Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein

The 2.1-Å resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 → Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore’s excited state to Glu-222. Loss of the Ser-205–Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.     

Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein.

FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control. The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic filament forming proteins such as tubulin. In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity. A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleotides. A lethal ftsZ mutation, ftsZ3 (Rsa), that leads to an amino acid alteration in this homologous segment decreased GTP binding and hydrolysis, suggesting that interaction with GTP is essential for ftsZ function.     

FtsA acts through FtsW to promote cell wall synthesis during cell division in Escherichia coli

In Escherichia coli, FtsQLB is required to recruit the essential septal peptidoglycan (sPG) synthase FtsWI to FtsA, which tethers FtsZ filaments to the membrane. The arrival of FtsN switches FtsQLB in the periplasm and FtsA in the cytoplasm from a recruitment role to active forms that synergize to activate FtsWI. Genetic evidence indicates that the active form of FtsQLB has an altered conformation with an exposed domain of FtsL that acts on FtsI to activate FtsW. However, how FtsA contributes to the activation of FtsW is not clear, as it could promote the conformational change in FtsQLB or act directly on FtsW. Here, we show that the overexpression of an activated FtsA (FtsA*) bypasses FtsQ, indicating it can compensate for FtsQ’s recruitment function. Consistent with this, FtsA* also rescued FtsL and FtsB mutants deficient in FtsW recruitment. FtsA* also rescued an FtsL mutant unable to deliver the periplasmic signal from FtsN, consistent with FtsA* acting on FtsW. In support of this, an FtsW mutant was isolated that was rescued by an activated FtsQLB but not by FtsA*, indicating it was specifically defective in activation by FtsA. Our results suggest that in response to FtsN, the active form of FtsA acts on FtsW in the cytoplasm and synergizes with the active form of FtsQLB acting on FtsI in the periplasm to activate FtsWI to carry out sPG synthesis.

