Differential Conformational Changes Research Articles

ATP-competitive and allosteric inhibitors induce differential conformational changes at the autoinhibitory interface of Akt1

Akt is a master regulator of pro-growth signaling in the cell. Akt is activated by phosphoinositides that disrupt the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains and then is phosphorylated at T308 and S473. Akt hyperactivation is oncogenic, which has spurred development of potent and selective inhibitors as therapeutics. Using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding Akt ATP-competitive and allosteric inhibitors. We compared inhibitors against three different states of Akt1. The allosteric inhibitor caused substantive conformational changes and restricts membrane binding. ATP-competitive inhibitors caused extensive allosteric conformational changes, altering the autoinhibitory interface and leading to increased membrane binding, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and conformational changes induced by Akt inhibitors and has important implications for the design of Akt targeted therapeutics.

Abstract 1395: ATP-competitive and allosteric inhibitors induce differential conformational changes at the autoinhibitory interface of Akt1

Abstract 1395: ATP-competitive and allosteric inhibitors induce differential conformational changes at the autoinhibitory interface of Akt1

Counteractive functions are encrypted in the residues of CD154

CD40, as a single receptor that binds CD154 (CD40-ligand or CD40L), regulates counteractive effector functions such as production of pro- and anti-inflammatory cytokines. Therefore, we examined whether such dual messages are encrypted in CD40L. As such message encryption was never investigated, we hypothesized that mutation of certain amino acid residues should in principle enhance pro-inflammatory cytokine production whereas mutation of some others would enhance anti-inflammatory cytokine secretion. We mutated six such residues, which were previously showed to participate in CD40L function. Here, we report that the mutant CD154 129E→V was superior to the wild-type CD154 in killing of Leishmania donovani, induction of inducible nitric oxide synthase (iNOS) and production of IL-12 and relative phosphorylation of p38MAPK and ERK-1/2 in PBMC-derived macrophages. By contrast, 128S→V promoted L. donovani survival, reducing iNOS, but increasing IL-10 expression and predominant ERK-1/2 phosphorylation. The mutant 144G→V did not have significant effects. Other mutants (142E→V, 143K→A, 145Y→F) mimicked the wild-type CD154. Molecular dynamics simulation suggested that these mutations induced differential conformational changes in the CD40–CD154 complex. Therefore, assortment of the contrasting messages encrypted in a given ligand performing counteractive functions presents a novel fundamental biological principle that can be used for devising various therapies.

Protein dynamics of the HIF-2α PAS-B domain upon heterodimerization and ligand binding.

Hypoxia-Inducible Factor (HIF) transcription factors are heterodimeric proteins involved in the regulation of oxygen homeostatis. Their upregulation has been related to several tumors with a remarkably poor clinical outcome. The recent discovery of a druggable cavity in the HIF-2α PAS-B domain has opened an unprecedented opportunity for targeting the HIF-2α transcription factor in view of pharmaceutical strategies. Coincidentally, a novel compound able to selectively disrupt the HIF heterodimerization with a submicromolar activity has been reported. In this work, we investigated the molecular mechanisms responsible for the inhibition by comparing the dynamical features of the HIF-2α PAS-B monomer and the HIF-2α PAS-B/HIF-1β PAS-B complex, in the ligand-bound and -unbound states. Plain and biased Molecular Dynamics were used to characterize the differential conformational changes both structurally and energetically.

Transcription Factor-dependent DNA Bending Governs Promoter Recognition by the Mitochondrial RNA Polymerase

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

Amyloid β Peptide-(1–42) Induces Internalization and Degradation of β2 Adrenergic Receptors in Prefrontal Cortical Neurons

