The molecular puzzle of two-component signaling cascades.

Two-component systems constitute prevalent signaling pathways in bacteria and mediate a large variety of adaptative cellular responses. Signaling proceeds through His-Asp phosphorelay cascades that involve two central partners, the histidine protein kinase and the response regulator protein. Structural studies have provided insights into some design principles and activation mechanisms of these multi-domain proteins implicated in the control of virulence gene expression in several pathogens.

Effects of ligand binding on the association properties and conformation in solution of retinoic acid receptors RXR and RAR.

In higher eukaryotes, vitamin A derived metabolites such as 9-cis and all-trans retinoic acid (RA), are involved in the regulation of several essential physiological processes. Their pleiotropic physiological effects are mediated through direct binding to cognate nuclear receptors RXRs and RARs that act as regulated transcription factors belonging to the superfamily of nuclear hormone receptors. Hormone binding to the structurally conserved ligand-binding domain (LBD) of these receptors triggers a conformational change that principally affects the conserved C-terminal transactivation helix H12 involved in transcriptional activation. We report an extensive biophysical solution study of RAR alpha, RXR alpha LBDs and their corresponding RXR alpha/RAR alpha LBD heterodimers combining analytical ultracentrifugation (AUC), small-angle X-ray and neutron scattering (SAXS and SANS) and ab initio three-dimensional shape reconstruction at low resolution. We show that the crystal structures of RXRs and RARs LBDs correlate well with the average conformations observed in solution. Furthermore we demonstrate the effects of 9-cisRA and all-transRA binding on the association properties and conformations of RXR alpha and RAR alpha LBDs in solution. The present study shows that in solution RAR alpha LBD behaves as a monomer in both unliganded and liganded forms. It confirms the existence in solution of a ligand-induced conformational change towards a more compact form of the LBD. It also confirms the stability of the predicted RXR alpha/RAR alpha LBD heterodimers in solution. SAS measurements performed on three different types of RXR alpha/RAR alpha LBD heterodimers (apo/apo, apo/holo and holo/holo) with respect to their ligand-binding site occupancy show the existence of three conformational states depending on the progressive binding of RA stereoisomers on RAR alpha and RXR alpha LBD subunits in the heterodimeric context. These results suggest that the subunits are structurally independent within the heterodimers. Our study also underlines the particular behaviour of RXR alpha LBD. In solution unliganded RXR alpha LBD is observed as two species that are unambiguously identified as homotetramers and homodimers. Molecular modelling combined with SAS data analysis allows us to propose a structural model for this autorepressed apo-tetramer. In contrast to the monomeric state observed in the crystal structure, our data show that in solution active holo-RXR alpha LBD bound to 9-cisRA is a homodimer regardless of the protein concentration. This study demonstrates the crucial role of ligands in the regulation of homodimeric versus heterodimeric association state of RXR in the NR signalling pathways.

Further insights into the mechanism of function of the response regulator CheY from crystallographic studies of the CheY--CheA(124--257) complex.

New crystallographic structures of the response regulator CheY in association with CheA(124--257), its binding domain in the kinase CheA, have been determined. In all crystal forms, the molecular interactions at the heterodimer interface are identical. Soaking experiments have been performed on the crystals using acetyl phosphate as phosphodonor to CheY. No phosphoryl group attached to Asp57 of CheY is visible from the electron density, but the response regulator in the CheY-CheA(124--257) complex may have undergone a phosphorylation-dephosphorylation process. The distribution of water molecules and the geometry of the active site have changed and are now similar to those of isolated CheY. In a second soaking experiment, imido-diphosphate, an inhibitor of the phosphorylation reaction, was used. This compound binds in the vicinity of the active site, close to the N-terminal part of the first alpha-helix. Together, these results suggest that the binding of CheY to CheA(124--257) generates a geometry of the active site that favours phosphorylation and that imido-diphosphate interferes with phosphorylation by precluding structural changes in this region.

Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein.

The Nef protein of human immunodeficiency virus type I (HIV-1) is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.

Synergistic transcriptional activation by TATA-binding protein and hTAFII28 requires specific amino acids of the hTAFII28 histone fold.

Coexpression of the human TATA-binding protein (TBP)-associated factor 28 (hTAFII28) with the altered-specificity mutant TBP spm3 synergistically enhances transcriptional activation by the activation function 2 of the nuclear receptors (NRs) for estrogen and vitamin D3 from a reporter plasmid containing a TGTA element in mammalian cells. This synergy is abolished by mutation of specific amino acids in the alpha2-helix of the histone fold in the conserved C-terminal region of hTAFII28. Critical amino acids are found on both the exposed hydrophilic face of this helix and the hydrophobic interface with TAFII18. This alpha-helix of hTAFII28 therefore mediates multiple interactions required for coactivator activity. We further show that mutation of specific residues in the H1' alpha-helix of TBP either reduces or increases interactions with hTAFII28. The mutations which reduce interactions with hTAFII28 do not affect functional synergy, whereas the TBP mutation which increases interaction with hTAFII28 is defective in its ability to synergistically enhance activation by NRs. However, this TBP mutant supports activation by other activators and is thus specifically defective for its ability to synergize with hTAFII28.

