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Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects. Characterizations of beta-barrel (H43R) and dimer interface (A4V) FALS mutants reveal reduced stability and drastically increased aggregation propensity. Moreover, electron and atomic force microscopy indicate that these defects promote the formation of filamentous aggregates. The filaments resemble those seen in neurons of FALS patients and bind both Congo red and thioflavin T, suggesting the presence of amyloid-like, stacked beta-sheet interactions. These results support free-cysteine-independent aggregation of FALS mutant SOD as an integral part of FALS pathology. They furthermore provide a molecular basis for the single FALS disease phenotype resulting from mutations of diverse side-chains throughout the protein: many FALS mutations reduce structural integrity, lowering the energy barrier for fibrous aggregation.

Mutations in human superoxide dismutase (HSOD) have been linked to the familial form of amyotrophic lateral sclerosis (FALS). Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is one of the most common neurodegenerative disorders in humans. In ALS patients, selective killing of motor neurons leads to progressive paralysis and death within one to five years of onset. The most frequent FALS mutation in HSOD, Ala4-->Val, is associated with the most rapid disease progression. Here we identify and characterize key differences in the stability between the A4V mutant protein and its thermostable parent (HSOD-AS), in which free cysteine residues were mutated to eliminate interferences from cysteine oxidation. Denaturation studies reveal that A4V unfolds at a guanidine-HCl concentration 1M lower than HSOD-AS, revealing that A4V is significantly less stable than HSOD-AS. Determination and analysis of the crystallographic structures of A4V and HSOD-AS reveal structural features likely responsible for the loss of architectural stability of A4V observed in the denaturation experiments. The combined structural and biophysical results presented here argue that architectural destabilization of the HSOD protein may underlie the toxic function of the many HSOD FALS mutations.

Among catalytic antibodies, the well-characterized antibody 43C9 is unique in its ability to catalyze the difficult, but desirable, reaction of selective amide hydrolysis. The crystallographic structures that we present here for the single-chain variable fragment of the 43C9 antibody, both with and without the bound product p -nitrophenol, strongly support and extend the structural and mechanistic information previously provided by a three-dimensional computational model, together with extensive biochemical, kinetics, and mutagenesis results. The structures reveal an unexpected extended beta-sheet conformation of the third complementarity determining region of the heavy chain, which may be coupled to the novel indole ring orientation of the adjacent Trp H103. This unusual conformation creates an antigen-binding site that is significantly deeper than predicted in the computational model, with a hydrophobic pocket that encloses the p -nitrophenol product. Despite these differences, the previously proposed roles for Arg L96 in transition-state stabilization and for His L91 as the nucleophile that forms a covalent acyl-antibody intermediate are fully supported by the crystallographic structures. His L91 is now centered at the bottom of the antigen-binding site with the imidazole ring poised for nucleophilic attack. His L91, Arg L96, and the bound p -nitrophenol are linked into a hydrogen-bonding network by two well-ordered water molecules. These water molecules may mimic the positions of the phosphonamidate oxygen atoms of the antigen, which in turn mimic the transition state of the reaction. This network also contains His H35, suggesting that this residue may also stabilize the transition-states. A possible proton-transfer pathway from His L91 through two tyrosine residues may assist nucleophilic attack. Although transition-state stabilization is commonly observed in esterolytic antibodies, nucleophilic attack appears to be unique to 43C9 and accounts for the unusually high catalytic activity of this antibody., (Copyright 1999 Academic Press.)

The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.

The repair of DNA requires the removal of abasic sites, which are constantly generated in vivo both spontaneously and by enzymatic removal of uracil, and of bases damaged by active oxygen species, alkylating agents and ionizing radiation. The major apurinic/apyrimidinic (AP) DNA-repair endonuclease in Escherichia coli is the multifunctional enzyme exonuclease III, which also exhibits 3'-repair diesterase, 3'-->5' exonuclease, 3'-phosphomonoesterase and ribonuclease activities. We report here the 1.7 A resolution crystal structure of exonuclease III which reveals a 2-fold symmetric, four-layered alpha beta fold with similarities to both deoxyribonuclease I and RNase H. In the ternary complex determined at 2.6 A resolution, Mn2+ and dCMP bind to exonuclease III at one end of the alpha beta-sandwich, in a region dominated by positive electrostatic potential. Residues conserved among AP endonucleases from bacteria to man cluster within this active site and appear to participate in phosphate-bond cleavage at AP sites through a nucleophilic attack facilitated by a single bound metal ion.

