M. Sax

Domain Organization in Clostridium botulinum Neurotoxin Type E Is Unique: Its Implication in Faster Translocation

Clostridium botulinum produces seven antigenically distinct neurotoxins [C. botulinum neurotoxins (BoNTs) A-G] sharing a significant sequence homology. Based on sequence and functional similarity, it was believed that their three-dimensional structures will also be similar. Indeed, the crystal structures of BoNTs A and B exhibit similar fold and domain association where the translocation domain is flanked on either side by binding and catalytic domains. Here, we report the crystal structure of BoNT E holotoxin and show that the domain association is different and unique, although the individual domains are similar to those of BoNTs A and B. In BoNT E, both the binding domain and the catalytic domain are on the same side of the translocation domain, and all three have mutual interfaces. This unique association may have an effect on the rate of translocation, with the molecule strategically positioned in the vesicle for quick entry into cytosol. Botulism, the disease caused by BoNT E, sets in faster than any other serotype because of its speedy internalization and translocation, and the present structure offers a credible explanation. We propose that the translocation domain in other BoNTs follows a two-step process to attain translocation-competent conformation as in BoNT E. We also suggest that this translocation-competent conformation in BoNT E is a probable reason for its faster toxic rate compared to BoNT A. However, this needs further experimental elucidation.

A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct.

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate α-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

Effect of Baclofen Enantiomorphs on the Spinal Trigeminal Nucleus and Steric Similarities of Carbamazepine

The seemingly structurally different drugs, baclofen and carbamazepine, have a similar neurophysiologic effect on the cat spinal trigeminal nucleus and a similar clinical effect in the amelioration of trigeminal neuralgia pain. In this investigation, we report on the enhancement of segmental inhibition by carbamazepine and l-baclofen; d-baclofen produced no effect on segmental inhibition. Doses of l-baclofen one fifth its equivalent racemic dosage produced a much greater enhancement of segmental inhibition. d-Baclofen, when given prior to l-baclofen, blocked the effect of l-baclofen on segmental inhibition and the unconditioned response at previously effective doses. Pretreatment with d-baclofen also blocked the effect of subsequent carbamazepine on segmental inhibition, but had no effect on the unconditioned response. Crystallographic evaluation of carbamazepine and the enantiomorphs of baclofen revealed a surprisingly good fit of baclofen isomers to moieties of the carbamazepine molecule. The results suggest that the baclofen enantiomorphs and carbamazepine have a common mechanism of action in the cat spinal trigeminal nucleus, and that d-baclofen, though inactive, is capable of interfering with the effect of l-baclofen and to a lesser extent with carbamazepine.

Crystallographic computing on an array processor

To date, six computationally intense but frequently needed crystallographic applications programs have been developed for an array processor (Floating Point Systems model AP 190L) driven by a DEC 10 computer. In all of the applications attempted, it was possible to reduce the required processing time to at least an order of magnitude below that required by a large university scale computer (DEC 10) for the same problem. In fact, the rate-determining step in full-matrix least-squares refinement can be made to run 20–30 times faster in the array processor. For the refinement of proteins, one cycle with a space-group-general algorithm executes faster in the array processor than a typical small-molecule refinement cycle executes on the DEC 10. We conclude that many of the time-consuming operations frequently encountered in crystallographic computer programs can be handled very efficiently on an inexpensive array processor attached to a host computer.

Refined structure of rat Clara cell 17 kDa protein at 3.0 Å resolution

The rat Clara cell 17 kDa protein (previously referred to as the rat Clara cell 10 kDa protein) has been reported to inhibit phospholipase A2 and papain, and to also bind progesterone. It has been isolated from rat lung lavage fluid and crystallized in the space group P6(5)22. The structure has been determined to 3.0 A resolution using the molecular replacement method. Uteroglobin, whose amino acid sequence is 55.7% identical, was used as the search model. The structure was then refined using restrained least-squares and simulated annealing methods. The R-factor is 22.5%. The protein is a covalently bound dimer. Two disulfide bonds join the monomers together in an antiparallel manner such that the dimer encloses a large internal hydrophobic cavity. The hydrophobic cavity is large enough to serve as the progesterone binding site, but access to the cavity is limited. Each monomer is composed of four alpha-helices. The main-chain structure of the Clara cell protein closely resembles that of uteroglobin, but the nature of many of the exposed side-chains differ. This is true, particularly in a hypervariable region between residues 23 and 36, and in the H1H4 pocket.

