Andrzej Weichsel

Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer.

BACKGROUND Human thioredoxin reduces the disulfide bonds of numerous proteins in vitro, and can activate transcription factors such as NFkB in vivo. Thioredoxin can also act as a growth factor, and is overexpressed and secreted in certain tumor cells. RESULTS Crystal structures were determined for reduced and oxidized wild type human thioredoxin (at 1.7 and 2.1 A nominal resolution, respectively), and for reduced mutant proteins Cys73-->Ser and Cys32-->Ser/Cys35-->Ser (at 1.65 and 1.8 A, respectively). Surprisingly, thioredoxin is dimeric in all four structures; the dimer is linked through a disulfide bond between Cys73 of each monomer, except in Cys73-->Ser where a hydrogen bond occurs. The thioredoxin active site is blocked by dimer formation. Conformational changes in the active site and dimer interface accompany oxidation of the active-site cysteines, Cys32 and Cys35. CONCLUSIONS It has been suggested that a reduced pKa in the first cysteine (Cys32 in human thioredoxin) of the active-site sequence is important for modulation of the redox potential in thioredoxin. A hydrogen bond between the sulfhydryls of Cys32 and Cys35 may reduce the pKa of Cys32 and this pKa depression probably results in increased nucleophilicity of the Cys32 thiolate group. This nucleophilicity, in tum, is thought to be necessary for the role of thioredoxin in disulfide-bond reduction. The physiological role, if any, of thioredoxin dimer formation remains unknown. It is possible that dimerization may provide a mechanism for regulation of the protein, or a means of sensing oxidative stress.

Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli

CueO (YacK), a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli. The crystal structure of CueO has been determined to 1.4-Å resolution by using multiple anomalous dispersion phasing and an automated building procedure that yielded a nearly complete model without manual intervention. This is the highest resolution multicopper oxidase structure yet determined and provides a particularly clear view of the four coppers at the catalytic center. The overall structure is similar to those of laccase and ascorbate oxidase, but contains an extra 42-residue insert in domain 3 that includes 14 methionines, nine of which lie in a helix that covers the entrance to the type I (T1, blue) copper site. The trinuclear copper cluster has a conformation not previously seen: the Cu-O-Cu binuclear species is nearly linear (Cu-O-Cu bond angle = 170°) and the third (type II) copper lies only 3.1 Å from the bridging oxygen. CueO activity was maximal at pH 6.5 and in the presence of >100 μM Cu(II). Measurements of intermolecular and intramolecular electron transfer with laser flash photolysis in the absence of Cu(II) show that, in addition to the normal reduction of the T1 copper, which occurs with a slow rate (k = 4 × 107 M−1⋅s−1), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k = 9 × 107 M−1⋅s−1, followed by a slow intramolecular electron transfer to T1 copper (k ∼10 s−1). These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper.

Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods.

Recent gene sequence and crystal structure determinations of salivary proteins from several blood-sucking arthropods have revealed an unusual evolutionary relationship: many such proteins derive their functions from lipocalin protein folds. Many blood-sucking arthropods have independently evolved the ability to overcome a host organisms means of preventing blood loss (called hemostasis). Most blood feeders have proteins that induce vasodilation, inhibit blood coagulation, and reduce inflammation, but do so by distinctly different mechanisms. Despite this diversity, in many cases the antihemostatic activities in such organisms reside in proteins with lipocalin folds. Thirteen such lipocalins are described in this review, with a particular focus on the heme-containing nitrophorins from Rhodnius prolixus, which transport nitric oxide, sequester histamine, and disrupt blood coagulation. Also described are the antiplatelet compounds RPAI, moubatin, and pallidipin from R. prolixus, Ornithodoros moubata, and Triatoma pallidipennis; the antithrombin protein triabin from T. pallidipennis; and the tick histamine binding proteins from Rhipicephalus appendiculatus.

Nitric oxide binding to nitrophorin 4 induces complete distal pocket burial

The nitrophorins comprise an unusual family of proteins that use ferric (Fe(III)) heme to transport highly reactive nitric oxide (NO) from the salivary gland of a blood sucking bug to the victim, resulting in vasodilation and reduced blood coagulation. We have determined structures of nitrophorin 4 in complexes with H2O, cyanide and nitric oxide. These structures reveal a remarkable feature: the nitrophorins have a broadly open distal pocket in the absence of NO, but upon NO binding, three or more water molecules are expelled and two loops fold into the distal pocket, resulting in the packing of hydrophobic groups around the NO molecule and increased distortion of the heme. In this way, the protein apparently forms a ‘hydrophobic trap’ for the NO molecule. The structures are very accurate, ranging between 1.6 and 1.4 Å resolutions.

NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III 1

The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III1 region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III1 region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III1 and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III1 in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg88 to Ala88 (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression. [Mol Cancer Ther 2009;8(5):1363–77]

S-nitrosylation-induced conformational change in blackfin tuna myoglobin.

S-Nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by “wedging” apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-Å structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

Crystal Structure of the Bacterial Luciferase/Flavin Complex Provides Insight into the Function of the β Subunit

Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

Crystal Structures of Multicopper Oxidase CueO Bound to Copper(I) and Silver(I) FUNCTIONAL ROLE OF A METHIONINE-RICH SEQUENCE

Background: The multicopper oxidase CueO allows Escherichia coli to survive in aqueous solutions with a high copper concentration. Results: Cu(I) and Ag(I) ions coordinate to a flexible, methionine-rich sequence. Conclusion: The methionine-rich insert and a substrate-binding site ensure that only Cu(I) is oxidized. Significance: Understanding how bacteria survive in the presence of normally toxic concentrations of metal ions may lead to better antibacterial agents. The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 Å with the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a ∼4-fold reduction in kcat for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis.

Molecular Model of a Soluble Guanylyl Cyclase Fragment Determined by Small-Angle X-ray Scattering and Chemical Cross-Linking

Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The β1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

Evolution in an Ancient Detoxification Pathway Is Coupled with a Transition to Herbivory in the Drosophilidae

Chemically defended plant tissues present formidable barriers to herbivores. Although mechanisms to resist plant defenses have been identified in ancient herbivorous lineages, adaptations to overcome plant defenses during transitions to herbivory remain relatively unexplored. The fly genus Scaptomyza is nested within the genus Drosophila and includes species that feed on the living tissue of mustard plants (Brassicaceae), yet this lineage is derived from microbe-feeding ancestors. We found that mustard-feeding Scaptomyza species and microbe-feeding Drosophila melanogaster detoxify mustard oils, the primary chemical defenses in the Brassicaceae, using the widely conserved mercapturic acid pathway. This detoxification strategy differs from other specialist herbivores of mustard plants, which possess derived mechanisms to obviate mustard oil formation. To investigate whether mustard feeding is coupled with evolution in the mercapturic acid pathway, we profiled functional and molecular evolutionary changes in the enzyme glutathione S-transferase D1 (GSTD1), which catalyzes the first step of the mercapturic acid pathway and is induced by mustard defense products in Scaptomyza. GSTD1 acquired elevated activity against mustard oils in one mustard-feeding Scaptomyza species in which GstD1 was duplicated. Structural analysis and mutagenesis revealed that substitutions at conserved residues within and near the substrate-binding cleft account for most of this increase in activity against mustard oils. Functional evolution of GSTD1 was coupled with signatures of episodic positive selection in GstD1 after the evolution of herbivory. Overall, we found that preexisting functions of generalized detoxification systems, and their refinement by natural selection, could play a central role in the evolution of herbivory.