A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme.

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the beta subunit and conserved region 4 of the sigma subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the sigma region 4/beta-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the beta-flap for binding to sigma region 4, thereby inhibiting transcription initiation by disrupting the sigma region 4/beta-flap interaction.

Conserved regions 4.1 and 4.2 of sigma(70) constitute the recognition sites for the anti-sigma factor AsiA, and AsiA is a dimer free in solution.

The association of the bacteriophage T4-encoded AsiA protein with the final sigma(70) subunit of the Escherichia coli RNA polymerase is one of the principal events governing transcription of the T4 genome. Analytical ultracentrifugation and NMR studies indicate that free AsiA is a symmetric dimer and the dimers can exchange subunits. Using NMR, the mutual recognition sites on AsiA and final sigma(70) have been elucidated. Residues throughout the N-terminal half of AsiA are involved either directly or indirectly in binding to final sigma(70) whereas the two highly conserved C-terminal regions of final sigma(70), denoted 4.1 and 4.2, constitute the entire AsiA binding domain. Peptides corresponding to these regions bind tightly to AsiA individually and simultaneously. Simultaneous binding promotes structural changes in AsiA that mimic interaction with the complete AsiA binding determinant of final sigma(70). Moreover, the results suggest that a significant rearrangement of the dimer accompanies peptide binding. Thus, both conserved regions 4.1 and 4.2 are intimately involved in recognition of AsiA by final sigma(70). The interaction of AsiA with 4.1 provides a potential explanation of the differential abilities of DNA and AsiA to bind to free final sigma(70) and a mechanistic alternative to models of AsiA function that rely on binding to a single site on final sigma(70).

The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3.

The ubiquitin fold is a versatile and widely used targeting signal that is added post-translationally to a variety of proteins. Covalent attachment of one or more ubiquitin domains results in localization of the target protein to the proteasome, the nucleus, the cytoskeleton or the endocytotic machinery. Recognition of the ubiquitin domain by a variety of enzymes and receptors is vital to the targeting function of ubiquitin. Several parallel pathways exist and these must be able to distinguish among ubiquitin, several different types of polymeric ubiquitin, and the various ubiquitin-like domains. Here we report the first molecular description of the binding site on ubiquitin for ubiquitin C-terminal hydrolase L3 (UCH-L3). The site on ubiquitin was experimentally determined using solution NMR, and site-directed mutagenesis. The site on UCH-L3 was modeled based on X-ray crystallography, multiple sequence alignments, and computer-aided docking. Basic residues located on ubiquitin (K6, K11, R72, and R74) are postulated to contact acidic residues on UCH-L3 (E10, E14, D33, E219). These putative interactions are testable and fully explain the selectivity of ubiquitin domain binding to this enzyme.

Synthesis of (2R,3R)-erythro- and (2R,3S)-threo-fluoromalate using malic dehydrogenase; stereoselectivity of malic dehydrogenase.

3-Fluorooxalacetate is a substrate for malic dehydrogenase. When enzymatic reduction is slower than the rate of epimerization of the two enantiomers, only (2R,3R)-erythro-fluoromalate is formed. Conversely, when a high enzyme level and excess of NADH lead to reduction that is fast relative to the epimerization rate, equal amounts of (2R,3R)-erythro- and (2R,3S)-threo-fluoromalate are formed. These data suggest that the V/K value for reduction of the R enantiomer to give the erythro isomer is approximately 100 times greater than for reduction of the S enantiomer to give the threo isomer. The equilibrium constant for the oxidation of fluoromalate is an order of magnitude less favorable than for oxidation of malate, while the equilibrium deuterium isotope effect from deuteration at C-2 of the substrate is 1.09 for fluoromalate versus 1.18 for malate. These effects reflect the inductive effect of fluorine at the 3-position.

Determination of the kinetic and chemical mechanism of malic enzyme using (2R,3R)-erythro-fluoromalate as a slow alternate substrate.