The structure of myo-inositol hexaphosphate dodecasodium salt octatriacontahydrate: A single crystal X-ray analysis

Abstract Sodium phytate·38H2O crystals from water belonging to space group Cc gave cell dimensions a = 23.082, b = 12.203, c = 22.885 A, β = 108.30°, DC = 1.7457 gm/cm3 with four molecules per unit cell. Phytate has the hexaorthophosphate ester structure. The phosphates at positions C-1, 3, 4, 5 and 6 are axially disposed with that at C-2 equatorial rather than the reverse stereochemistry reported by Johnson and Tate. The twelve sodium atoms are coordinated with phosphate and water oxygens in 2-hexa, 8-octa and 2-decahedral arrangements.

Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures.

The crystal structures of pyruvate decarboxylase from the yeast Saccharomyces uvarum and Saccharomyces cerevisiae have been determined at 2.4 and 2.3 A resolution, respectively. These structures provide details about the protein fold and domain assembly within subunits, about subunit assembly to form dimers and about dimer assembly to form tetramers. They also provide a clear picture of the active site centered on the thiamin diphosphate cofactor, and have allowed amino acids critical for catalysis and involved in stabilization of the unusual cofactor conformation to be identified. The structural information has enabled identification of the site of allosteric activation to be centered on Cys-221, and suggests that a six residue segment leading from the regulatory site to the catalytic site may be involved in transmission of a binding signal. The importance of several amino acids within this segment in the regulatory process, as well as some involved in stabilizing and activating the cofactor has been confirmed by analyzing the behavior of recombinant enzymes with single point mutations introduced at these sites. Additional structures have been determined for pyruvate decarboxylase in multiple crystal forms, some of which were obtained from crystals grown with known allosteric activators present in the media. Currently four distinct types of tetramers have been observed, with each showing a different mode of association of dimers to form the tetramers. In some of the cases involving the presence of allosteric activators drastic changes in the mode of dimer assembly to form tetramers is seen.

Insight to the Interaction of the Dihydrolipoamide Acetyltransferase (E2) Core with the Peripheral Components in the Escherichia coli Pyruvate Dehydrogenase Complex via Multifaceted Structural Approaches.

Background: The pyruvate dehydrogenase complex produces acetyl-CoA and NADH, utilizing three protein components whose structural interactions need elucidation. Results: The E3 structure and interaction loci between E2 and E3 were determined. Conclusion: The peripheral subunit-binding domain of E2 establishes key interactions with E1 and E3. Significance: The multifaceted approach used could help delineate interaction surfaces in other 2-oxoacid dehydrogenase complexes. Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 Å resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected reductive acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.

Crystal structure of Bence Jones protein rhe (3 A) and its unique domain-domain association.