Emerging evidence indicates that amyloid β peptide (Aβ) initially induces subtle alterations in synaptic function in Alzheimer disease. We have recently shown that Aβ binds to β(2) adrenergic receptor (β(2)AR) and activates protein kinase A (PKA) signaling for glutamatergic regulation of synaptic activities. Here we show that in the cerebrums of mice expressing human familial mutant presenilin 1 and amyloid precursor protein genes, the levels of β(2)AR are drastically reduced. Moreover, Aβ induces internalization of transfected human β(2)AR in fibroblasts and endogenous β(2)AR in primary prefrontal cortical neurons. In fibroblasts, Aβ treatment also induces transportation of β(2)AR into lysosome, and prolonged Aβ treatment causes β(2)AR degradation. The Aβ-induced β(2)AR internalization requires the N terminus of the receptor containing the peptide binding sites and phosphorylation of β(2)AR by G protein-coupled receptor kinase, not by PKA. However, the G protein-coupled receptor kinase phosphorylation of β(2)AR and the receptor internalization are much slower than that induced by βAR agonist isoproterenol. The Aβ-induced β(2)AR internalization is also dependent on adaptor protein arrestin 3 and GTPase dynamin, but not arrestin 2. Functionally, pretreatment of primary prefrontal cortical neurons with Aβ induces desensitization of β(2)AR, which leads to attenuated response to subsequent stimulation with isoproterenol, including decreased cAMP levels, PKA activities, PKA phosphorylation of serine 845 on α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor subunit 1 (GluR1), and AMPA receptor-mediated miniature excitatory postsynaptic currents. This study indicates that Aβ induces β(2)AR internalization and degradation leading to impairment of adrenergic and glutamatergic activities.

In silico analysis of the binding of agonists and blockers to the β 2-adrenergic receptor

Activation of G protein-coupled receptors (GPCRs) is a complex phenomenon. Here, we applied Induced Fit Docking (IFD) in tandem with linear discriminant analysis (LDA) to generate hypotheses on the conformational changes induced to the β 2-adrenergic receptor by agonist binding, preliminary to the sequence of events that characterize activation of the receptor. This analysis, corroborated by a follow-up molecular dynamics study, suggested that agonists induce subtle movements to the fifth transmembrane domain (TM5) of the receptor. Furthermore, molecular dynamics also highlighted a correlation between movements of TM5 and the second extracellular loop (EL2), suggesting that freedom of motion of EL2 is required for the agonist-induced TM5 displacement. Importantly, we also showed that the IFD/LDA procedure can be used as a computational means to distinguish agonists from blockers on the basis of the differential conformational changes induced to the receptor. In particular, the two most predictive models obtained are based on the RMSD induced to Ser207 and on the counterclockwise rotation induced to TM5.

Differential signal transduction via TrmB, a sugar sensing transcriptional repressor of Pyrococcus furiosus

TrmB is a transcriptional repressor of the hyperthermophilic archaeon Pyrococcus furiosus serving at least two operons. TrmB represses genes encoding an ABC transporter for trehalose and maltose (the TM system) with trehalose and maltose as inducers. TrmB also represses genes encoding another ABC transporter for maltodextrins (the MD system) with maltotriose and sucrose as inducers. Here we report that glucose which was also bound by TrmB acted as a corepressor (causing stronger repression) for both the TM and the MD system. Binding of glucose by TrmB was increased in the presence of TM promoter DNA. Maltose which acted as inducer for the TM system acted as a corepressor for the MD system intensifying repression. We propose that the differential conformational changes of TrmB in response to binding the different sugars governs the ability of TrmB to interact with the promoter region and represents a simple mechanism for selecting the usage of one carbon source over the other, reminiscent of catabolite repression in bacteria.

Molecular mechanism of activation and nuclear translocation of the mineralocorticoid receptor upon binding of pregnanesteroids

The mineralocorticoid receptor (MR) is primarily localized in the cytoplasm of the cell in the absence of ligand. The first step in the genomic-dependent mechanism of action of mineralocorticoids is the binding of steroid to the MR, which in turn triggers MR nuclear translocation. The regulation of hormone-binding to MR is complex and involves a multifactorial mechanism, making it difficult to determine the optimal structure of a steroid for activating the MR and promoting its nuclear translocation. Here we review the structure–activity relationship for several pregnanesteroids that possess various functional groups, and suggest that a flat conformation of the ligand rather than the presence of particular chemical groups is a critical parameter for the final biological effect in vivo. We also discuss how the MR undergoes differential conformational changes according to the nature of the bound ligand, which in turn affects the dynein-dependent retrograde rate of movement for the steroid/receptor complex.

Asymmetric recognition of nonconsensus AP-1 sites by Fos-Jun and Jun-Jun influences transcriptional cooperativity with NFAT1.

