The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase.

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism.

Substrate pathways in the nitrogenase MoFe protein by experimental identification of small molecule binding sites.

In the nitrogenase molybdenum-iron (MoFe) protein, we have identified five potential substrate access pathways from the protein surface to the FeMo-cofactor (the active site) or the P-cluster using experimental structures of Xe pressurized into MoFe protein crystals from Azotobacter vinelandii and Clostridium pasteurianum. Additionally, all published structures of the MoFe protein, including those from Klebsiella pneumoniae, were analyzed for the presence of nonwater, small molecules bound to the protein interior. Each pathway is based on identification of plausible routes from buried small molecule binding sites to both the protein surface and a metallocluster. Of these five pathways, two have been previously suggested as substrate access pathways. While the small molecule binding sites are not conserved among the three species of MoFe protein, residues lining the pathways are generally conserved, indicating that the proposed pathways may be accessible in all three species. These observations imply that there is unlikely a unique pathway utilized for substrate access from the protein surface to the active site; however, there may be preferred pathways such as those described here.

Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis.

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, ß, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and ß domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg(2+) complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This "loop-in" conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the "loop-out" conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.

Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures.

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis-trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis-trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the ß-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively.

Crystallization and preliminary X-ray diffraction analysis of the barley yellow dwarf virus cap-independent translation element.

Barley yellow dwarf virus (BYDV) RNA lacks a 5' m(7)GTP cap, yet it is translated efficiently because it contains a 105-base BYDV-like cap-independent translation element (BTE) in the 3' untranslated region (UTR). To understand how the BTE outcompetes the host mRNA for protein-synthesis machinery, its three-dimensional structure is being determined at high resolution. The purification using transcription from DNA containing 2'-O-methyl nucleotides and preliminary crystallographic analyses of the BTE RNA are presented here. After varying the BTE sequence and crystallization-condition optimization, crystals were obtained that diffracted to below 5 Å resolution, with a complete data set being collected to 6.9 Å resolution. This crystal form indexes with an R(merge) of 0.094 in the monoclinic space group C2, with unit-cell parameters a = 316.6, b = 54.2, c = 114.5 Å, α = γ = 90, ß = 105.1°.

Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase.

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X-Pro imide bond readily undergoes cis-trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis-trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis-trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.

Tertiary structure and characterization of a glycoside hydrolase family 44 endoglucanase from Clostridium acetobutylicum.

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-A resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and beta-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55 degrees C and an optimal pH of 5.0.

Computational and experimental analyses converge to reveal a coherent yet malleable aptamer structure that controls chemical reactivity.

As short nucleic acids, aptamers in solution are believed to be structurally flexible. Consistent with this view, most aptamers examined for this property have been shown to bind their target molecules by mechanisms that can be described as "induced fit". But, it is not known to what extent this structural flexibility affects the integrity of the target-aptamer interaction. Using the malachite green aptamer (MGA) as a model system, we show that the MGA can protect its bound target, malachite green (MG), from oxidation over several days. Protection is reversed by an oligonucleotide complementary to the MGA binding pocket. Computational cavity analysis of the MGA-MG structure predicted that MG oxidation is protected because a molecule as small as an OH(-) is sterically excluded from the C1 position of the bound MG. These results suggest that, while the MGA-MG interface is sufficiently coherent to prevent OH(-) penetration, the bases involved in the interaction are sufficiently mobile that they can exchange out of the MG binding interface to hybridize with a complementary oligonucleotide. The computational predictions were confirmed experimentally using variants of the MGA with single base changes in the binding pocket. This work demonstrates the successful application of molecular dynamics simulations and cavity analysis in determining the effects of sequence variations on the structure of a small single-stranded nucleic acid. It also shows that a nucleic acid aptamer can control access to specific chemical groups on its target, which suggests that aptamers might be applied for selectively protecting small molecules from modification.

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport.

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.

The structure and function of plant hemoglobins.