(2R,3R)-erythro-Fluoromalate, but not the threo isomer, is a slow substrate for chicken liver malic enzyme with either NADP or 3-acetylpyridine-NADP (APADP) as the other substrate. The Km for erythro-fluoromalate is similar to that of malate, but the turnover number with NADP is 3300-fold slower, although 5.5-fold faster with APADP than with NADP. Deuteration of fluoromalate at C-2 gave an isotope effect on V/K of 1.39 with NADP and 3.32 with APADP. With NADP, the 13C isotope effects at C-4 were 1.0490 with unlabeled and 1.0364 with deuterated fluoromalate. With APADP, the corresponding values were 1.0138 and 1.0087. These data show that the mechanism is stepwise with both nucleotide substrates, in contrast to the reaction of malate and APADP, which was postulated to be concerted by Karsten et al. [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103], a conclusion recently shown to be correct by Edens et al. [Edens, W. A., Urbauer, J. L., and Cleland, W. W. (1997) Biochemistry 36, 1141-1147]. To explain the effect of deuteration on the 13C isotope effect with APADP, it is necessary to assume a secondary 13C isotope effect at C-4 on the hydride transfer step of approximately 1.0064 (assuming 5.7 as the intrinsic primary deuterium isotope effect and 1.054 as the product of the 13C equilibrium isotope effect on hydride transfer and the intrinsic 13C isotope effect on decarboxylation). The secondary 13C isotope effect on hydride transfer is thought to result from hyperconjugation between the carbonyl group and C-4 of the enzyme-bound fluorooxaloacetate intermediate.

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins.

Selective incorporation of 13C into the methyl groups of protein side chains is described as a means for simplifying the measurement and interpretation of 13C relaxation parameters. High incorporation (> 90%) is accomplished by using pyruvate (3-13C, 99%) as the sole carbon source in the growth media for protein overexpression in E. coli. This improved labeling scheme increases the sensitivity of the relaxation experiments by approximately fivefold when compared to randomly fractionally 13C-labeled protein, allowing high-quality measurements on relatively dilute (< 1 mM) protein samples at a relatively low cost.

Determination of the chemical mechanism of malic enzyme by isotope effects.

Carbon-13 isotope effects have been determined for all four carbons of L-malate as a substrate for chicken liver malic enzyme, using either NADP or acetylpyridine-NADP as the other substrate. The effect of deuteration at C2 of malate was then used to tell whether the chemical mechanism of this oxidative decarboxylation was stepwise, with oxaloacetate as an intermediate, or concerted. With NADP, the 13C isotope effects at C3 and C4 both decrease with deuteration of malate, showing a stepwise mechanism, as previously determined [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114]. With acetylpyridine-NADP, however, the 13C isotope effects at both C3 and C4 increase with deuteration of malate. While the increase at C4 could be explained by a secondary 13C isotope effect on hydride transfer, the increase at C3 proves that the chemical mechanism has changed to a concerted one, presumably because hydride transfer is more rate-limiting and the overall equilibrium constant is more favorable by 2 orders of magnitude. The transition state for this concerted reaction is asynchronous, however, with an intrinsic deuterium isotope effect of approximately 5 and a 13C isotope effect of only 1.010-1.015. Equilibrium 13C isotope effects for conversion of carbons 2, 3, and 4 of malate to pyruvate or CO2 are 1.010, 1.011, and 0.988, respectively. Measured 13C isotope effects at C2 of malate are slightly inverse, but no explanation for this is obvious. With NADP, deuterium isotope effects at C3 of 1.17 and 1.08 for di- and monodeuteration and an increase in the 13C isotope effect at C4 upon dideuteration at C3 are consistent with a stepwise mechanism with the deuterium isotope effect at C3 being only on the decarboxylation step. Smaller deuterium isotope effects of 1.03-1.04 from dideuteration at C3 with acetylpyridine-NADP are consistent with a concerted but asynchronous mechanism where C-C cleavage is not far advanced in the transition state.

Internal dynamics of human ubiquitin revealed by 13C-relaxation studies of randomly fractionally labeled protein.

The use of random, fractional 13C-enrichment combined with low pass filtration has allowed the determination of NMR relaxation parameters at an unprecedented number of sites within recombinant human ubiquitin. Essentially complete 1H, 13C, and 15N resonance assignments for the protein are reported. Carbon spin lattice and heteronuclear NOE relaxation data have been analyzed in the context of the Lipari-Szabo "model free" formalism. The generalized order parameters for 56 main chain alpha C-H vectors have been determined and are found to correspond to the highly restricted motion seen in previous studies of the motion of amide N-H vectors. In distinct contrast, the analysis presented here indicates an unexpected range of dynamics within the interior of the protein. The generalized order parameters of 45 methyl groups of human ubiquitin have been determined. The methyl groups of Thr and Ala residues show generalized order parameters ranging from the Woessner limit (0.111) to below 0.01. Generalized order parameters for all methyl groups of the seven isoleucine residues were determined. With one exception, the generalized order parameters of the gamma methyls were equal to or greater than the corresponding delta methyls, indicating higher mobility away from the main chain. Generalized order parameters for 11 methyl groups of leucine residues were also determined. In six of the seven cases where the generalized order parameters of both prochiral methyl groups were determined, the pro-R methyl consistently shows a higher value than the pro-S methyl group. Generalized order parameters for seven methyl groups of four valines were also determined. There is no apparent correlation of methyl group prochirality with the value of the generalized order parameter. These data have several implications and generally indicate that the interior of the protein is heterogeneously dynamic.

Structural analysis of a novel interaction by calmodulin: high-affinity binding of a peptide in the absence of calcium.

The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of both apocalmodulin and its 1:1 complex with the Neurop peptide have been assigned by triple resonance and nuclear Overhauser effect-(NOE-) based strategies. ApoCaM displays many of the same basic structural features as calcium-saturated calmodulin. Analysis of observed chemical shifts and patterns of NOEs on the main chain indicates extensive and regular secondary structure throughout the N-terminal domain. In contrast, the helices of the C-terminal domain are somewhat irregular and are dynamically averaged. The EF-hands are intact in the N-terminal domain with the loops forming a short antiparallel beta sheet. Under low-salt conditions, two helix-loop-helix EF-hand motifs are present in the C-terminal domain of apoCaM but do not show interstrand NOEs. The spectral perturbations of apoCaM upon complexation with the Neurop peptide are relatively small with the larger chemical shift perturbations occurring in the C-terminal domain. The general secondary structure and tertiary organization appears to remain roughly the same as in free apoCaM. Stoichiometric titration of the apoCaM.Neurop complex with calcium indicates that the C-terminal domain EF-hands have a higher affinity for calcium than N-terminal domain EF-hands. Thus, this complex offers a unique opportunity to examine the structural and energetic consequences of calcium-dependent and calcium-independent binding of peptide to calmodulin.

The energetics and dynamics of molecular recognition by calmodulin.

Amide hydrogen exchange has been used to examine the structural dynamics and energetics of the interaction of a peptide corresponding to the calmodulin binding domain of smooth muscle myosin light chain kinase with calcium-saturated calmodulin. Heteronuclear NMR 15N-1H correlation techniques were used to quantitate amide proton exchange rates of both 15N-labeled and unlabeled amide protons of the smMLCK peptide complexed to calmodulin. Hydrogen exchange slowing factors were determined for 18 of the 19 amide hydrogens and found to span 6 orders of magnitude. The first six residues of the bound peptide were found to have slowing factors near 1 and are considered not to be hydrogen bonded, consistent with the previously reported model for the structure of the peptide. The pattern of hydrogen exchange of hydrogen-bonded amide hydrogens is indicative of end-fraying behavior characteristic of helix-coil transitions. The effective statistical mechanical parameters revealed by the end fraying are consistent with exchange from a highly solvated state. However, the slowing factors of the first hydrogen-bonded amide hydrogens are large, indicating the requirement for a reorganization of the calmodulin-peptide complex before the helix-coil transitions leading to exchange can occur. Taken together, these observations suggest that the collapsed complex reorganizes with an associated free energy change of 5.5 kcal/mol to a more open state where the helical peptide is highly solvated and undergoes helix-coil transitions leading to exchange. The free energy difference between the most and least stable intrahelical amide hydrogen bonds of the bound peptide is estimated to be approximately 2.5 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance assignment strategies for the analysis of NMR spectra of proteins.