DNA repair proteins act to correct mutagenic and toxic DNA damage, which can lead to cancer, aging and death. These proteins and their mechanisms of action have been found to be widely conserved between species, often from bacteria to man. Structural and biochemical studies on several bacterial enzymes involved in direct reversal and base excision repair have provided insights into the molecular basis of the recognition of damaged DNA and have also highlighted the novel roles that transition metals play in DNA repair.

The three-dimensional structure of exonuclease III, the major AP DNA repair endonuclease of Escherichia coli, has been determined using x-ray crystallographic methods at 2.7 A resolution. The atomic model was fit to an electron density map calculated with phases obtained from three isomorphous heavy atom derivatives. The overall chain fold of exonuclease III is that of a compact alpha,beta-protein of dimensions 55 by 50 by 45 A. The pair of extended beta-pleated sheets pack against each other in an approximately parallel fashion to form the hydrophobic core of a four-layered sandwich structure. These beta sheets are flanked by four alpha-helices that form the outer two layers of the fold. The individual strands of the beta-sheets are in a mostly antiparallel configuration and are linked by extensive loop regions that connect adjoining strands. The structure contains internal symmetry with the two extended beta-sheets and four alpha-helices related by a pseudo-twofold axis running approximately down the center of the two sheets. This internal symmetry is not mirrored in the structure of the loop regions, nor is it detectable within the amino acid sequence. There is a "groove" between the beta-sheets at one end of the molecule that is bordered by several of the exposed loop regions and may be significant for DNA binding.

In order to develop a more complete understanding of urea induced protein denaturation we have investigated the crystal structure of urea with the cyclic dipeptide diketopiperazine. This structure, determined to an R factor of 8.1%, shows extensive hydrogen bonding between urea and the peptide groups of diketopiperazine. These studies support a model where hydrogen bonding plays an important contribution in urea-induced protein denaturation. In the companion paper we present thermodynamic data for urea-peptide interactions in aqueous solution that further support this model.

The elastase protein of Pseudomonas aeruginosa is a zinc metalloprotease which has been shown to be a member of the bacterial neutral protease family. Its overall tertiary structure is similar to that of thermolysin. The x-ray crystallographic structure of the elastase has been solved to high resolution in three different crystal forms. Substantial conformational differences are observed in the protein in different crystal forms. In the absence of ligand, and independently in the presence of a covalent noncompetitive inhibitor, the elastase is observed to have a relatively "open" substrate binding cleft, while in the presence of tight-binding competitive inhibitors, the active site cleft is "closed".

Pseudomonas aeruginosa elastase (PAE) is a zinc metalloprotease with 301 amino acids. We have crystallized and solved the three-dimensional structure of PAE, using data to 1.5-A resolution, and have refined the native molecular structure to R = 0.188. The overall tertiary structure of the PAE molecule is similar to that of thermolysin, with which it shares 28% amino acid sequence identity. Nearly all of the active site residues that might potentially interact with substrates are identical in the two proteins. However, the active site cleft is significantly more "open" in PAE than in thermolysin.

We have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [3H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O4; the total HC1O4-soluble, Cl3CCO2H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns [Gurley, L. R., D'Anna, J. A., Blumenfeld, M., Valdez, J. G., Sebring, R. J., Donahue, D. K., Prentice, D. A., & Spall, W. D. (1984) J. Chromatogr. 297, 147-165]. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases (3.6 +/- 0.5)-fold as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases (3.1 +/- 0.2)-fold so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure.