Sister chromatid cohesion: the beginning of a long and beautiful relationship.

Our understanding of the mechanism of sister chromatid cohesion has advanced significantly with the recent identification and characterization of important regulatory factors, structural factors and chromosomal cohesion sites. These analyses reveal a surprisingly complex mechanism of cohesion that is just beginning to be elucidated and exciting connections between cohesion, cell-cycle regulation and other forms of DNA metabolism.

A response regulator model in a simple sensory system.

Bacterial behavior is shown to be modulated through a simple on-off switching device which directs migration toward favorable conditions and away from unfavorable ones. The behavioral response is controlled by a rudimentary memory which allows the bacteria to sense gradients over time. The memory can be explained by a biochemical system involving a response regulator whose level relative to a threshold controls flagellar function. The level of the response regulator is itself controlled by factors such as enzyme levels and environmental stimuli. The molecular basis of the model appears to be relevant to more complex hormonal and neural signaling systems.

NO news is good news.

A startlingly simple molecule unites neuroscience, physiology, and immunology, and revises scientists' understanding of how cells communicate and defend themselves.

Intrasubunit signal transduction by the aspartate chemoreceptor.

Receptors that transmit signals across cell membranes are typically composed of multiple subunits. To test whether subunit interactions are required for transmembrane signaling by the bacterial aspartate receptor, dimers were constructed with (i) two full-length subunits, (ii) one full-length subunit and one subunit lacking the cytoplasmic domain, or (iii) one full-length subunit and one subunit lacking both the cytoplasmic and the transmembrane domains. Methylation of the cytoplasmic domain of all three receptor constructs was stimulated by the binding of aspartate. These findings demonstrate that transmembrane signaling does not require interactions between cytoplasmic or transmembrane domains of adjacent subunits and suggest that signaling occurs via conformational changes transduced through a single subunit.

Global flexibility in a sensory receptor: a site-directed cross-linking approach.

The aspartate receptor of Escherichia coli and Salmonella typhimurium is a cell surface sensory transducer that binds extracellular aspartate and sends a transmembrane signal to the inside of the bacterium. The flexibility and allostery of this receptor was examined by placing sulfhydryl groups as potential cross-linking sites at targeted locations in the protein. Seven different mutant receptors were constructed, each containing a single cysteine residue at a different position in the primary structure. Intramolecular disulfide bond formation within oligomers of these mutant receptors is shown to trap structural fluctuations and to detect ligand-induced changes in structure. The results indicate that the receptor oligomer has a flexible, dynamic structure which undergoes a global change upon aspartate binding.

Common mechanism for repellents and attractants in bacterial chemotaxis.

The migrational response of Salmonella typhimurium away from compounds such as phenol, indole, acetic acid, and leucine occurs because the bacteria tumble less frequently while descending gradients of repellents. This contrasts with their response of tumbling less frequently while ascending gradients of attractants. The results of competition experiments suggest that repellents, like attractants, operate through specific receptors, and the algebraic additivity experiments indicate that repellents and attractants utilize a common memory mechanism for taxis.

Covalent enzyme-substrate intermediates.

A literature search reveals 60 cases in which there is strong evidence for covalent enzyme-substrate intermediates.

Separation of signal transduction and adaptation functions of the aspartate receptor in bacterial sensing.

In order to investigate the functions of stimulus recognition, signal transduction, and adaptation, the aspartate receptor gene for bacterial chemotaxis in Salmonella typhimurium has been sequenced and modified. A carboxyl-terminal truncated receptor was shown to bind aspartate and to transmit a signal to change motility behavior. However, the truncated receptor showed greatly reduced methyl-accepting capacity, and did not allow adaptation to the sensory stimulation. The separation of receptor functions by alteration of primary structure emphasizes that the receptor is directly involved in adaptation and is not solely a device for transmitting a signal across a membrane.

Mg2+, Ca2+-dependent adenosine triphosphatase as receptor for divalent cations in bacterial sensing.

The Mg2+, Ca2+-dependent adenosine triphosphatase responsible for interconversion of the energized membrane state to adenosine triphosphate is the receptor for divalent metal ions in bacterial chemotaxis. The receptors controlling bacterial behavior show a common pattern of dual functions.

Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase.

The isocitrate dehydrogenase of Escherichia coli is regulated by covalent modification at the active site rather than, as expected, at an allosteric site. As a means of evaluating the mechanism of regulation, the kinetics of the substrate, 2R,3S-isocitrate, and a substrate analog, 2R-malate, were compared for the native, phosphorylated, and mutant enzymes. Phosphorylation decreases activity by more than a factor of 10(6) for the true substrate, but causes minor changes in the activity of the substrate analog. The kinetic results indicate that electrostatic repulsion and steric hindrance between the phosphoryl moiety and the gamma carboxyl group of 2R,3S-isocitrate are the major causes of the inactivation, with a lesser contribution from the loss of a hydrogen bond.

Orbital steering in the catalytic power of enzymes: small structural changes with large catalytic consequences.

Small structural perturbations in the enzyme isocitrate dehydrogenase (IDH) were made in order to evaluate the contribution of precise substrate alignment to the catalytic power of an enzyme. The reaction trajectory of IDH was modified (i) after the adenine moiety of nicotinamide adenine dinucleotide phosphate was changed to hypoxanthine (the 6-amino was changed to 6-hydroxyl), and (ii) by replacing Mg2+, which has six coordinating ligands, with Ca2+, which has eight coordinating ligands. Both changes make large (10(-3) to 10(-5)) changes in the reaction velocity but only small changes in the orientation of the substrates (both distance and angle) as revealed by cryocrystallographic trapping of active IDH complexes. The results provide evidence that orbital overlap produced by optimal orientation of reacting orbitals plays a major quantitative role in the catalytic power of enzymes.

Autophosphorylation of protein kinase C at three separated regions of its primary sequence.

The major autophosphorylation sites of the rat beta II isozyme of protein kinase C were identified. The modified threonine and serine residues were found in the amino-terminal peptide, the carboxyl-terminal tail, and the hinge region between the regulatory lipid-binding domain and the catalytic kinase domain. Because this autophosphorylation follows an intrapeptide mechanism, extraordinary flexibility of the protein is necessary to phosphorylate the three regions. Comparison of the sequences surrounding the modified residues showed no obvious recognition motif nor any similarity to substrate phosphorylation sites, suggesting that proximity to the active site may be the primary criterion for their phosphorylation.

A piston model for transmembrane signaling of the aspartate receptor.

To characterize the mechanism by which receptors propagate conformational changes across membranes, nitroxide spin labels were attached at strategic positions in the bacterial aspartate receptor. By collecting the electron paramagnetic resonance spectra of these labeled receptors in the presence and absence of the ligand aspartate, ligand binding was shown to generate an approximately 1 angstrom intrasubunit piston-type movement of one transmembrane helix downward relative to the other transmembrane helix. The receptor-associated phosphorylation cascade proteins CheA and CheW did not alter the ligand-induced movement. Because the piston movement is very small, the ability of receptors to produce large outcomes in response to stimuli is caused by the ability of the receptor-coupled enzymes to detect small changes in the conformation of the receptor.

Mutagenesis and Laue structures of enzyme intermediates: isocitrate dehydrogenase.

Site-directed mutagenesis and Laue diffraction data to 2.5 A resolution were used to solve the structures of two sequential intermediates formed during the catalytic actions of isocitrate dehydrogenase. Both intermediates are distinct from the enzyme-substrate and enzyme-product complexes. Mutation of key catalytic residues changed the rate determining steps so that protein and substrate intermediates within the overall reaction pathway could be visualized.

A small-molecule modulator of poly-alpha 2,8-sialic acid expression on cultured neurons and tumor cells.

Poly-alpha2,8-sialic acid (PSA) has been implicated in numerous normal and pathological processes, including development, neuronal plasticity, and tumor metastasis. We report that cell surface PSA expression can be reversibly inhibited by a small molecule, N-butanoylmannosamine (ManBut). Inhibition occurs through a metabolic mechanism in which ManBut is converted to unnatural sialic acid derivatives that effectively act as chain terminators during cellular PSA biosynthesis. N-Propanoylmannosamine (ManProp), which differs from ManBut by a single methylene group, did not inhibit PSA biosynthesis. Modulation of PSA expression by chemical means has a role complementary to genetic and biochemical approaches in the study of complex PSA-mediated events.

