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.

Propagating conformational changes over long (and short) distances in proteins.

The problem of the propagation of conformational changes over long distances or through a closely packed protein is shown to fit a model of a ligand-induced conformational change between two protein states selected by evolution. Moreover, the kinetics of the pathway between these states is also selected so that the energy of ligand binding and the speed of the transition between conformational states are physiologically appropriate. The crystallographic data of a wild-type aspartate receptor that has negative cooperativity and a mutant that has no cooperativity but has native transmembrane signaling are shown to support this model.

Structural basis for a change in substrate specificity: crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP.

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K(M)) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg(2+) to 2.0 A resolution. On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The S113E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site. In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the S113E substitution and the binding of substrates act together to promote catalysis of isopropylmalate. Ligand binding results in reorientation of the active site helix that contains residues 113 through 116. E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP. In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specificity.

Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae.

The PDS5 gene (precocious dissociation of sisters) was identified in a genetic screen designed to identify genes important for chromosome structure. PDS5 is an essential gene and homologues are found from yeast to humans. Pds5p function is important for viability from S phase through mitosis and localizes to chromosomes during this cell cycle window, which encompasses the times when sister chromatid cohesion exists. Pds5p is required to maintain cohesion at centromere proximal and distal sequences. These properties are identical to those of the four cohesion complex members Mcd1p/Scc1p, Smc1p, Smc3p, and Scc3p/Irr1p (Guacci, V., D. Koshland, and A. Strunnikov. 1997. Cell. 91:47-57; Michaelis, C., R. Ciosk, and K. Nasmyth. 1997. Cell. 91:35-45; Toth, A., R. Ciosk, F. Uhlmann, M. Galova, A. Schleiffer, and K. Nasmyth. 1999. Genes Dev. 13:307-319). Pds5p binds to centromeric and arm sequences bound by Mcd1p. Furthermore, Pds5p localization to chromosomes is dependent on Mcd1p. Thus, Pds5p, like the cohesin complex members, is a component of the molecular glue that mediates sister chromatid cohesion. However, Mcd1p localization to chromosomes is independent of Pds5p, which may reflect differences in their roles in cohesion. Finally, Pds5p is required for condensation as well as cohesion, which confirms the link between these processes revealed through analysis of Mcd1p (Guacci, V., D. Koshland, and A. Strunnikov. 1997. Cell. 91:47-57). Therefore, the link between cohesion and condensation is a general property of yeast chromosomes.

Chromosomal addresses of the cohesin component Mcd1p.

We identified the chromosomal addresses of a cohesin subunit, Mcd1p, in vivo by chromatin immunoprecipitation coupled with high resolution PCR-based chromosomal walking. The mapping of new Mcd1p-binding sites (cohesin-associated regions [CARs]) in single-copy sequences of several chromosomes establish their spacing ( approximately 9 kb), their sequestration to intergenic regions, and their association with AT-rich sequences as general genomic properties of CARs. We show that cohesins are not excluded from telomere proximal regions, and the enrichment of cohesins at the centromere at mitosis reflects de novo loading. The average size of a CAR is 0.8-1.0 kb. They lie at the boundaries of transcriptionally silenced regions, suggesting they play a direct role in defining the silent chromatin domain. Finally, we identify CARs in tandem (rDNA) and interspersed repetitive DNA (Ty2 and subtelomeric repeats). Each 9-kb rDNA repeat has a single CAR proximal to the 5S gene. Thus, the periodicity of CARs in single-copy regions and the rDNA repeats is conserved. The presence and spacing of CARs in repetitive DNA has important implications for genomic stability and chromosome packaging/condensation.

Redesigning the substrate specificity of an enzyme: isocitrate dehydrogenase.

Despite the structural similarities between isocitrate and isopropylmalate, isocitrate dehydrogenase (IDH) exhibits a strong preference for its natural substrate. Using a combination of rational and random mutagenesis, we have engineered IDH to use isopropylmalate as a substrate. Rationally designed mutations were based on comparison of IDH to a similar enzyme, isopropylmalate dehydrogenase (IPMDH). A chimeric enzyme that replaced an active site loop-helix motif with IPMDH sequences exhibited no activity toward isopropylmalate, and site-directed mutants that replaced IDH residues with their IPMDH equivalents only showed small improvements in k(cat). Random mutants targeted the IDH active site at positions 113 (substituted with glutamate), 115, and 116 (both randomized) and were screened for activity toward isopropylmalate. Six mutants were identified that exhibited up to an 8-fold improvement in k(cat) and increased the apparent binding affinity by as much as a factor of 80. In addition to the S113E mutation, five other mutants contained substitutions at positions 115 and/or 116. Most small hydrophobic substitutions at position 116 improved activity, possibly by generating space to accommodate the isopropyl group of isopropylmalate; however, substitution with serine yielded the most improvement in k(cat). Only two substitutions were identified at position 115, which suggests a more specific role for the wild-type asparagine residue in the utilization of isopropylmalate. Since interactions between neighboring residues in this region greatly influenced the effects of each other in unexpected ways, structural solutions were best identified in combinations, as allowed by random mutagenesis.

Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells.

In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.

Sites of binding and orientation in a four-location model for protein stereospecificity.

The stereospecificity of the enzyme isocitrate dehydrogenase was examined by steady-state kinetics and x-ray crystallography. The enzyme has the intriguing property that the apoenzyme in the absence of divalent metal showed a selectivity for the inactive l-enantiomer of the substrate isocitrate, whereas the enzyme containing magnesium showed selectivity for the physiologically active d-enantiomer. The hydrogen atom on the C2 carbon that is transferred during the reaction was, in both the d- and l-isocitrate complexes, in an orientation very close to that expected for delivery of a hydride ion to the cosubstrate NADP+. The beta-carboxylate that is eliminated as a CO2 molecule during the reaction occupied the same site on the protein in both the d- and l-isocitrate complexes. In addition, the C3 carbon was in the same protein site in both the d- and l-enantiomers. Only the fourth group, the OH atom, was in a very different position in the apo enzyme and in the metal-containing complexes. A four-location model is necessary to explain the enantiomeric specificity of IDH in contrast to the conventional three-point attachment model. The thermodynamic and kinetic ramifications of this model are explored.

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.

Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren.

In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of the Xenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally, brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics.

Active site water molecules revealed in the 2.1 A resolution structure of a site-directed mutant of isocitrate dehydrogenase.

Isocitrate dehydrogenase catalyses the two step, acid base, oxidative decarboxylation of isocitrate to alpha-ketoglutarate. Lysine 230 was suggested to act as proton donor based on geometry and spatial proximity to isocitrate. To clarify further the role of lysine 230, we co-crystallized the lysine-to-methionine mutant (K230M) with isocitrate and with alpha-ketoglutarate. Crystals were flash-frozen and the two structures were determined and refined to 2. 1 A. Several new features were identified relative to the wild-type structure. Seven side-chains previously unplaced in the wild-type structure were identified and included in the model, and the amino acid terminus was extended by an alanine residue. Many additional water molecules were identified. Examination of the K230M active sites (K230M isocitrate and K230M-ketoglutarate) revealed that tyrosine 160 protrudes further into the active site in the presence of either isocitrate or alpha-ketoglutarate in K230 M than it does in the wild-type structure. Also, methionine 230 was not as fully extended, and asparagine 232 rotates approximately 30 degrees toward the ligand permitting polar interactions. Outside the active site cleft a tetragonal volume of density was identified as a sulfate molecule. Its location and interactions suggest it may influence the equilibrium between the tetragonal and the orthorhombic forms of isocitrate dehydrogenase. Differences observed in the active site water structure between the wild-type and K230M structures were due to a single point mutation. A water molecule was located in the position equivalent to that occupied by the wild-type epsilon-amine of lysine 230; a water molecule in that location in K230M suggests it may influence catalysis in the mutant. Comparison of K230M complexed with isocitrate and alpha-ketoglutarate illuminates the influence a ligand has on active site water structure.

The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis.

The Escherichia coli aspartate receptor is a dimer with two transmembrane sequences per monomer that connect a periplasmic ligand binding domain to a cytoplasmic signaling domain. The method of 'hydrophobic-biased' random mutagenesis, that we describe here, was used to construct mutant aspartate receptors in which either the entire transmembrane sequence or seven residues near the center of the transmembrane sequence were replaced with hydrophobic and polar random residues. Some of these receptors responded to aspartate in an in vivo chemotaxis assay, while others did not. The acceptable substitutions included hydrophobic to polar residues, small to larger residues, and large to smaller residues. However, one mutant receptor that had only a few hydrophobic substitutions did not respond to aspartate. These results add to our understanding of sequence specificity in the transmembrane regions of proteins with more than one transmembrane sequence. This work also demonstrates a method of constructing families of mutant proteins containing random residues with chosen characteristics.

The centromeric sister chromatid cohesion site directs Mcd1p binding to adjacent sequences.

Cohesion between sister chromatids occurs along the length of chromosomes, where it plays essential roles in chromosome segregation. We show here that the centromere, a cis-acting cohesion factor, directs the binding of Mcd1p, a cohesin subunit, to at least 2 kb regions flanking centromeres in a sequence-independent manner. The centromere is essential for the maintenance as well as the establishment of this cohesin domain. The efficiency of Mcd1p binding within the cohesin domain is independent of the primary nucleotide sequence of the centromere-flanking DNA but correlates with high A + T DNA content. Thus, the function of centromeres in the cohesion of centromere-proximal regions may be analogous to that of enhancers, nucleating cohesin complex binding over an extended chromosomal domain of A + T-rich DNA.

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.

Pds1p of budding yeast has dual roles: inhibition of anaphase initiation and regulation of mitotic exit.

Progression through mitosis is controlled by protein degradation that is mediated by the anaphase-promoting complex/cyclosome (APC/C) and its associated specificity factors. In budding yeast, APC/C(Cdc20) promotes the degradation of the Pds1p anaphase inhibitor at the metaphase-to-anaphase transition, whereas APC/C(Cdh1) promotes the degradation of the mitotic cyclins at the exit from mitosis. Here we show that Pds1p has a novel activity as an inhibitor of mitotic cyclin destruction, apparently by preventing the activation of APC/C(Cdh1). This activity of Pds1p is independent of its activity as an anaphase inhibitor. We propose that the dual role of Pds1p as an inhibitor of anaphase and of cyclin degradation allows the cell to couple the exit from mitosis to the prior completion of anaphase. Finally, these observations provide a novel regulatory paradigm in which the sequential degradation of two substrates is determined by the substrates themselves, such that an early substrate inhibits the degradation of a later one.

A functional assay for centromere-associated sister chromatid cohesion.

Cohesion of sister chromatids occurs along the entire length of chromosomes, including the centromere where it plays essential roles in chromosome segregation. Here, minichromosomes in the budding yeast Saccharomyces cerevisiae are exploited to generate a functional assay for DNA sequences involved in cohesion. The centromeric DNA element CDEIII was found to be necessary but not sufficient for cohesion. This element was shown previously to be required for assembly of the kinetochore, the centromere-associated protein complex that attaches chromosomes to the spindle. These observations establish a link between centromere-proximal cohesion and kinetochore assembly.

Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle.

A genetic synthetic dosage lethality (SDL) screen using CTF13 encoding a known kinetochore protein as the overexpressed reference gene identified two chromosome transmission fidelity (ctf) mutants, YCTF58 and YCTF26. These mutant strains carry independent alleles of a novel gene, which we have designated CTF19. In light of its potential role in kinetochore function, we have cloned and characterized the CTF19 gene in detail. CTF19 encodes a nonessential 369-amino acid protein. ctf19 mutant strains display a severe chromosome missegregation phenotype, are hypersensitive to benomyl, and accumulate at G2/M in cycling cells. CTF19 genetically interacts with kinetochore structural mutants and mitotic checkpoint mutants. In addition, ctf19 mutants show a defect in the ability of centromeres on minichromosomes to bind microtubules in an in vitro assay. In vivo cross-linking and chromatin immunoprecipitation demonstrates that Ctf19p specifically interacts with CEN DNA. Furthermore, Ctf19-HAp localizes to the nuclear face of the spindle pole body and genetically interacts with a spindle-associated protein. We propose that Ctf19p is part of a macromolecular kinetochore complex, which may function as a link between the kinetochore and the mitotic spindle.

Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery.

CTF7 (chromosome transmission fidelity) gene in budding yeast encodes an essential protein that is required for high-fidelity chromosome transmission and contains regions of identity conserved from yeast to man. ctf7 mutant cells arrested prior to anaphase onset contain separated sister chromatids. Thus, Ctf7p is essential for cohesion. Cohesion is established during S phase and then maintained until mitosis. However, Ctf7p activity is required only during S phase, suggesting that Ctf7p functions in the establishment of cohesion. In addition, ctf7 genetically interacts with DNA metabolism mutations pol30 (PCNA) and ctf18 (an RF-C like protein) and ctf7 temperature sensitivity and chromosome loss are rescued by high levels of POL30. These findings provide the first evidence that links the establishment of sister chromatid cohesion to the DNA replication machinery and suggest that the assembly of cohesion (and possibly condensation) complexes are coupled to PCNA-dependent DNA replication. The analysis of Ctf7p also reveals an important connection between sister chromatid cohesion, spindle integrity and the spindle assembly checkpoint.

Conformational changes in the cytoplasmic domain of the Escherichia coli aspartate receptor upon adaptive methylation.

By using targeted disulfide cross-linking, we have characterized structural changes that the Escherichia coli aspartate receptor undergoes upon modification of the four specific residues that are reversibly methylated during sensory adaptation. Cysteine residues were introduced at specific positions either in the cytoplasmic domain or in the periplasmic domain, and the rates of disulfide cross-linking were used to probe for conformational changes upon covalent modification. Conversion of the methylation sites from glutamates to glutamines greatly reduced the rate of disulfide formation between residues 265 and 265' and residues 250 and 250' in the cytoplasmic domain but not between residues 36 and 36' in the periplasmic domain. (Primes are used to indicate the second of the two identical subunits in the homologous dimer.) The covalent modification of the cytoplasmic domain induces conformational changes that are detectable in the cytoplasmic domain but none that are detectable in the periplasmic domain.