Conformational changes induced by phosphorylation of the FixJ receiver domain.

BACKGROUND: A variety of bacterial adaptative cellular responses to environmental stimuli are mediated by two-component signal transduction pathways. In these phosphorelay cascades, histidine kinases transphosphorylate a conserved aspartate in the receiver domain, a conserved module in the response regulator superfamily. The main effect of this phosphorylation is to alter the conformation of the response regulator in order to modulate its biological function. The response regulator FixJ displays a typical modular arrangement, with a phosphorylatable N-terminal receiver domain and a C-terminal DNA-binding domain. In the symbiotic bacterium Sinorhizobium meliloti, phosphorylation of this response regulator activates transcription of nitrogen-fixation genes. RESULTS: The crystal structures of the phosphorylated and of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) were solved at 2.3 A and 2.4 A resolution, respectively. They reveal the environment of the phosphoaspartate in the active site and the specific conformational changes leading to activation of the response regulator. Phosphorylation of the conserved aspartate induces major structural changes in the beta 4-alpha 4 loop, and in the signaling surface alpha 4-beta 5 that mediates dimerization of the phosphorylated full-length response regulator. A site-directed mutant at this protein-protein interface decreases the affinity of the phosphorylated response regulator for the fixK promoter tenfold. CONCLUSIONS: The cascade of phosphorylation-induced conformational changes in FixJN illustrates the role of conserved residues in stabilizing the phosphoryl group in the active site, triggering the structural transition and achieving the post-phosphorylation signaling events. We propose that these phosphorylation-induced conformational changes underly the activation of response regulators in general.

Structural transitions in the FixJ receiver domain.

BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.

Human TAF(II)28 and TAF(II)18 interact through a histone fold encoded by atypical evolutionary conserved motifs also found in the SPT3 family.

Determination of the crystal structure of the human TBP-associated factor (hTAF(II))28/hTAF(II)18 heterodimer shows that these TAF(II)s form a novel histone-like pair in the TFIID complex. The histone folds in hTAF(II)28 and hTAF(II)18 were not predicted from their primary sequence, indicating that these TAF(II)s define a novel family of atypical histone fold sequences. The TAF(II)18 and TAF(II)28 histone fold motifs are also present in the N- and C-terminal regions of the SPT3 proteins, suggesting that the histone fold in SPT3 may be reconstituted by intramolecular rather than classical intermolecular interactions. The existence of additional histone-like pairs in both the TFIID and SAGA complexes shows that the histone fold is a more commonly used motif for mediating TAF-TAF interactions than previously believed.

Crystal structure of the arcelin-1 dimer from Phaseolus vulgaris at 1.9-A resolution.

Arcelin-1 is a glycoprotein from kidney beans (Phaseolus vulgaris) which displays insecticidal properties and protects the seeds from predation by larvae of various bruchids. This lectin-like protein is devoid of monosaccharide binding properties and belongs to the phytohemagglutinin protein family. The x-ray structure determination at 1.9-A resolution of native arcelin-1 dimers, which correspond to the functional state of the protein in solution, was solved using multiple isomorphous replacement and refined to a crystallographic R factor of 0.208. The three glycosylation sites on each monomer are all covalently modified. One of these oligosaccharide chains provides interactions with protein atoms at the dimer interface, and another one may act by preventing the formation of higher oligomeric species in the arcelin variants. The dimeric structure and the severe alteration of the monosaccharide binding site in arcelin-1 correlate with the hemagglutinating properties of the protein, which are unaffected by simple sugars and sugar derivatives. Sequence analysis and structure comparisons of arcelin-1 with the other insecticidal proteins from kidney beans, arcelin-5, and alpha-amylase inhibitor and with legume lectins, yield insights into the molecular basis of the different biological functions of these proteins.

Structure of the CheY-binding domain of histidine kinase CheA in complex with CheY.

Bacterial adaptation to the environment is accomplished through the coordinated activation of specific sensory receptors and signal processing proteins. Among the best characterized of these pathways are those which employ the two-component paradigm. In these systems, signal transmission is mediated by Mg(2+)-dependent phospho-relay reactions between histidine auto-kinases and phospho-accepting receiver domains in response-regulator proteins. Although this mechanism of activation is common to all response-regulators, detrimental cross-talk between different two-component pathways within the same cell is minimized through the use of specific recognition domains. Here, we report the crystal structure, at 2.95 A resolution, of the response regulator of bacterial chemotaxis, CheY, bound to the recognition domain from its cognate histidine kinase, CheA. The structure suggests that molecular recognition, in this low affinity complex (KD = 2 microM), may also contribute to the mechanism of CheY activation.

Functional and structural analysis of the subunits of human transcription factor TFIID.