Plants, like humans, contain hemoglobin. Three distinct types of hemoglobin exist in plants: symbiotic, non-symbiotic, and truncated hemoglobins. Crystal structures and other structural and biophysical techniques have revealed important knowledge about ligand binding and conformational stabilization in all three types. In symbiotic hemoglobins (leghemoglobins), ligand binding regulatory mechanisms have been shown to differ dramatically from myoglobin and red blood cell hemoglobin. In the non-symbiotic hemoglobins found in all plants, crystal structures and vibrational spectroscopy have revealed the nature of the structural transition between the hexacoordinate and ligand-bound states. In truncated hemoglobins, the abbreviated globin is porous, providing tunnels that may assist in ligand binding, and the bound ligand is stabilized by more than one distal pocket residue. Research has implicated these plant hemoglobins in a number of possible functions differing among hemoglobin types, and possibly between plant species.

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation.

Synechocystis hemoglobin contains an unprecedented covalent bond between a nonaxial histidine side chain (H117) and the heme 2-vinyl. This bond has been previously shown to stabilize the ferric protein against denaturation, and also to affect the kinetics of cyanide association. However, it is unclear why Synechocystis hemoglobin would require the additional degree of stabilization accompanying the His117-heme 2-vinyl bond because it also displays endogenous bis-histidyl axial heme coordination, which should greatly assist heme retention. Furthermore, the mechanism by which the His117-heme 2-vinyl bond affects ligand binding has not been reported, nor has any investigation of the role of this bond on the structure and function of the protein in the ferrous oxidation state. Here we report an investigation of the role of the Synechocystis hemoglobin His117-heme 2-vinyl bond on structure, heme coordination, exogenous ligand binding, and stability in both the ferrous and ferric oxidation states. Our results reveal that hexacoordinate Synechocystis hemoglobin lacking this bond is less stable in the ferrous oxidation state than the ferric, which is surprising in light of our understanding of pentacoordinate Hb stability, in which the ferric protein is always less stable. It is also demonstrated that removal of the His117-heme 2-vinyl bond increases the affinity constant for intramolecular histidine coordination in the ferric oxidation state, thus presenting greater competition for the ligand binding site and lowering the observed rate and affinity constants for exogenous ligands.

Crystallographic analysis of synechocystis cyanoglobin reveals the structural changes accompanying ligand binding in a hexacoordinate hemoglobin.

The crystal structures of cyanide and azide-bound forms of the truncated hemoglobin from Synechocystis are presented at 1.8 angstroms resolution. A comparison with the structure of the endogenously liganded protein reveals a conformational shift unprecedented in hemoglobins, and provides the first picture of a hexacoordinate hemoglobin in both the bis-histidyl and the exogenously coordinated states. The structural changes between the different conformations are confined to two regions of the protein; the B helix, and the E helix, including the EF loop. A molecular "hinge" controlling movement of the E helix is observed in the EF loop, which is composed of three principal structural elements: Arg64, the heme-d-propionate, and a three-residue extension of the F helix. Additional features of the structural transition between the two protein conformations are discussed as they relate to the complex ligand-binding behavior observed in hexacoordinate hemoglobins, and the potential physiological function of this class of proteins.

Review: correlations between oxygen affinity and sequence classifications of plant hemoglobins.

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full-length globins with the classical eight helix Mb-like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O(2) binding ( approximately 250 muM(-1) s(-1)), moderately high rates of O(2) dissociation ( approximately 5-15 s(-1)), and high oxygen affinity (K(d) or P(50) approximately 50 nM). These properties both facilitate O(2) diffusion to respiring N(2) fixing bacteria and reduce O(2) tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (K(HisE7) approximately 2), moderate rates of O(2) binding ( approximately 25 muM(-1) s(-1)), very small rates of O(2) dissociation ( approximately 0.16 s(-1)), and remarkably high O(2) affinities (P(50) approximately 2 nM), suggesting a function involving O(2) and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (K(HisE7) approximately 100), low rates of O(2) binding ( approximately 1 muM(-1) s(-1)), moderately low O(2) dissociation rate constants ( approximately 1 s(-1)), and moderate, Mb-like O(2) affinities (P(50) approximately 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.

