The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott-Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. Here we report the autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that 'intrinsically unstructured' peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context.

We have used a combination of FTIR, VCD, ECD, Raman, and NMR spectroscopies to probe the solution conformations sampled by H-(AAKA)-OH by utilizing an excitonic coupling model and constraints imposed by the 3JCalphaHNH coupling constants of the central residues to simulate the amide I' profile of the IR, isotropic Raman, anisotropic Raman, and VCD spectra in terms of a mixture of three conformations, i.e., polyproline II, beta-strand and right-handed helical. The representative coordinates of the three conformations were obtained from published coil libraries. Alanine was found to exhibit PPII fractions of 0.60 or greater, mixed with smaller fractions of helices and beta-strand conformations. Lysine showed no clear conformational propensity in that it samples polyproline II, beta-strand, and helical conformations with comparable probability. This is at variance with results obtained earlier for ionized polylysine, which suggest a high polyproline II propensity. We reanalyzed previously investigated tetra- and trialanine by combining published vibrational spectroscopy data with 3JCalphaHNH coupling constants and obtained again blends dominated by PPII with smaller admixtures of beta-strand and right-handed helical conformations. The polyproline II propensity of alanine was found to be higher in tetraalanine than in trialanine. For all peptides investigated, our results rule out a substantial population of turn-like conformations. Our results are in excellent agreement with MD simulations on short alanine peptides by Gnanakaran and Garcia [(2003) J. Phys. Chem. B 107, 12555-12557] but at variance with multiple MD simulations particularly for the alanine dipeptide.

1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner, and is the first step in the essential pathway to isoprenoids in human pathogens. Understanding the mechanism of this unique enzyme is critical for developing new anti-infective agents that selectively target isoprenoid biosynthesis. The present study uses mutagenesis and a combination of protein fluorescence, circular dichroism and kinetics experiments to investigate the roles of Arg-420, Arg-478 and Tyr-392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism and predict Arg-420 and Arg-478 are involved in binding of the acceptor substrate, d-GAP. d-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg-420 and Arg-478, also accelerates decarboxylation of the pre-decarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating this binding interaction is not absolutely required, and the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr-392 contributes to d-GAP affinity and is not required for LThDP formation or its d-GAP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate LThDP is significantly stabilized on R420A and Y392F variants compared to wild type DXP synthase in the absence of acceptor substrate, yet these substitutions do not appear to impact the rate of d-GAP-promoted LThDP decarboxylation in the presence of high d-GAP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues to promote d-GAP binding which in turn facilitates decarboxylation, and further highlight interesting differences between DXP synthase and other ThDP-dependent enzymes.

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Summary The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.

Appropriate compounds were synthesized to create models for the 1',4'-imino tautomer of the 4'-aminopyrimidine ring of thiamin diphosphate recently found to exist on the pathway of enzymatic reactions requiring this cofactor (Jordan, F., and Nemeria, N.S. (2005) Bioorganic Chemistry, 33, 190−215). The N1-methyl-4-aminopyrimidinium compounds synthesized on treatment with a strong base produce the 1,4-imino tautomer whose UV spectrum indicates a maximum between 300−320 nm, depending on the absence or presence of a methyl group at the 4-amino nitrogen. The λ max found is in the same wavelength range as the positive circular dichroism band observed on several enzymes and showed a very strong dependence on solvent dielectric constant. To help with the 15 N chemical shift assignments, the model compounds were specifically labeled with 15 N at the amino nitrogen atom. The chemical shift of the amino nitrogen was deshielded by N1-methylation, then dramatically further deshielded by more than 100 ppm on formation of the 1,4-iminopyrimidine tautomer. Both the UV spectroscopic values and the 15 N chemical shift for the 1,4-iminopyrimidine tautomer should serve as useful guides to the assignment of enzyme-bound signals.The notion that the 4'-aminopyrimidine group of thiamin diphosphate (ThDP) undergoes tautomerization to the 1',4'-imino form (1',4'-iminoThDP) during the catalytic cycle of enzymes that utilize it has gained wider acceptance since the appearance of X-ray crystal structural data (1). The role and likelihood of tautomerization is suggested by two totally conserved structural features on all ThDP enzymes: (a) the V coenzyme conformation ensuring that the C2 thiazolium atom and the N4' atom of the 4'-aminopyrimidine ring are within less than 3.5Å from each other (2), potentially enabling the 1',4'-imino tautomer to participate in proton transfers; and (b) the presence of a glutamate within hydrogen bonding distance of the N1' atom of the 4-aminopyrimidine ring, as a potential catalyst for the tautomerization (Scheme 1, as exemplified with the reaction of yeast pyruvate decarboxylase, YPDC). As illustrated in the active site structure of YPDC in Figure 1, the residue E51 probably carries out this function (3). Chemical evidence for the importance of the 4'-aminopyrimidine moiety of ThDP in catalysis was obtained from ThDP analogues in which one or another nitrogen atom was replaced by carbon (4), while a model for activation of the ring for catalysis via tautomerization by N1-protonation was suggested from our laboratories (5-7). Notwithstanding the attractive features of this hypothesis, until recently no direct spectroscopic or structural evidence was available on any ThDP enzymes for the presence of the 1',4'-imino tautomer. In rapid-scan stopped flow experiments, mixing slow active-center variants of YPDC with pyruvate, a UV # Supported by National Institutes of Health grants GM050380 and GM062330. In these examples, the keto group of the substrate analog phosphonate forms a covalent adduct with the t...

Spectroscopic identification and characterization of covalent and non-covalent intermediates on large enzyme complexes is an exciting and challenging area of modern enzymology. The Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), consisting of multiple copies of enzymic components and coenzymes, performs the oxidative decarboxylation of pyruvate to acetyl-CoA and is central to carbon metabolism linking glycolysis to the Krebs cycle. Based on earlier studies we hypothesized that the dynamic regions of the E1p component, which undergo a disorder-order transition upon substrate binding to thiamin diphosphate (ThDP), play a critical role in modulation of the catalytic cycle of PDHc. To test our hypothesis, we kinetically characterized ThDP-bound covalent intermediates on the E1p component, and the lipoamide-bound covalent intermediate on the E2p component in PDHc and in its variants with disrupted active-site loops. Our results suggest that formation of the first covalent pre-decarboxylation intermediate, C2α-lactylthiamin diphosphate (LThDP), is rate limiting for the series of steps culminating in acetyl-CoA formation. Substitutions in the active center loops produced variants with up to 900-fold lower rates of formation of the LThDP demonstrating that these perturbations directly affected covalent catalysis. This rate was rescued by up to 5-fold upon assembly to PDHc of the E401K variant. The E1p loop dynamics control covalent catalysis with ThDP and are modulated by PDHc assembly, presumably by selection of catalytically competent loop conformations. This mechanism could be a general feature of 2-oxoacid dehydrogenase complexes since such interfacial dynamic regions are highly conserved.

Background: The E. coli pyruvate dehydrogenase complex catalyzes conversion of pyruvate to acetyl-CoA and comprises E1p, E2p, and E3 components. Results: The structure of the E2 core domain was solved and shown to efficiently catalyze acetyl transfer between domains. Conclusion: Mass spectrometry revealed hitherto unrecognized domain-induced interactions between E1 and E2 core domain. Significance: A multifaceted approach is required to understand communication between intact multidomain components.