Many regulatory elements in eukaryotic promoters do not correspond to optimal recognition sequences for the transcription factors that regulate promoter function by binding to the elements. The sequence of the binding site may influence the structural and functional properties of regulatory protein complexes. Fos-Jun heterodimers were found to bind nonconsensus AP-1 sites in a preferred orientation. Oriented Fos-Jun heterodimer binding was attributed to nonidentical recognition of the two half-sites by Fos and Jun. Jun bound preferentially to the consensus half-site, whereas Fos was able to bind nonconsensus half-sites. The orientation of heterodimer binding affected the transcriptional cooperativity of Fos-Jun-NFAT1 complexes at composite regulatory elements in mammalian cells. Jun dimerization with Fos versus ATF2 caused it to bind opposite half-sites at nonconsensus AP-1 elements. Similarly, ATF2 bound to opposite half-sites in Fos-ATF2-NFAT1 and ATF2-Jun-NFAT1 complexes. The orientations of nonconsensus AP-1 sites within composite regulatory elements affected the cooperativity of Fos-Jun as well as Jun-Jun binding with NFAT1. Since Jun homodimers cannot bind to AP-1 sites in a preferred orientation, the effects of the orientations of nonconsensus AP-1 sites on the stabilities of Jun-Jun-NFAT1 complexes are likely to be due to asymmetric conformational changes in the two subunits of the homodimer. Nonconsensus AP-1 site orientation also affected the synergy of transcription activation between Jun homodimers and NFAT1 at composite regulatory elements. The asymmetric recognition of nonconsensus AP-1 sites can therefore influence the transcriptional activities of Fos and Jun both through effects on the orientation of heterodimer binding and through differential conformational changes in the two subunits of the dimer.

Contribution of Human Mlh1 and Pms2 ATPase Activities to DNA Mismatch Repair

MutLalpha, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLalpha and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLalpha proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLalpha demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLalpha activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.

Interactions of synthetic estrogens with human estrogen receptors

Synthetic estrogens have diverse chemical structures and may either positively or negatively affect the estrogenic signaling pathways through interactions with the estrogen receptors (ERs). Modeling studies suggest that 4-(1-adamantyl)phenol (AdP) and 4,4'-(1,3-adamantanediyl)diphenol (AdDP) can bind in the ligand binding site of ERalpha. We used fluorescence polarization (FP) to compare the binding affinities of AdP, AdDP and 2-(1-adamantyl)-4-methylphenol (AdMP) for human ERalpha and ERbeta with the binding affinities of the known ER ligands, diethylstilbestrol (DES) and 4hydroxytamoxifen (4OHT). Competition binding experiments show that AdDP has greater affinity for both ERs than does AdP, while AdMP does not bind the receptor proteins. The relative binding affinities of AdDP and AdP are weaker than the affinity of DES or 4OHT for both ERs with the exception of AdDP, which binds ERbeta with higher affinity than does 4OHT. We also found that AdDP and AdP cause differential conformational changes in ERalpha and ERbeta, which result in altered affinities of the ERs for fluorescein-labeled estrogen response elements (EREs) using a direct binding FP assay. The results show that ERbeta liganded with either AdDP or AdP has greater affinity for human pS2 ERE than the ERbeta-4OHT complex. The data suggest that synthetic molecules like adamantanes may function as biologically active ligands for human ERs. This demonstrates the importance of considering the potential of novel classes of synthetic compounds as selective ER modulators.

Conformation and template activity of human reovirus genome RNA.

In vitro activation of human reovirus transcriptase by alpha-chymotrypsin digestion of viral outer shell proteins was completely dependent on the ionic size of the monovalent cation in the medium. Cations with nonhydrated ionic radii larger than 1.3 A showed full potency of activation of chymotrypsin digestion, and produced transcriptionally active virus cores. Smaller cations having ionic radii of 0.6 A or 0.95 A, on the other hand, promoted the chymotrypsin digestion to lesser extents, and yielded subviral particles showing latent or very low transcriptase activities. Differential conformational changes would be induced in viral outer shell proteins by these monovalent cations, resulting in the varied accessibility to chymotrypsin. Electron microscopic analyses under denaturing conditions of the cross-linked reovirus core genome RNAs with the AMT photoreaction revealed that they were almost evenly cross-linked by the psoralen adducts forming no reproducible size of "bubbles." This result suggests that the double helical reovirus genome may not be bound tightly by the inner viral proteins forming such nucleoprotein structures as nucleosomes in eukaryotic chromatin.

A comparison of preparative techniques for the examination of the external morphology of fungal material with the scanning electron microscope

The external morphology of the ostiolar pore region in Sordaria humana was examined with the scanning electron microscope (SEM) in the vacuum-dried, air-dried, critical-point-dried, freeze-dried, partially frozen–hydrated, and fully frozen–hydrated states after a range of pretreatments. The appearance of this structure was dependent on the method of preparation and the operational conditions employed within the microscope. All the preparative procedures used produced artifacts. These secondary changes varied according to the method of preparation and the nature of the specimen (or part thereof). Common artifacts arising from freezing damage, slow chemical fixation, differential conformational changes, ice recrystallization, water condensation, and beam damage have been characterized. The examination of material in the frozen–hydrated condition is the closest we can get to viewing its structure under the SEM in a state of "suspended animation."

Baker's yeast adenylate kinase. Evidence of conformational change from intrinsic fluorescence and difference spectra. Determination of the structure of enzyme-bound metal-nucleotide by use of phosphorothioate analogues of ATP.

The diastereomers of adenosine 5′‐O‐(1‐thiotriphosphate) ATPαS) and adenosine 5′‐O‐(2‐thiotriphosphate) (ATPβS) were used to determine the stereochemical mode of coordination of Mg2+ to ATP when bound in the yeast adenylate kinase complex. ATPαS isomer A is the preferred substrate either in the presence of Mg2+ or Cd2+; ratios of the rate of reaction at infinite concentration of substrate, V, for A to B isomers of ATPαS with Mg2+ were 457/51.4 = 8.9 and with Cd2+ 124/12.8 = 9.7. Thus there is no reversal of stereospecificity on substituting Cd2+ for Mg2+ and this supports the view that metal does not chelate with the α‐P. In contrast the V ratios of ATPβS(A) to ATPβS(B) with Mg2+ were 1.33/0.15 = 8.9 and with Cd2+ 0.18/5.1 = 0.035. Thus this reversal of stereospecificity when Cd2+ is substituted for Mg2+ (in view of the fact that Mg2+ binds to O and Cd2+ to S) leads to the conclusion that a metal chelation must occur via the β‐phosphate. Since the enzyme prefers MgATPβS(A) and CdATPβS(B) (both isomers have Δ‐screw sense) to MgATPβS(B) and CdATPβS(A) (both isomers have Δ‐screw sense) and Mg2+ also coordinates with the γ‐phosphate (by analogy with other adenylate kinase isozymes), the interpretation is that baker' yeast adenylate kinase binds MgATP as Δ, β, γ. The fluorescence of the two residues of yeast adenylate kinase has been used to investigate conformational changes of the enzyme on binding its substrates. Fluorescene is maximal at 334 nm (λexx 295 nm, pH 7.5) and is reduced 10% with no shift in λmax in the presence of ATP and reduced a further 15% on the addition of Mg2+ or other metal acitvators to E‐ATP. In contrast non‐activating metals such as Ba2+ and Sr2+ increase quenching only slightly. With ITP, UTP, CTP, GTP the quenching is 4.1%, 5.2%, 8.7% and 6.6% with or without Mg2+. Differential conformational changes of yeast adenylate kinase on binding MgATP, ATP, and other nucleotides are shown by (a) fluorescence changes, (b) previously reported kinetic and binding studied and (c) by the fact that the Km for AMP in the presence of MgATP is 17 times less than in the presence of MgGTP. These differential conformational changes result in differing tendencies of AMP to bind to the enzyme. Ultraviolet difference spectra obtained on binding of ATP to the enzyme (shoulder at 271 nm and peak at 276 nm) have been compared with the difference spectra of the interaction of ATP, adenosine and 1‐methyl adensoine respectiively with methyl mercuric hydroxide and formaldehyde. On the basis of these comparisons, the shoulder at 271 nm reflects the interaction of N(1) and the peak at 276 nm the interaction of tthe 6‐amino group of ATP with the enyzme.The following mechanism of reaction is proposed: upon binding MgATP the enzyme undergoes a conformational change which closes the active‐site crevice by bringing the two lobes closer, expelling water from the crevice. AMP is bound with higher affinity and the α‐oxygen and the α‐oxygen attacks the γ‐phosphate of ATP in a nucleophilic reaction.