Determination of the high resolution solution structure of a protein using nuclear magnetic resonance (NMR) spectroscopy requires that resonances observed in the NMR spectra be unequivocally assigned to individual nuclei of the protein. With the advent of modern, two-dimensional NMR techniques arose methodologies for assigning the 1H resonances based on 2D, homonuclear 1H NMR experiments. These include the sequential assignment strategy and the main chain directed strategy. These basic strategies have been extended to include newer 3D homonuclear experiments and 2D and 3D heteronuclear resolved and edited methods. Most recently a novel, conceptually new approach to the problem has been introduced that relies on heteronuclear, multidimensional so-called triple resonance experiments for both backbone and sidechain resonance assignments in proteins. This article reviews the evolution of strategies for the assignment of resonances of proteins.

Structural water in oxidized and reduced horse heart cytochrome c.

The existence of structural water in the interior of both oxidized and reduced horse-heart cytochrome c in solution is demonstrated using nuclear magnetic resonance spectroscopy. Six water molecules have been located in ferrocytochrome c and five in ferricytochrome c, with residence times greater than a few hundred picoseconds. Two water molecules are located in the haem crevice, one of which is found to undergo a large change in position with a change of oxidation state. Both of these observations indicate that buried structural waters in the haem crevice have, by microscopic dielectric effects, significant roles in the setting of the solvent reorganization energy associated with electron transfer.

Secondary 18O and primary 13C isotope effects as a probe of transition-state structure for enzymatic decarboxylation of oxalacetate.

A new method for directly measuring 18O isotope effects on decarboxylation reactions has been developed. By running the reaction under high vacuum (10(-5) torr), CO2 leaves the solution before exchange with the oxygens of water to an extent greater than 2%. Thus, the method permits determination of 18O isotope effects with the precision of the isotope ratio mass spectrometer, and without the necessity of resorting to the remote label method and its attendant required syntheses. The method is used to determine 18O isotope effects for decarboxylation of oxalacetate (OAA) by Mg2+, and enzymatically by OAA decarboxylase from Pseudomonas putida; 13C isotope effects are also reported for this enzyme, as well as for decarboxylation of OAA by pyruvate kinase. Initial velocity patterns and pH profiles are reported for the P. putida enzyme, and all available data are used to discuss the kinetic and chemical mechanism of decarboxylation.

Calmodulin interacts with amphiphilic peptides composed of all D-amino acids.

Calmodulin binds to amphiphilic, helical peptides of a variety of amino-acid sequences. These peptides are usually positively charged, although there is spectroscopic evidence that at least one neutral peptide binds. The complex between calmodulin and one of its natural target peptides, the binding site for calmodulin on smooth muscle myosin light-chain kinase (RS20), has been investigated by crystallography and NMR which have characterized the interactions between the ligand and the protein. From these data, it appears that the calmodulin-binding surface is sterically malleable and van der Waals forces probably dominate the binding. To explore further this apparently permissive binding, we investigated the chiral selectivity of calmodulin using synthesized analogues of melittin and RS20 that consisted of only D-amino acids. Fluorescence and NMR measurements show that D-melittin and D-RS20 both bind avidly to calmodulin, probably in the same general binding site as that for peptides having all L-amino acids. The calmodulin-peptide binding surface is therefore remarkably tolerant sterically. Our results suggest a potentially useful approach to the design of non-hydrolysable or slowly hydrolysable intracellular inhibitors of calmodulin.

Design and synthesis of multi-haem proteins.

A water-soluble, 62-residue, di-alpha-helical peptide has been synthesized which accommodates two bis-histidyl haem groups. The peptide assembles into a four-helix dimer with 2-fold symmetry and four parallel haems that closely resemble native haems in their spectral and electrochemical properties, including haem-haem redox interaction. This protein is an essential intermediate in the synthesis of molecular 'maquettes', a novel class of simplified versions of the metalloproteins involved in redox catalysis and in energy conversion in respiratory and photosynthetic electron transfer.

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)- and (E)-3-fluorophosphoenolpyruvate as substrates.

The catalytic mechanism of phosphoenolpyruvate (PEP) carboxylase from Zea mays has been studied using (Z)- and (E)-3-fluorophosphoenolpyruvate (F-PEP) as substrates. Both (Z)- and (E)-F-PEP partition between carboxylation to produce 3-fluorooxalacetate and hydrolysis to produce 3-fluoropyruvate. Carboxylation accounts for 3% of the reaction observed with (Z)-F-PEP, resulting in the formation of (R)-3-fluorooxalacetate, and for 86% of the reaction of (E)-F-PEP forming (S)-3-fluorooxalacetate. Carboxylation of F-PEP occurs on the 2-re face, which corresponds to the 2-si face of PEP. The partitioning of F-PEP between carboxylation and hydrolysis is insensitive to pH but varies with metal ion. Use of 18O-labeled bicarbonate produces phosphate that is multiply labeled with 18O; in addition, 18O is also incorporated into residual (Z)- and (E)-F-PEP. The 13(V/K) isotope effect on the carboxylation of F-PEP catalyzed by PEP carboxylase at pH 8.0, 25 degrees C, is 1.049 +/- 0.003 for (Z)-F-PEP and 1.009 +/- 0.006 for (E)-F-PEP. These results are consistent with a mechanism in which carboxylation of PEP occurs via attack of the enolate of pyruvate on CO2 rather than carboxy phosphate. In this mechanism phosphorylation of bicarbonate to give carboxy phosphate and decarboxylation of the latter are reversible steps. An irreversible step, however, precedes partitioning between carboxylation to give oxalacetate and release of CO2, which results in hydrolysis of PEP.

Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction.

Deuterium isotope effects and 13C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the 13C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. When dinucleotide substrates such as thio-NAD, 3-acetylpyridine adenine dinucleotide, and 3-pyridinealdehyde adenine dinucleotide that contain modified nicotinamide rings are used, the 13C effect increases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the 13C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a beta-secondary 13C isotope effect accompanies hydride transfer as a result of hyperconjugation of the beta-carboxyl of malate as the transition state for the hydride transfer step is approached.(ABSTRACT TRUNCATED AT 250 WORDS)

Dependence of the activity of beef heart mitochondrial adenosinetriphosphatase on the properties of the catalytic metal ion.

Several divalent metal ions were used as kinetic probes of the beef heart mitochondrial adenosinetriphosphatase (F1) under a variety of conditions, and the relationship between the properties of the catalytic metal ion and the catalytic activity of the enzyme was examined. Vmax for ATP hydrolysis was largest when metal ions characterized by intermediate values of acidity of coordinated water molecules (pKa) and metal-nucleotide stability constants (Kstab) were present. As temperature increased, the peak of Vmax vs. pKa (or Kstab) shifted to lower initial values of pKa or Kstab. The solvent deuterium isotope effect on Vmax (DV) was normal and largest when the metal ion present during F1-catalyzed ATP hydrolysis was most acidic and the metal nucleotide stability constant was large. When an active site tyrosine on F1 was nitrated, Vmax was most affected when the metal ion present was least acidic and the metal nucleotide stability constant was small. The isotope effect on V/K (DV/K) was normal, small, and apparently independent of the metal ion present. ADP inhibition of F1-catalyzed ATP hydrolysis is competitive, and the Ki is independent of the metal ion present. The degree of Pi inhibition of F1 is dependent on the metal ion present. The inhibition by Pi is competitive at low temperature and becomes noncompetitive as temperature increases. These and previous results support a mechanism whereby a water molecule coordinated to the metal ion of an enzyme-bound gamma-monodentate metal-ATP complex is deprotonated to begin a series of events whereby a beta,gamma-bidentate metal-ATP complex is produced. Upon hydrolysis, the bond between the metal ion and the beta-phosphate of ADP in the Pi-metal-ADP complex is broken before products (ADP and metal-Pi) are released.