Abstract The crystal structure of protein Rhe, a lambda type VL dimer, has been determined at a resolution of 3 A by the method of multiple isomorphous replacement supplemented with anomalous scattering data. A crystallographic sequence was assigned from an interpretation of the electron density map in an optical comparator and is compared with a chemically determined partial amino acid sequence. The monomeric unit of Rhe, as determined crystallographically, contains 113 amino acids, 110 belonging to the variable region and three belonging to the constant segment of a light chain. The single polypeptide chain constituting the monomer forms a nine-stranded β-barrel characteristic of V domains. The β-pleated sheet surrounds an ellipsoidally shaped interior core of approximately 10 A × 15 A × 25 A in size. The monomers that are related by the crystallographic dyad are held together as dimers by interdomain hydrogen bonds and hydrophobic interactions. At one end of the dimer is an opening which is lined exclusively by residues from the hypervariable regions. A comparison of Rhe with Rei, a kappa type VL dimer (Epp et al., 1975), revealed that monomers of Rhe and Rei dimerized differently. Their respective dyad and pseudodyad of dimerization are not the same, and this causes a variation in the overall steric arrangement of the hypervariable regions in the two cavities. In adition a dissimilarity was observed in the non-hypervariable segment linking the first and second hypervariable regions. This segment is in the form of a loop and it includes most of the residues participating in the interdomain interactions stabilizing dimer formation in both proteins and these loop positions differ by as much as 7 A. Our results also show that there is a good correlation between the dissimilarity of the loop position and the difference in the domain-association. Our preliminary analysis indicates that the positions of the corresponding non-hypervariable loops in V domains may be determined in part by the residues in the hypervariable regions. Accordingly, the three-dimensional structure of Rhe suggests that this nonhypervariable loop in VL and its counterpart in VH may have an important biological function in antibody specificity and variability by virtue of their influence over the architecture of the complementarity site.

Structure and Absolute Configuration of (R)-Baclofen Monohydrochloride

C~oHI3C1NO+.C1 -, M, = 250.13, ortho- rhombic, P2~2t2 ~, a = 6.373 (1), b = 7.318 (2), c = 25.699 (5) A, 2(Cu) = 1.54180 A, V= 1198.5 A 3, Z = 4, D c- 1.386 g cm -3, ~ = 47.35 cm-k The phase problem was solved by the direct method (MULTAN 78); R(F) -- 0.029 for 1169 reflections. The molecules are linked into infinite chains along the b axis by hydrogen bonding. There is no significant ring stacking. Introduction. Baclofen (y-amino-fl-(p-chlorophenyl)- butyric acid) is a derivative of the inhibitory neuro- transmitter y-aminobutyric acid (GABA) but, unlike GABA, it can cross the blood/brain barrier (Birkmayer, 1972). It has been shown that baclofen reduces excitatory transmitter effects, especially sub- stance P (Pier & Zimmerman, 1973; Polc & Haefely, 1976; Potashner, 1979; Saito, Konishi & Otsuka, 1975). Baclofen has become the drug of choice in the treatment of spasticity of spinal origin due to its antispastic efficacy at doses which do not produce Cl sedation, its low frequency of serious side effects and its 0(1) lack of organ toxicity (Sachais & Logue, 1977). Recent o(2) studies have shown baclofen to be a promising new N drug in the treatment of the paroxysmal pain of c(1) trigeminal neuralgia (Fromm, Terrence, Chattha & c(2) Glass, 1980). c(4) White, tabular crystals were grown from water by c(5) slow evaporation. The space group was uniquely C(6) determined from Weissenberg photographs as P212~21. C(7) The unit-cell parameters were obtained from a least- c(8) squares fitting of the setting angles for 12 reflections c(10) measured on a Picker FACS-1 diffractometer. The H(O) intensity data were collected using graphite-mono- H 1(2) chromated Cu Ka radiation with the 0:20 scan H2(2) technique. Within the 20 range of 5.0 ° to 125.0 °, 68 Hl(4) out of 1 169 reflections were considered unobserved by H2(4) the criterion I < 3o(1). The E map generated by H(6) MULTAN 78 (Main, 1978) revealed positions of two H(7) CI atoms. All C and N atoms were located from a H(10) subsequent Fourier synthesis. The structure was refined by the full-matrix least-squares refinement procedures and all H atoms (with the exception of those of the NH 3 group) were located in a difference Fourier map. A close look at the difference Fourier map around N showed vague positions of three H atoms. The coordinates of these H atoms were fixed from consideration of the hydrogen bonding to CI-. In the final stages of the refinement all atoms except H were refined with anisotropic temperature factors. The final R factor is 0.029 while the weighted R factor,