Cell division in Escherichia coli starts with the assembly of the Z-ring, which forms when FtsZ filaments, tethered to the membrane by FtsA and ZipA, coalesce at midcell (1–3). FtsEX joins the ring as it is formed and acts on FtsA to initiate the recruitment of the late cell division proteins (4). Various studies indicate a hierarchy in the recruitment of the late proteins with the order FtsK, FtsQ, FtsLB, FtsW, FtsI, and FtsN (Fig. 1A) (5–7). Despite this hierarchy, there is a web of interactions among these proteins and FtsQ, FtsL, and FtsB, each a bitopic membrane protein, are in a complex, even in the absence of the Z-ring (8). FtsQ is responsible for the localization of the complex to the Z-ring, and FtsL is required to recruit FtsWI (9, 10), which synthesizes septal peptidoglycan (sPG) (11–13). FtsW is a glycosyltransferase and a member of the SEDS family (septation, elongation, division, and sporulation) that functions with its cognate transpeptidase (FtsI [PBP3]) to carry out the two enzymatic steps necessary to incorporate peptidoglycan precursors into the cell wall.Open in a separate windowFig. 1.Recruitment and activation of FtsWI in E. coli. (A) The hierarchical divisome assembly pathway and activation mechanism. The FtsQLB complex lies between FtsK and FtsWI in the pathway. FtsWI is recruited to the Z-ring in an FtsQ-dependent manner with the cytoplasmic domain of FtsL being required to recruit FtsWI. The arrival of FtsN (E domain in yellow and cyto domain in red) switches FtsA in the cytoplasm and FtsQLB in the periplasm to the “on” state, which synergize to activate FtsWI to carry out sPG synthesis. Part of this activation is due to a conformational change in FtsQLB, which exposes a domain of FtsL (AWI) that interacts with FtsI that affects FtsW. The work presented in this study indicates that FtsA* acts directly on FtsW. Note that some cell division proteins are not depicted, including ZipA and FtsEX. Also, the deletion of FtsQ, loss of FtsL^cyto or disruption of the FtsB–FtsQ interaction prevents the recruitment of FtsWI. (B) Bypassing ftsQ with the FtsW–FtsK^cyto fusion and FtsA*. The FtsW–FtsK^cyto fusion was used to bypass the requirement for FtsQ for the recruitment to the Z-ring. This fusion bypasses ftsQ, provided it carries an ftsW* mutation and ftsA* is present. It is likely that FtsL and FtsB are back recruited by the FtsWI complex since they cannot be deleted. (C) Bypass of ftsQ by overexpression of ftsA* in the presence of ftsW*. The results presented herein indicate that, in the absence of FtsQ, FtsA* rescues the recruitment of FtsW by interacting with it in the cytoplasm. FtsA* can also activate FtsWI in the absence of the signal from FtsL^AWI.The FtsQLB complex is conserved in peptidoglycan containing bacteria and, in addition to a role in recruitment, is also involved in regulating sPG synthesis (10). FtsQLB, along with FtsA and FtsWI, is required to recruit FtsN, which leads to the activation of FtsWI to synthesize sPG, which is essential for cell division (14–16). FtsN may also stimulate PBP1b, which contributes to sPG synthesis, but this activity is not essential (17–20). In the current model, the arrival of FtsN switches both FtsQLB and FtsA to an active state, which synergize to activate FtsWI (21, 22). This step requires two domains of FtsN; FtsN^cyto acts on FtsA in the cytoplasm and FtsN^E, a short sequence of ∼20 amino acts in the periplasmic domain, likely acts on FtsQLB (21) (Fig. 1A). How FtsA contributes to the activation is not clear.Although signaling by both domains of FtsN is normally required, hyperactivation of either signal alone is sufficient for viability. Such cells are filamentous in rich media however, suggesting that activation is not fully restored (21, 22). The periplasmic signal can be hyperactivated by overexpressing FtsN^E in the periplasm or by “activation (superfission)” mutations in ftsB or ftsL (4, 21). These activation mutations appear to mimic FtsN action, causing a region of FtsL (called AWI) to contact FtsI, which activates FtsW (23). The cytoplasmic signal can be hyperactivated by the overexpression of certain “activated” ftsA alleles (ftsA^E124A and ftsA^I143L), which may act through the FtsQLB complex or act directly on FtsWI (21, 24). A mutant of FtsEX that is unable to hydrolyze ATP blocks the cytoplasmic signal, but this can be overcome by the hyperactivation of the periplasmic signal (4, 25) or a mutation in ftsW, which is thought to produce a constitutively active FtsW that is less dependent upon FtsN (4).Beckwith’s group provided evidence that the cytoplasmic signal generated by FtsN does not go through FtsQ. They isolated FtsA^I143L as a suppressor of an FtsQ mutant (V92D) defective in localization, and surprisingly, it restored localization, even in the absence of the cytoplasmic domain of FtsQ (the only region of FtsQ that could contact FtsA) (9). Although FtsA^I143L was unable to bypass ftsQ, it rescued another FtsQ mutant (FtsQ^A252P) unable to bind FtsB and therefore recruit FtsW. Another FtsA allele (FtsA^R286W) also suppressed these deficiencies but to different degrees. As a result, it was concluded that these activated FtsA mutants do not interact with FtsQ but somehow stabilize the divisome, possibly by interacting with another late division protein. Many additional activated FtsA mutants have been isolated, and most show a reduction in self-interaction (26).Other attempts to bypass ftsQ in E. coli have been unsuccessful, although it has been bypassed in several gram-positive bacteria (27–30). Overexpression of ftsA^R286W (hereafter ftsA*), which bypasses ftsK, the gene immediately upstream of ftsQ in the recruitment pathway (Fig. 1A), did not bypass ftsQ (25). Attempts to bypass ftsL or ftsB using the same conditions that bypass ftsK also failed (27). Nonetheless, a ZapA–FtsL fusion, which targets FtsL directly to the Z-ring, recruits FtsB in the absence of FtsQ and also recruits FtsWI, indicating that FtsL and FtsB retain some function in the absence of FtsQ as in gram-positive bacteria (14).Although FtsQLB is involved in activating FtsWI in response to FtsN, how an activated FtsA (FtsA*) contributes to this activation is not clear (21, 22). In vitro reconstitution confirmed that FtsWI was a PG synthase and was activated by FtsQLB; however, the system did not fully recapitulate the in vivo regulation, as FtsN had no effect (12, 13). Furthermore, FtsA was not included. In this study, we set out to explore the role of FtsA in activation. Our results indicate that an FtsA* contributes to the activation of FtsWI by acting on FtsW.     

