrole ring and amide modes are made on the basis of frequency shifts observed in D2O. When distamycin is dissolved in dimethyl sulfoxide or dimethylformamide, large (30 cm-1) upshifts are seen for the band assigned to amide I, while amides II and III shift down appreciably. Similar but smaller shifts are seen when distamycin is bound to poly(dA-dT) and poly(dA)-poly(dT). Examination of literature data for N-methylacetamide in various solvents shows that the amide I frequencies correlate well with solvent acceptor number but poorly with solvent donor number. This behavior implies that acceptor interactions with the C = O group are more important than donor interactions with the N-H group in polarizing the amide bond and stabilizing the zwitterionic resonance form. The resonance Raman spectra therefore imply that the distamycin C = O groups, despite being exposed to solvent, are less strongly H-bonded in the polynucleotide complexes than in aqueous distamycin, perhaps because of orienting influences of the nearby backbone phosphate groups. In this respect, the poly(dA-dT) and poly(dA)-poly(dT) complexes are the same, showing the same RR frequencies. Resonance Raman spectra were also obtained at 200-nm excitation, where modes of the DNA residues are enhanced. The spectra were essentially the same with and without distamycin, except for a perceptable narrowing of the adenine modes of poly(dA-dT), suggesting a reduction in conformational flexibility of the polymer upon drug binding.