We have designed a heterodimerizing leucine zipper system to target a radionuclide to prelocalized noninternalizing tumor-specific antibodies. The modular nature of the leucine zipper allows us to iteratively use design rules to achieve specific homodimer and heterodimer affinities. We present circular-dichroism thermal denaturation measurements on four pairs of heterodimerizing leucine zippers. These peptides are 47 amino acids long and contain four or five pairs of electrostatically attractive g ↔ eЈ (i, iЈ +5) interhelical heterodimeric interactions. The most stable heterodimer consists of an acidic leucine zipper and a basic leucine zipper that melt as homodimers in the micro (T m ‫ס‬ 28°C) or nanomolar (T m ‫ס‬ 40°C) range, respectively, but heterodimerize with a T m >90°C, calculated to represent femtamolar affinities. Modifications to this pair of acidic and basic zippers, designed to destabilize homodimerization, resulted in peptides that are unstructured monomers at 4 M and 6°C but that heterodimerize with a T m ‫ס‬ 74°C or K d(37) ‫ס‬ 1.1 × 10 −11 M. A third heterodimerizing pair was designed to have a more neutral isoelectric focusing point (pI) and formed a heterodimer with T m ‫ס‬ 73°C. We can tailor this heterodimerizing system to achieve pharmacokinetics aimed at optimizing targeted killing of cancer cells.

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.

Biosynthesis of subtilisin is dependent on a 77 amino acid, N-terminal prodomain, which is autocatalytically processed to create the mature form of the enzyme [Ikemura, H., Takagi, H., & Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864]. In order to better understand the role of the prodomain in subtilisin folding, we have determined the structure of the processed complex between the prodomain and subtilisin Sbt-70, a mutant engineered for facilitated folding. The prodomain is largely unstructured by itself but folds into a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices when complexed with subtilisin. The Ka of the complex is 2 x 10(8) M-1 at 25 degrees C. The prodomain binds on subtilisin's two parallel surface alpha-helices and supplies caps to the N-termini of the two helices. The C-terminal strand of the prodomain binds in the subtilisin substrate binding cleft. While Sbt-70 is capable of independent folding, the prodomain accelerates the process by a factor of > 10(7) M-1 of prodomain in 30 mM Tris-HCl, pH 7.5, at 25 degrees C. X-ray structures of the mutant subtilisin folded in vitro either with or without the prodomain are compared and show that the identical folded state is achieved in either case. A model of the folding reaction of Sbt-70 and the prodomain is described as the following equilibria: P + Su<-->Pf--SI<-->Pf--Sf, where Su and P are Sbt-70 and prodomain, respectively, which are largely unstructured at the start of the reaction, Pf--SI is a collision complex of a partially folded Sbt-70 and folded prodomain, and Pf--Sf is the complex of folded Sbt-70 and prodomain.(ABSTRACT TRUNCATED AT 250 WORDS)

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, M r ‫؍‬ 50,513) is a unique 6-phosphoryl-O-␣-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-␣-glucosidase) that requires both NAD(H) and divalent metal (Mn 2؉ , Fe 2؉ , Co 2؉ , or Ni 2؉) for activity. 6-Phospho-␣-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the M r of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-␣-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-␣-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp 41 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-␣-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn 2؉ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-␣-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.The serendipitous discovery in 1964 (1, 2) of the bacterial phosphoenol pyruvate-dependent sugar phosphotransferase system (PEP-PTS) 1 by Roseman and colleagues represents a landmark in our understanding of carbohydrate transport by microorganisms (3, 4). Since the initial description in Escherichia coli, this phosphoryl group-transfer system (5, 6) has been established as the primary mechanism for the accumulation of sugars by bacteria from both Gram-negative (7, 8) and Gram-positive genera (9 -12). Operationally, the multi-component PEP-PTS (13) comprises both membrane-localized and cytoplasmic proteins that in concert catalyze the simultaneous phosphorylation and vectorial translocation of sugar across the cytoplasmic membrane. Catalytically, each PEP-PTS requires two general components (Enzyme I and HPr) that, allied with sugar-specific proteins (IIA, -B, and -C; for discussion, see Ref. 14), promote the sequential transfer of the high energy, phosphoryl moiety from PEP to the incoming sugar. Prior to catabolism via energy-yielding pathways, the intracellular disaccharide phosphates must first be hydrolyzed to their constituent hexose 6-phosphate and aglycone moieties. Several phosphoglycosylhydrolases (whose ge...

