Two binding modes reveal flexibility in kinase/response regulator interactions in the bacterial chemotaxis pathway

The crystal structure at 2.0-Å resolution of the complex of the Escherichia coli chemotaxis response regulator CheY and the phosphoacceptor-binding domain (P2) of the kinase CheA is presented. The binding interface involves the fourth and fifth helices and fifth β-strand of CheY and both helices of P2. Surprisingly, the two heterodimers in the asymmetric unit have two different binding modes involving the same interface, suggesting some flexibility in the binding regions. Significant conformational changes have occurred in CheY compared with previously determined unbound structures. The active site of CheY is exposed by the binding of the kinase domain, possibly to enhance phosphotransfer from CheA to CheY. The conformational changes upon complex formation as well as the observation that there are two different binding modes suggest that the plasticity of CheY is an essential feature of response regulator function.

A transcriptional activating region with two contrasting modes of protein interaction

A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions of cysteine and proline substitutions. We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro. Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function. Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP. Thus, the same stretch of amino acids are involved in two quite different protein–protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure.

Norepinephrine transporters have channel modes of conduction.

Neurotransmitter transporters couple to existing ion gradients to achieve reuptake of transmitter into presynaptic terminals. For coupled cotransport, substrates and ions cross the membrane in fixed stoichiometry. This is in contrast to ion channels, which carry an arbitrary number of ions depending on the channel open time. Members of the gamma-aminobutyric acid transporter gene family presumably function with fixed stoichiometry in which a set number of ions cotransport with one transmitter molecule. Here we report channel-like events from a presumably fixed stoichiometry [norepinephrine (NE)+, Na+, and Cl-], human NE (hNET) in the gamma-aminobutyric acid transporter gene family. These events are stimulated by NE and by guanethidine, an hNET substrate, and they are blocked by cocaine and the antidepressant desipramine. Voltage-clamp data combined with NE uptake data from these same cells indicate that hNETs have two functional modes of conduction: a classical transporter mode (T-mode) and a novel channel mode (C-mode). Both T-mode and C-mode are gated by the same substrates and antagonized by the same blockers. T-mode is putatively electrogenic because the transmitter and cotransported ions sum to one net charge. However, C-mode carries virtually all of the transmitter-induced current, even though it occurs with low probability. This is because each C-mode opening transports hundreds of charges per event. The existence of a channel mode of conduction in a previously established fixed-stoichiometry transporter suggests the appearance of an aqueous pore through the transporter protein during the transport cycle and may have significance for transporter regulation.

Two modes of transvection: Enhancer action in trans and bypass of a chromatin insulator in cis

Ed Lewis introduced the term “transvection” in 1954 to describe mechanisms that can cause the expression of a gene to be sensitive to the proximity of its homologue. Transvection since has been reported at an increasing number of loci in Drosophila, where homologous chromosomes are paired in somatic tissues, as well as at loci in other organisms. At the Drosophila yellow gene, transvection can explain intragenic complementation involving the yellow2 allele (y2). Here, transvection was proposed to occur by enhancers of one allele acting in trans on the promoter of a paired homologue. In this report, we describe two yellow alleles that strengthen this model and reveal an unexpected, second mechanism for transvection. Data suggest that, in addition to enhancer action in trans, transvection can occur by enhancer bypass of a chromatin insulator in cis. We propose that bypass results from the topology of paired genes. Finally, transvection at yellow can occur in genotypes not involving y2, implying that it is a feature of yellow itself and not an attribute of one particular allele.

Four primordial modes of tRNA-synthetase recognition, determined by the (G,C) operational code

In distinction to single-stranded anticodons built of G, C, A, and U bases, their presumable double-stranded precursors at the first three positions of the acceptor stem are composed almost invariably of G-C and C-G base pairs. Thus, the “second” operational RNA code responsible for correct aminoacylation seems to be a (G,C) code preceding the classic genetic code. Although historically rooted, the two codes were destined to diverge quite early. However, closer inspection revealed that two complementary catalytic domains of class I and class II aminoacyl-tRNA synthetases (aaRSs) multiplied by two, also complementary, G2-C71 and C2-G71 targets in tRNA acceptors, yield four (2 × 2) different modes of recognition. It appears therefore that the core four-column organization of the genetic code, associated with the most conservative central base of anticodons and codons, was in essence predetermined by these four recognition modes of the (G,C) operational code. The general conclusion follows that the genetic code per se looks like a “frozen accident” but only beyond the “2 × 2 = 4” scope. The four primordial modes of tRNA–aaRS recognition are amenable to direct experimental verification.

Two-dimensional infrared spectroscopy of peptides by phase-controlled femtosecond vibrational photon echoes

Two-dimensional infrared spectra of peptides are introduced that are the direct analogues of two- and three-pulse multiple quantum NMR. Phase matching and heterodyning are used to isolate the phase and amplitudes of the electric fields of vibrational photon echoes as a function of multiple pulse delays. Structural information is made available on the time scale of a few picoseconds. Line narrowed spectra of acyl-proline-NH2 and cross peaks implying the coupling between its amide-I modes are obtained, as are the phases of the various contributions to the signals. Solvent-sensitive structural differences are seen for the dipeptide. The methods show great promise to measure structure changes in biology on a wide range of time scales.