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.     

GTP-dependent polymerization of Escherichia coli FtsZ protein to form tubules.

The FtsZ protein is a GTPase that is essential for cell division in Escherichia coli. During cytokinesis, FtsZ localizes to a ring at the leading edge of septum synthesis. We report the GTP-dependent polymerization of purified FtsZ measured by sedimentation and light scattering. Electron microscopy of polymerized FtsZ revealed structures including tubules 14-20 nm in diameter with longitudinal arrays of protofilaments. FtsZ depolymerized upon removal of GTP and repolymerized after subsequent GTP addition. Mutant FtsZ84 protein polymerized inefficiently, suggesting that polymerization is important for the cellular role of FtsZ in division. The possibility that tubules of FtsZ protein form a cytoskeleton involved in septum synthesis is consistent with our data.     

Role of the SulB (FtsZ) protein in division inhibition during the SOS response in Escherichia coli: FtsZ stabilizes the inhibitor SulA in maxicells.

Induction of the SOS response in Escherichia coli by DNA-damaging treatments results in the synthesis of the SulA polypeptide, and this is sufficient to cause the resulting inhibition of cell division. Mutations at either sulA (sfiA) or sulB (sfiB) suppress this division inhibition. The SulB protein is identical to FtsZ, a protein required for normal division in E. coli. In the presence of FtsZ, the half-life of SulA synthesized in maxicells is approximately 12 min. In contrast, in the absence of FtsZ or in the presence of a mutant form of FtsZ (SulB114) that prevents division inhibition in vivo, SulA is extremely unstable with a half-life of only 3 min. Both FtsZ and SulA are isolated with the inner membrane of E. coli maxicells in the presence of MgCl2. We propose that the SulA inhibitor interacts directly with FtsZ in vivo to block the essential division function of this protein.     

The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization

Positioning of the Z ring at the midcell site in Escherichia coli is assured by the min system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.     

Force generation by a dynamic Z-ring in Escherichia coli cell division

FtsZ, a bacterial homologue of tubulin, plays a central role in bacterial cell division. It is the first of many proteins recruited to the division site to form the Z-ring, a dynamic structure that recycles on the time scale of seconds and is required for division to proceed. FtsZ has been recently shown to form rings inside tubular liposomes and to constrict the liposome membrane without the presence of other proteins, particularly molecular motors that appear to be absent from the bacterial proteome. Here, we propose a mathematical model for the dynamic turnover of the Z-ring and for its ability to generate a constriction force. Force generation is assumed to derive from GTP hydrolysis, which is known to induce curvature in FtsZ filaments. We find that this transition to a curved state is capable of generating a sufficient force to drive cell-wall invagination in vivo and can also explain the constriction seen in the in vitro liposome experiments. Our observations resolve the question of how FtsZ might accomplish cell division despite the highly dynamic nature of the Z-ring and the lack of molecular motors.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Identification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein

We describe a method for identifying genes encoding proteins with stereospecific intracellular localizations in the fission yeast Schizosaccharomyces pombe. Yeast are transformed with a gene library in which S. pombe genomic sequences are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP), and intracellular localizations are subsequently identified by rapid fluorescence screening in vivo. In a model application of these methods to the fission yeast nucleus, we have identified several novel genes whose products are found in specific nuclear regions, including chromatin, the nucleolus, and the mitotic spindle, and sequence similarities between some of these genes and previously identified genes encoding nuclear proteins have validated the approach. These methods will be useful in identifying additional components of the S. pombe nucleus, and further extensions of this approach should also be applicable to a more comprehensive identification of the elements of intracellular architecture in fission yeast.     

YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae, localizes in codependent fashion with FtsL to the division site

YgbQ is a cell division protein in Escherichia coli and Vibrio cholerae. In E. coli the ygbQ gene was discovered as a result of a computer search of the E. coli genome designed to find potential interacting partners for cell division protein FtsL. In V. cholerae, ygbQ was identified as an essential gene by using a transposon that fuses genes to an arabinose promoter. The role of YgbQ in cell division is supported by the following. Cells depleted of YgbQ in both organisms form long filaments, but DNA segregation is not affected. YgbQ localizes to the constriction site in wild-type E. coli cells. Localization of E. coli YgbQ to the constriction site depends on cell division proteins FtsQ and FtsL but not FtsW and FtsI, placing YgbQ in the sequential dependency order of proteins localizing to the division site. Localization of green fluorescent protein-FtsL also depends on YgbQ, indicating that FtsL and YgbQ colocalize to the division site in E. coli. Our results show colocalization of proteins to the bacterial midcell in E. coli and raise the possibility that these proteins interact in a coiled-coil structure.     

Robustness and accuracy of cell division in Escherichia coli in diverse cell shapes

Cell division in typical rod-shaped bacteria such as Escherichia coli shows a remarkable plasticity in being able to adapt to a variety of irregular cell shapes. Here, we investigate the roles of the Min system and the nucleoid-occlusion factor SlmA in supporting this adaptation. We study "squeezed" E. coli in narrow nanofabricated channels where these bacteria exhibit highly irregular shapes and large volumes. Despite the severely anomalous morphologies we find that most of these bacteria maintain their ability to divide into two equally sized daughters with an accuracy comparable to that of normal rod-shaped cells (about 4%). Deletion of either slmA or minC shows that the molecular systems associated with these genes are largely dispensable for accurate cell division in these irregular cell shapes. Using fluorescence time-lapse microscopy, we determine that the functionality of the Min system is affected by the cell shape, whereas the localization of a nucleoid relative to the cell division proteins (the divisome) remains unperturbed in a broad spectrum of morphologies, consistent with nucleoid occlusion. The observed positioning of the nucleoid relative to the divisome appears not to be affected by the nucleoid-occlusion factor SlmA. The current study underscores the importance of nucleoid occlusion in positioning the divisome and shows that it is robust against shape irregularities.     

Recombinational construction in Escherichia coli of infectious adenoviral genomes

A two-step gene replacement procedure was developed that generates infectious adenoviral genomes through homologous recombination in Escherichia coli. As a prerequisite, a human adenovirus serotype 5 (Ad5)-derived genome was first introduced as a PacI restriction fragment into an incP-derived replicon which, in contrast to ColE1-derivatives (e.g., pBR322 or pUC plasmids), is functional in a polA mutant of E. coli. Any modification can be introduced at will following two consecutive homologous recombinations between the incP/Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the in vitro engineering of the ColE1-derivative by flanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP/Ad5 replicon and the kanamycin-resistant ColE1-derivative is selected by growing the polA host in the presence of both antibiotics. Resolution of this cointegrate is further selected in sucrose growth conditions due to the loss of a conditional suicide marker (the sacB gene of Bacillus subtilis) present in the ColE1 plasmid, leading to unmodified and modified incP/Ad5 replicons that can be differentiated upon restriction analysis. Consecutive rounds of this two-step cloning procedure allowed the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure is demonstrated by the recovery of several E1E3E4-deleted adenoviruses following transfection of the corresponding E. coli-derived genomes in IGRP2 cells.     