Regulation of an enzyme by phosphorylation at the active site.

The isocitrate dehydrogenase of Escherichia coli is an example of a ubiquitous class of enzymes that are regulated by covalent modification. In the three-dimensional structure of the enzyme-substrate complex, isocitrate forms a hydrogen bond with Ser113, the site of regulatory phosphorylation. The structures of Asp113 and Glu113 mutants, which mimic the inactivation of the enzyme by phosphorylation, show minimal conformational changes from wild type, as in the phosphorylated enzyme. Calculations based on observed structures suggest that the change in electrostatic potential when a negative charge is introduced either by phosporylation or site-directed mutagenesis is sufficient to inactivate the enzyme. Thus, direct interaction at a ligand binding site is an alternative mechanism to induced conformational changes from an allosteric site in the regulation of protein activity by phosphorylation.

Three-dimensional structures of the ligand-binding domain of the bacterial aspartate receptor with and without a ligand.

The three-dimensional structure of an active, disulfide cross-linked dimer of the ligand-binding domain of the Salmonella typhimurium aspartate receptor and that of an aspartate complex have been determined by x-ray crystallographic methods at 2.4 and 2.0 angstrom (A) resolution, respectively. A single subunit is a four-alpha-helix bundle with two long amino-terminal and carboxyl-terminal helices and two shorter helices that form a cylinder 20 A in diameter and more than 70 A long. The two subunits in the disulfide-bonded dimer are related by a crystallographic twofold axis in the apo structure, but by a noncrystallographic twofold axis in the aspartate complex structure. The latter structure reveals that the ligand binding site is located more than 60 A from the presumed membrane surface and is at the interface of the two subunits. Aspartate binds between two alpha helices from one subunit and one alpha helix from the other in a highly charged pocket formed by three arginines. The comparison of the apo and aspartate complex structures shows only small structural changes in the individual subunits, except for one loop region that is disordered, but the subunits appear to change orientation relative to each other. The structures of the two forms of this protein provide a step toward understanding the mechanisms of transmembrane signaling.

Induction and expression of long- and short-term neurosecretory potentiation in a neural cell line.

In a neural cell line, the secretion of excitatory amino acids in response to a depolarizing stimulus is potentiated by the addition of serotonin. The duration of this potentiation is dependent on the strength of the stimulus. Persistent secretory potentiation induced by a strong stimulus requires the activation of both serotonin and NMDA receptors. Inhibiting the NMDA receptor during serotonin presentation prevented the induction of potentiation. The temporal characteristic of the potentiation is correlated with the elevation of cAMP levels. Serotonin exposure while inhibiting NMDA receptors results in a transient elevation of cAMP levels, whereas coactivation with NMDA and serotonin results in a persistent elevation of cAMP. Thus, it is possible to obtain potentiation of secretion in a single cell either transiently or persistently. The timing of potentiated responses in this system is of the same magnitude as that in similar systems used as models for short-term and long-term memory.

Parallel pathways for habituation in repetitively stimulated PC12 cells.

Habituation was investigated in neuronally differentiated PC12 cells by measuring the decrease in cellular secretion of norepinephrine with repetitive stimulation by either membrane depolarization or acetylcholine. When these two types of repetitive stimulation were applied to the same cells, habituation occurred independently to each, showing that two pathways for habituation can be utilized simultaneously in single cells. The two pathways apparently process stimuli in parallel, without competition between common intermediates.

The structural basis of negative cooperativity: receptors and enzymes.

Crystal structures of the negatively cooperative aspartate receptor caught at intermediate stages in the binding process help to elucidate structural factors involved in ligand binding. The frequency of occurrence of negatively cooperative proteins suggests that sequential changes in binding patterns will be extensive in positively cooperative as well as in negatively cooperative and no cooperativity proteins.