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.

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.

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.