The past few years have brought many new insights concerning the structure and function of TAFII proteins. In the future, further biochemical and structural studies will no doubt lead to a greater understanding of the molecular organization of TFIID complexes. A better understanding of the function of metazoan, in particular, mammalian, TAFIIs in cell cycle progression and gene activation will, however, require the use of novel genetic techniques in addition to the biochemical analyses.

Is the function of the cdc2 kinase subunit proteins tuned by their propensities to oligomerize? Conformational states in solution of the cdc2 kinase partners p13suc1 and p9cksphy.

The cdc2 kinase subunit (cks) proteins play an essential function in the control of mitosis through their molecular complexes with the cdc2 kinase. In this work, we characterize the conformational state(s) in solution of the cks proteins p13suc1 from Schizosaccharomyces pombe and p9cksphy from Physarum polycephalum. Monomers of p13suc1 and p9cksphy were found to be markedly nonglobular, presumably with a long, nonfolded C-terminal moiety. This was in contrast to the previously published structure of p13suc1, derived from crystallographic studies on a zinc-promoted p13suc1 dimer, in which the individual p13suc1 subunits had a globular conformation. This disparity was resolved when we found that the globular p13suc1 fold undergoes a conformational transition into nonglobular monomers upon dissociation of the dimers following chelation of the zinc ions by ethylenediaminetetraacetic acid (EDTA). We also found that p13suc1, but not p9cksphy, forms stable dimers in the absence of metal ions. The topology of these EDTA-insensitive dimers likely resembles that of the human p9ckshs2 protein, characterized by beta 4 strand exchange from each nonglobular monomer.

Characterisation of human cdc2 lysine 33 mutations expressed in the fission yeast Schizosaccharomyces pombe.

The mammalian p34cdc2 protein kinase, a universal cell cycle regulator, complements cdc2/CDC28 temperature-sensitive mutations in yeasts. We report the biochemical characterisation of two substitutions of human cdc2 at lysine 33, a residue involved in nucleotide binding, that differently alter the fission yeast cell cycle. K33A-hscdc2 and K33R-hscdc2 mutants are both catalytically inactive, but overexpression of K33R-cdc2 is lethal while K33A-cdc2 is not. We show that human K33R-cdc2 acts as a dominant negative allele that associates yeast cdc13/cyclinB and therefore renders endogeneous Schizosaccharomyces pombe cdc2 unactivatable. These results are discussed on the light of the molecular modeling of the mutants in the cdc2 model structure.

Oligomerization state in solution of the cell cycle regulators p13suc1 from the fission yeast and p9cksphy from the myxomycete Physarum, two members of the cks family.

The cks proteins (for cdc2 kinase subunit) are essential cell cycle regulators. They interact strongly with the mitotic cdc2 kinase, but the mechanism and the biological function of this association still await understanding. The oligomerization state in solution of two members of this ubiquitous protein family, the suc1 gene product from the fission yeast and the newly cloned cksphy gene product from the myxomycete Physarum, was investigated by small-angle X-ray scattering (SAXS) and biochemical methods. We found that the major molecular species are monodispersed monomeric proteins. Minor amounts of dimeric suc1 proteins were also found, but no equilibrium between the two forms was observed and surprisingly, the hexameric assemblies observed in the crystal structure of the human ckshs2 homolog were not detected. These apparent discrepancies between proteins that display cross-complementation address the question of the control of the cks oligomerization process and its link to the biological function.

High cell density-dependent resistance and P-glycoprotein-mediated multidrug resistance in mitoxantrone-selected Chinese hamster cells.

Mitoxantrone (MIT) resistance has been studied in a colony selected from the CHO AA8 parental line in one step under a low degree of selective pressure (9 nM). The cells of the clonal isolate AA8/MIT C1(0) were sensitive to 9 nM MIT at low cell density but able to grow at high density. Parental AA8 cells were not able to grow under the latter condition. Decreased MIT accumulation (-20%) was observed at this step (step 0) in the absence of overexpression of mdr RNA coding for the drug efflux pump P-glycoprotein. Furthermore, AA8/MIT C1(0) did not exhibit cross resistance to vincristine, Adriamycin and etoposide at low cell density. During subsequent controlled growth for 2 months at high cell density in the presence of 9 nM drug, an additional selection occurred leading to a 4-fold MIT-resistant subline AA8/MIT C1(+). This subline was characterized at this step (step I) and after an additional 4 months of culture in the presence of 9 nM MIT (step II). Analysis of mdr gene expression and gene copy number showed an increase in mdr RNA and a pattern of mdr gene amplification which changed between step I and II. AA8/MIT C1(+)II exhibited a classical multidrug resistance phenotype with decreased accumulation of [14C]MIT and cross-resistance to vincristine, Adriamycin and etoposide. The ability to form the cleavable complex in the presence of etoposide in DNA topoisomerase II-containing nuclear extracts was identical in AA8/MIT C1(+)II and AA8 cell lines. These results demonstrate a new sequence of events in MIT resistance: low level of drug resistance at high cell density followed by mdr gene amplification.