Crystal structure of the membrane fusion protein CusB from Escherichia coli.

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu(+) and Ag(+) ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly beta-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the alpha-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first beta-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu(+) and Ag(+) were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.

Role of phenylalanine B10 in plant nonsymbiotic hemoglobins.

All plants contain an unusual class of hemoglobins that display bis-histidyl coordination yet are able to bind exogenous ligands such as oxygen. Structurally homologous hexacoordinate hemoglobins (hxHbs) are also found in animals (neuroglobin and cytoglobin) and some cyanobacteria, where they are thought to play a role in free radical scavenging or ligand sensing. The plant hxHbs can be distinguished from the others because they are only weakly hexcacoordinate in the ferrous state, yet no structural mechanism for regulating hexacoordination has been articulated to account for this behavior. Plant hxHbs contain a conserved Phe at position B10 (Phe(B10)), which is near the reversibly coordinated distal His(E7). We have investigated the effects of Phe(B10) mutation on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states. Kinetic and equilibrium constants for hexacoordination and ligand binding along with CO-FTIR spectroscopy, midpoint reduction potentials, and the crystal structures of two key mutant proteins (F40W and F40L) reveal that Phe(B10) is an important regulatory element in hexacoordination. We show that Phe at this position is the only amino acid that facilitates stable oxygen binding to the ferrous Hb and the only one that promotes ligand binding in the ferric oxidation states. This work presents a structural mechanism for regulating reversible intramolecular coordination in plant hxHbs.

The crystal structure of Synechocystis hemoglobin with a covalent heme linkage.

The x-ray crystal structure of Synechocystis hemoglobin has been solved to a resolution of 1.8 A. The conformation of this structure is surprisingly different from that of the previously reported solution structure, probably due in part to a covalent linkage between the heme 2-vinyl and His117 that is present in the crystal structure but not in the structure solved by NMR. Synechocystis hemoglobin is a hexacoordinate hemoglobin in which the heme iron is coordinated by both the proximal and distal histidines. It is also a member of the "truncated hemoglobin" family that is much shorter in primary structure than vertebrate and plant hemoglobins. In contrast to other truncated hemoglobins, the crystal structure of Synechocystis hemoglobin displays no "ligand tunnel" and shows that several important amino acid side chains extrude into the solvent instead of residing inside the heme pocket. The stereochemistry of hexacoordination is compared with other hexacoordinate hemoglobins and cytochromes in an effort to illuminate factors contributing to ligand affinity in hexacoordinate hemoglobins.

Bis-histidyl hexacoordination in hemoglobins facilitates heme reduction kinetics.

Hexacoordinate hemoglobins are a class of proteins that exhibit reversible bis-histidyl coordination of the heme iron while retaining the ability to bind exogenous ligands. One hypothesis for their physiological function is that they scavenge nitric oxide, a reaction that oxidizes the protein and requires reduction of the heme iron to continue. Reduction kinetics of hexacoordinate hemoglobins, including human neuroglobin and cytoglobin, and those from Synechocystis and rice, are compared to myoglobin, soybean leghemoglobin, and several relevant mutant proteins. In all cases, bis-histidyl coordination greatly increases the rate of reduction by sodium dithionite when compared to pentacoordinate hemoglobins. In myoglobin and leghemoglobin, reduction is limited by the rate constant for electron transfer, whereas in the hexacoordinate hemoglobins reduction is limited only by bimolecular binding of the reductant. These results can be explained by differences in the reorganization energy for reduction between hexacoordinate and pentacoordinate hemoglobins.

Direct measurement of equilibrium constants for high-affinity hemoglobins.

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) << 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.