Structural analysis of the binding modes of minor groove ligands comprised of disubstituted benzenes

Two-dimensional homonuclear NMR was used to characterize synthetic DNA minor groove-binding ligands in complexes with oligonucleotides containing three different A-T binding sites. The three ligands studied have a C2 axis of symmetry and have the same general structural motif of a central para-substituted benzene ring flanked by two meta-substituted rings, giving the molecules a crescent shape. As with other ligands of this shape, specificity seems to arise from a tight fit in the narrow minor groove of the preferred A-T-rich sequences. We found that these ligands slide between binding subsites, behavior attributed to the fact that all of the amide protons in the ligand backbone cannot hydrogen bond to the minor groove simultaneously.

Hybrid characteristics of oligonucleotides consisting of isonucleoside 2′,5′-anhydro-3′-deoxy-3′-(thymin-1-yl)-d-mannitol with different linkage modes

Oligonucleotides consisting of the isonucleoside repeating unit 2′,5′-anhydro-3′-deoxy-3′-(thymin-1-yl)-d-mannitol (4) were synthesized with the monomeric unit 4 incorporated into oligonucleotides as 1′→4′ linkage 4a (oligomer I) or 6′→4′ linkage 4b (oligomer II). The hybrid properties of the two oligonucleotides I and II with their complementary strands were investigated by thermal denaturation and CD spectra. Oligonucleotide I (4a) formed a stable duplex with d(A)14 with a slightly reduced Tm value of 36.6°C, relative to 38.2°C for the control duplex d(T)14/d(A)14, but oligomer II (4b) failed to hybridize with a DNA complementary single strand. The spectrum of the duplex oligomer I/d(A)14 showed a positive CD band at 217 nm and a negative CD band at 248 nm attributable to a B-like conformation. Molecular modeling showed that in the case of oligomer I the C6′ hydroxy group of each unit could be located in the groove area when hybridized to the DNA single strand, which might contribute additional hydrogen bonding to the stability of duplex formation.

Distinct Modes of Macrophage Recognition for Apoptotic and Necrotic Cells Are Not Specified Exclusively by Phosphatidylserine Exposure

The distinction between physiological (apoptotic) and pathological (necrotic) cell deaths reflects mechanistic differences in cellular disintegration and is of functional significance with respect to the outcomes that are triggered by the cell corpses. Mechanistically, apoptotic cells die via an active and ordered pathway; necrotic deaths, conversely, are chaotic and passive. Macrophages and other phagocytic cells recognize and engulf these dead cells. This clearance is believed to reveal an innate immunity, associated with inflammation in cases of pathological but not physiological cell deaths. Using objective and quantitative measures to assess these processes, we find that macrophages bind and engulf native apoptotic and necrotic cells to similar extents and with similar kinetics. However, recognition of these two classes of dying cells occurs via distinct and noncompeting mechanisms. Phosphatidylserine, which is externalized on both apoptotic and necrotic cells, is not a specific ligand for the recognition of either one. The distinct modes of recognition for these different corpses are linked to opposing responses from engulfing macrophages. Necrotic cells, when recognized, enhance proinflammatory responses of activated macrophages, although they are not sufficient to trigger macrophage activation. In marked contrast, apoptotic cells profoundly inhibit phlogistic macrophage responses; this represents a cell-associated, dominant-acting anti-inflammatory signaling activity acquired posttranslationally during the process of physiological cell death.

The relaxation dynamics of the excited electronic states of retinal in bacteriorhodopsin by two-pump-probe femtosecond studies

We present the results of two-pump and probe femtosecond experiments designed to follow the relaxation dynamics of the lowest excited state (S1) populated by different modes. In the first mode, a direct (S0 → S1) radiative excitation of the ground state is used. In the second mode, an indirect excitation is used where the S1 state is populated by the use of two femtosecond laser pulses with different colors and delay times between them. The first pulse excites the S0 → S1 transition whereas the second pulse excites the S1 → Sn transition. The nonradiative relaxation from the Sn state populates the lowest excited state. Our results suggest that the S1 state relaxes faster when populated nonradiatively from the Sn state than when pumped directly by the S0 → S1 excitation. Additionally, the Sn → S1 nonradiative relaxation time is found to change by varying the delay time between the two pump pulses. The observed dependence of the lowest excited state population as well as its dependence on the delay between the two pump pulses are found to fit a kinetic model in which the Sn state populates a different surface (called S′1) than the one being directly excited (S1). The possible involvement of the Ag type states, the J intermediate, and the conical intersection leading to the S0 or to the isomerization product (K intermediate) are discussed in the framework of the proposed model.

Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA.

Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125-bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle.

Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley.

We describe a complete gene family encoding phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in one particular plant species. In parsley (Petroselinum crispum), the PAL gene family comprises two closely related members, PAL1 and PAL2, whose TATA-proximal promoter and coding regions are almost identical, and two additional members, PAL3 and PAL4, with less similarity to one another and to the PAL1 and PAL2 genes. Using gene-specific probes derived from the 5' untranslated regions of PAL1/2, PAL3, and PAL4, we determined the respective mRNA levels in parsley leaves and cell cultures treated with UV light or fungal elicitor and in wounded leaves and roots. For comparison, the functionally closely related cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL) mRNAs were measured in parallel. The results indicate various degrees of differential responsiveness of PAL4 relative to the other PAL gene family members, in contrast to a high degree of coordination in the overall expression of the PAL, C4H, and 4CL genes. The only significant sequence similarities shared by all four PAL gene promoters are a TATA-proximal set of three putative cis-acting elements (boxes P, A, and L). None of these elements alone, or the promoter region containing all of them together, conferred elicitor or light responsiveness on a reporter gene in transient expression assays. The elements appear to be necessary but not sufficient for elicitor- or light-mediated PAL gene activation, similar to the situation previously reported for 4CL.