The crystal structure of the cyclic phosphodiesterase (CPDase) from Arabidopsis thaliana, an enzyme involved in the tRNA splicing pathway, was determined at 2.5 A Ê resolution. CPDase hydrolyzes ADPribose 1¢¢,2¢¢-cyclic phosphate (Appr>p), a product of the tRNA splicing reaction, to the monoester ADPribose 1¢¢-phosphate (Appr-1¢¢p). The 181 amino acid protein shows a novel, bilobal arrangement of two ab modules. Each lobe consists of two a-helices on the outer side of the molecule, framing a three-or fourstranded antiparallel b-sheet in the core of the protein. The active site is formed at the interface of the two b-sheets in a water-®lled cavity involving residues from two H-X-T/S-X motifs. This previously noticed motif participates in coordination of a sulfate ion. A solvent-exposed surface loop (residues 100±115) is very likely to play a¯ap-like role, opening and closing the active site. Based on the crystal structure and on recent mutagenesis studies of a homologous CPDase from Saccharomyces cerevisiae, we propose an enzymatic mechanism that employs the nucleophilic attack of a water molecule activated by one of the active site histidines. Keywords: ADP-ribose 1¢¢,2¢¢-cyclic phosphate/ Arabidopsis/2¢,3¢-cyclic nucleotide phosphodiesterase/ tRNA splicing/X-ray crystallography

We are using the tryptophan synthase alpha 2 beta 2 complex as a model system to investigate how ligands, protein-protein interaction, and mutations regulate enzyme activity, reaction specificity, and substrate specificity. The rate of conversion of L-serine and indole to L-tryptophan by the beta 2 subunit alone is quite low, but is activated by certain monovalent cations or by association with alpha subunit to form an alpha 2 beta 2 complex. Since monovalent cations and alpha subunit appear to stabilize an active conformation of the beta 2 subunit, we have investigated the effects of monovalent cations on the activities and spectroscopic properties of a mutant form of alpha 2 beta 2 complex having beta 2 subunit glutamic acid 109 replaced by alanine (E109A). The E109A alpha 2 beta 2 complex is inactive in reactions with L-serine but active in reactions with beta-chloro-L-alanine. Parallel experiments show effects of monovalent cations on the properties of wild type beta 2 subunit and alpha 2 beta 2 complex. We find that CsCl stimulates the activity of the E109A alpha 2 beta 2 complex and of wild type beta 2 subunit with L-serine and indole and alters the equilibrium distribution of L-serine reaction intermediates. The results indicate that CsCl partially repairs the deleterious effects of the E109A mutation on the activity of the alpha 2 beta 2 complex by stabilizing a conformation with catalytic properties more similar to those of the wild type alpha 2 beta 2 complex. This conclusion is consistent with observations that monovalent cations alter the catalytic and spectroscopic properties of several pyridoxal phosphate-dependent enzymes by stabilizing alternative conformations.

In complex with subtilisin BPN', the 77 amino acid prodomain folds into a stable compact structure comprising a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated from subtilisin, the prodomain is 97% unfolded even under optimal folding conditions. Traditionally, to study stable proteins, denaturing cosolvents or temperatures are used to shift the equilibrium from folded to unfolded. Here we manipulate the folding equilibrium of the unstable prodomain by introducing stabilizing mutations generated by design. By sequentially introducing three stabilizing mutations into the prodomain we are able to shift the equilibrium for independent folding from 97% unfolded to 65% folded. Spectroscopic and thermodynamic analysis of the folding reaction was carried out to assess the effect of stability on two-state behavior and the denatured state. The denatured states of single and combination mutants are not discernably different in spite of a range of DeltaGunfolding from -2.1 to 0.4 kcal/mol. Conclusions about the nature of the denatured state of the prodomain are based on CD spectral data and calorimetric data. Two state folding is observed for a combination mutant of marginal stability (DeltaG = 0). Evidence for its two-state folding is based on the observed additivity of individual mutations to the overall DeltaGunfolding and the conformity of DeltaGunfolding vs T to two-state assumptions as embodied in the Gibbs-Helmholz equation. We believe our success in stabilizing the two-state folding reaction of the prodomain originates from the selection of mutations with improved ability to fold subtilisin rather than selection for increase in secondary structure content. The fact that a small number of mutations can stabilize the independent folding of the prodomain implies that most of the folding information already exists in the wild-type amino acid sequence in spite of the fact that the unfolded state predominates.

The bacterial tryptophan synthase alpha 2 beta 2 complex contains an unusual structural feature: an intramolecular tunnel that channels indole from the active site of the alpha subunit to the active site of the beta subunit 25 A away. Here we investigate the role of the tunnel in communication between the alpha and beta subunits using the polarity-sensitive fluorescent probe, Nile Red. Interaction of Nile Red in the nonpolar tunnel near beta subunit residues Cys-170 and Phe-280 is supported by studies with enzymes altered at these positions. Restricting the tunnel by enlarging Cys-170 by chemical modification or mutagenesis decreases the fluorescence of Nile Red by 30-70%. Removal of a partial restriction in the tunnel by replacing Phe-280 by Cys or Ser increases the fluorescence of Nile Red more than 2-fold. A binding site for Nile Red in this region near the pyridoxal phosphate coenzyme of the beta subunit is further supported by iodide quenching and fluorescence energy transfer experiments and by molecular modeling based on the three-dimensional structure of the alpha 2 beta 2 complex. Finally, studies using Nile Red as a sensitive probe of conformational changes in the tunnel reveal that allosteric ligands (alpha subunit) or active site ligands (beta subunit) decrease the fluorescence of Nile Red. We speculate that allosteric and active site ligands induce a tunnel restriction near Phe-280 that serves as a gate to control passage of indole through the tunnel.