A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase.

The three-dimensional X-ray structure of a complex of the potent neuraminidase inhibitor 4-guanidino-Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 A resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half-chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X-ray structure concurs with that obtained by theoretical calculations and validates the structure-based design of the inhibitor. Comparison of known high-resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4-guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase.     

Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.     

Chemical modification of enzymes: Critical evaluation of the graphical correlation between residual enzyme activity and number of groups modified

In the study of chemical modification of enzymes and other biologically active proteins, plots of fractional residual activity as a function of number of groups modified per enzyme molecule are often used to establish a correlation between the chemical modification and enzyme inactivation reactions and to determine the stoichiometry of the modification reaction. This paper presents a critical examination of the underlying theoretical framework of such graphs. Whereas these plots are usually presented as linear functions, it is shown here that the general equation describing the relationship between inactivation and modification contains an exponential term; therefore, in the general case, the plot is actually a curve. It is suggested that caution be exercised in the interpretation of such plots and that equations such as those derived in the text be used to fit theoretical curves to the data, in order to maximize the information gained from chemical modification experiments.     

Crystal structure of the human Fab fragment Kol and its comparison with the intact Kol molecule

The crystal structure of the Fab³ fragment of the IgG protein Kol was analysed and an electron density map calculated at 3 Å resolution based on phases obtained from multiple isomorphous replacement. An atomic model was constructed but the lack of amino acid sequence data causes some uncertainty, in particular concerning the amino acid side-chains. Several cycles of constrained crystallographic refinement (Deisenhofer &; Steigemann, 1975) produced an R value of 0.36. The resulting model was compared with the intact Kol IgG crystal structure (Colman et al., 1976), which had also been subjected to constrained crystallographic refinement, and two Fab fragments (Davies et al., 1975). The elbow angle was found open and only 8 ° more bent than in intact Kol, in contrast to the other Fab fragments, which show a strongly bent elbow angle. The intramolecular longitudinal V-C contacts are very similar in Kol Fab and Kol IgG and considerably fewer in number than in the other Fab fragments. The lateral V-V and C-C association is virtually identical in Kol Fab and Kol IgG. Although Kol Fab and Kol IgG crystallize in different lattices, they exhibit a nearly identical head to tail packing of the Fab parts, with the C modules touching the hypervariable segments of the V modules. This strongly conserved particular mode of aggregation might be reflected in the property of cold precipitation of the Kol cryoglobulin.     

Three-dimensional structure of the neuraminidase of influenza virus A/Tokyo/3/67 at 2.2 A resolution

An atomic model of the tetrameric surface glycoprotein neuraminidase of influenza virus A/Tokyo/3/67 has been built and refined based on X-ray diffraction data at 2.2 A resolution. The crystallographic residual is 0.21 for data between 6 and 2.2 A resolution and the r.m.s. deviations from ideal geometry are 0.02 A for bond lengths and 3.9 degrees for bond angles. The model includes amino acid residues 83 to 469, four oligosaccharide structures N-linked at asparagine residues 86, 146, 200 and 234, a single putative Ca2+ ion site, and 85 water molecules. One of the oligosaccharides participates in a novel crystal contact. The folding pattern is a beta-sheet propeller as described earlier and details of the intramolecular interactions between the six beta-sheets are presented. Strain-invariant residues are clustered around the propeller axis on the upper surface of the molecule where they line the wall of a cavity into which sialic has been observed to bind. Strain-variable residues implicated in binding to antibodies surround this site.     

Refined atomic structures of N9 subtype influenza virus neuraminidase and escape mutants

The crystal structure of the N9 subtype neuraminidase of influenza virus was refined by simulated annealing and conventional techniques to an R-factor of 0.172 for data in the resolution range 6.0 to 2.2 A. The r.m.s. deviation from ideal values of bond lengths is 0.014 A. The structure is similar to that of N2 subtype neuraminidase both in secondary structure elements and in their connections. The three-dimensional structures of several escape mutants of neuraminidase, selected with antineuraminidase monoclonal antibodies, are also reported. In every case, structural changes associated with the point mutation are confined to the mutation site or to residues that are spatially immediately adjacent to it. The failure of antisera to cross-react between N2 and N9 subtypes may be correlated with the absence of conserved, contiguous surface structures of area 700 A2 or more.