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.Plasmids serve as extrachromosomal DNA platforms for the reassortment, mobilization, and maintenance of antibiotic resistance genes in bacteria, enabling host cells to colonize environments flooded with antimicrobials and to take advantage of resources freed by the extinction of nonresistant competitors. Fueled by these selective forces and aided by their itinerant nature, plasmids disseminate resistance genes worldwide shortly after new antibiotics are developed, which is a major clinical concern (1–3). However, in antibiotic-free environments, such genes are dispensable. There, the cost that plasmid carriage imposes on cells constitutes a disadvantage in the face of competition from other cells and, because plasmids depend on their hosts to survive, also a threat to their own existence.Many plasmids keep low copy numbers (CNs) to minimize the problem above, because it reduces burdens to host cells. However, this also decreases their chances to fix in descendant cells, a new survival challenge (4). To counteract this, plasmids have evolved stability functions. Partition systems pull replicated plasmid copies to opposite poles in host cells, facilitating their inheritance by daughter cells (5). Plasmids also bear postsegregational killing (PSK) systems, which encode a stable toxin and a labile antitoxin (TA) pair that eliminates plasmid-free cells produced by occasional replication or partition failures. Regular production of the labile antitoxin protects plasmid-containing cells from the toxin. However, antitoxin replenishment is not possible in cells losing the plasmid, and this triggers their elimination (5).TA pairs are common in plasmids disseminating antibiotic resistance in bacterial pathogens worldwide (2, 6–10). The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5, 11–13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids. Although PSK systems encode TA pairs, not all TA pairs might function as PSK systems, as suggested by their abundance in bacterial chromosomes, where PSK seems unnecessary (14–16). Moreover, the observation that many plasmids bear several TA pairs (6–10) raises the intriguing question of why they would need more than one PSK system, particularly when they increase the metabolic burden that plasmids impose on host cells (17). Because PSK functions are not infallible, their gathering may provide a mechanism for reciprocal failure compensation, minimizing the number of cells that escape killing upon plasmid loss (5). Alternatively, some TA pairs may stabilize plasmids by mechanisms different from PSK, and their grouping might not necessarily reflect functional redundancy (18).This may be the case in plasmid R1, which encodes TA pairs hok-sok (host killing-suppressor of killing) and kis(pemI)-kid(pemK) (19–23). Inconsistent with PSK, we had noticed that activation of toxin Kid occurred in cells that still contained R1, and that this happened when CNs were insufficient to ensure plasmid transmission to descendant cells. We also found that Kid cleaved mRNA at UUACU sites, which appeared well suited to trigger a response that prevented plasmid loss and increased R1 CNs without killing cells, as suggested by our results. In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25, 26), a view that has prevailed in the literature (14, 16, 27–29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30, 31). Importantly, Kid, Kis, and other elements that we found essential for R1 rescue are conserved in plasmids conferring resistance to extended-spectrum β-lactamases, a worrying threat to human health (1, 6–10, 32). Therapeutic options to fight pathogens carrying these plasmids are limited, and activation of Kid may be perceived as a good antibiotic alternative. Because the potential involvement of this toxin in plasmid rescue advises against such approach, we aimed to ascertain here the mode of action; the effects on cells; and, ultimately, the function of Kid (and Kis) in R1.     

Probing protein conformational changes in living cells by using designer binding proteins: application to the estrogen receptor

A challenge in understanding the mechanism of protein function in biology is to establish the correlation between functional form in the intracellular environment and high-resolution structures obtained with in vitro techniques. Here we present a strategy to probe conformational changes of proteins inside cells. Our method involves: (i) engineering binding proteins to different conformations of a target protein, and (ii) using them to sense changes in the surface property of the target in cells. We probed ligand-induced conformational changes of the estrogen receptor alpha (ER alpha) ligand-binding domain (LBD). By using yeast two-hybrid techniques, we first performed combinatorial library screening of "monobodies" (small antibody mimics using the scaffold of a fibronectin type III domain) for clones that bind to ER alpha and then characterized their interactions with ER alpha in the nucleus, the native environment of ER alpha, in the presence of various ligands. A library using a highly flexible loop yielded monobodies that specifically recognize a particular ligand complex of ER alpha, and the pattern of monobody specificity was consistent with the structural differences found in known crystal structures of ER alpha-LBD. A more restrained loop library yielded clones that bind both agonist- and antagonist-bound ER alpha. Furthermore, we found that a deletion of the ER alpha F domain that is C-terminally adjacent to the LBD increased the crossreactivity of monobodies to the apo-ER alpha-LBD, suggesting a dynamic nature of the ER alpha-LBD conformation and a role of the F domain in restraining the LBD in an inactive conformation.     

Cell cycle regulation and cell type-specific localization of the FtsZ division initiation protein in Caulobacter.

Many genes involved in cell division and DNA replication and their protein products have been identified in bacteria; however, little is known about the cell cycle regulation of the intracellular concentration of these proteins. It has been shown that the level of the tubulin-like GTPase FtsZ is critical for the initiation of cell division in bacteria. We show that the concentration of FtsZ varies dramatically during the cell cycle of Caulobacter crescentus. Caulobacter produce two different cell types at each cell division: (i) a sessile stalked cell that can initiate DNA replication immediately after cell division and (ii) a motile swarmer cell in which DNA replication is blocked. After cell division, only the stalked cell contains FtsZ. FtsZ is synthesized slightly before the swarmer cells differentiate into stalked cells and the intracellular concentration of FtsZ is maximal at the beginning of cell division. Late in the cell cycle, after the completion of chromosome replication, the level of FtsZ decreases dramatically. This decrease is probably mostly due to the degradation of FtsZ in the swarmer compartment of the predivisional cell. Thus, the variation of FtsZ concentration parallels the pattern of DNA synthesis. Constitutive expression of FtsZ leads to defects in stalk biosynthesis suggesting a role for FtsZ in this developmental process in addition to its role in cell division.     

Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein

Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.     

Direct observation of the enhancement of noncooperative protein self-assembly by macromolecular crowding: indefinite linear self-association of bacterial cell division protein FtsZ

Recent measurements of sedimentation equilibrium and sedimentation velocity have shown that the bacterial cell division protein FtsZ self-associates to form indefinitely long rod-like linear aggregates in the presence of GDP and Mg(2+). In the present study, the newly developed technique of non-ideal tracer sedimentation equilibrium was used to measure the effect of high concentrations-up to 150 g/liter-of each of two inert "crowder" proteins, cyanmethemoglobin or BSA, on the thermodynamic activity and state of association of dilute FtsZ under conditions inhibiting (-Mg(2+)) and promoting (+Mg(2+)) FtsZ self-association. Analysis of equilibrium gradients of both FtsZ and crowder proteins indicates that, under the conditions of the present experiment, FtsZ interacts with each of the two crowder proteins essentially entirely via steric repulsion, which may be accounted for quantitatively by a simple model in which hemoglobin, albumin, and monomeric FtsZ are modeled as effective spherical hard particles, and each oligomeric species of FtsZ is modeled as an effective hard spherocylinder. The functional dependence of the sedimentation of FtsZ on the concentrations of FtsZ and either crowder indicates that, in the presence of high concentrations of crowder, both the weight-average degree of FtsZ self-association and the range of FtsZ oligomer sizes present in significant abundance are increased substantially.     

Cloning of Escherichia coli DNA that controls cell division and capsular polysaccharide synthesis

A 2 × 10⁶ dalton DNA fragment that controls cell division, capsular polysaccharide synthesis, and enzymes of capsular polysaccharide synthesis has been cloned.     

A GTP-binding protein of Escherichia coli has homology to yeast RAS proteins.

The DNA sequence of a gene (era) located immediately downstream of the gene (rnc) encoding ribonuclease III of Escherichia coli was determined and found to encode a protein of 316 amino acid residues. The amino acid sequence of this protein, Era, has significant similarity to the yeast RAS proteins. Overexpression of the Era protein was achieved and GTP cross-linking experiments demonstrated that the protein was indeed capable of binding GTP, as are the yeast and mammalian ras gene products. These data indicate that ras-related sequences occur not only in eukaryotes but also in prokaryotes.     

Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli

Accurate placement of the division septum at the midpoint of Escherichia coli cells requires the combined action of a general division inhibitor (MinC), a site-specific suppressor of division inhibition (MinE), and an ATPase (MinD) that is required for proper functioning of both MinC and MinE. We previously showed that a functional MinE-Gfp fusion accumulates in a ring structure at/near the middle of cells. Here we show that functional Gfp-MinD accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle imposed by MinE. The results indicate that MinD represents a novel type of dynamic cellular element in bacteria, with multiple roles in directing the division apparatus to the middle of the cell.