Underlying molecular alterations in human dihydrolipoamide dehydrogenase deficiency revealed by structural analyses of disease-causing enzyme variants.

Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency (OMIM# 246900) is an often prematurely lethal genetic disease usually caused by inactive or partially inactive hE3 variants. Here we report the crystal structure of wild-type hE3 at an unprecedented high resolution of 1.75 Å and the structures of six disease-causing hE3 variants at resolutions ranging from 1.44 to 2.34 Å. P453L proved to be the most deleterious substitution in structure as aberrations extensively compromised the active site. The most prevalent G194C-hE3 variant primarily exhibited structural alterations close to the substitution site, whereas the nearby cofactor-binding residues were left unperturbed. The G426E substitution mainly interfered with the local charge distribution introducing dynamics to the substitution site in the dimer interface; G194C and G426E both led to minor structural changes. The R460G, R447G and I445M substitutions all perturbed a solvent accessible channel, the so-called H+/H2O channel, leading to the active site. Molecular pathomechanisms of enhanced reactive oxygen species (ROS) generation and impaired binding to multienzyme complexes were also addressed according to the structural data for the relevant mutations. In summary, we present here for the first time a comprehensive study that links three-dimensional structures of disease-causing hE3 variants to residual hLADH activities, altered capacities for ROS generation, compromised affinities for multienzyme complexes and eventually clinical symptoms. Our results may serve as useful starting points for future therapeutic intervention approaches.

Discovery of stimulator binding to a conserved pocket in the heme domain of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the ß1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.

Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase: IMplications for Enzyme Maturation.

Heat shock protein 90 (hsp90) drives heme insertion into the ß1 subunit of soluble guanylate cyclase (sGC) ß1, which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular interactions between the specific human isoforms hsp90ß and apo-sGCß1. hsp90ß and its isolated M domain, but not its isolated N and C domains, bind with low micromolar affinity to a heme-free, truncated version of sGCß1 (sGCß1(1-359)-H105F). Surprisingly, hsp90ß and its M domain bound to the Per-Arnt-Sim (PAS) domain of apo-sGC-ß1(1-359), which lies adjacent to its heme-binding (H-NOX) domain. The interaction specifically involved solvent-exposed regions in the hsp90ß M domain that are largely distinct from sites utilized by other hsp90 clients. The interaction strongly protected two regions of the sGCß1 PAS domain and caused local structural relaxation in other regions, including a PAS dimerization interface and a segment in the H-NOX domain. Our results suggest a means by which the hsp90ß interaction could prevent apo-sGCß1 from associating with its partner sGCα1 subunit while enabling structural changes to assist heme insertion into the H-NOX domain. This mechanism would parallel that in other clients like the aryl hydrocarbon receptor and HIF1α, which also interact with hsp90 through their PAS domains to control protein partner and small ligand binding interactions.

Rational reprogramming of fungal polyketide first-ring cyclization.

Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-ß-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.

Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1.

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 µM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer-dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and (1)H{(15)N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The "closed loop" form of the A-B and G-H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.

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.

YC-1 binding to the ß subunit of soluble guanylyl cyclase overcomes allosteric inhibition by the α subunit.

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remain unknown. Using linked equilibrium binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the ß subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 µM, depending on the construct. In the presence of CO, these values decrease to 0.6-1.1 µM. Pfizer compound 25 bound ∼10-fold weaker than YC-1 in the absence of CO, whereas compound BAY 41-2272 bound particularly tightly in the presence of CO (Kd = 30-90 nM). Additionally, we found that CO binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 µM) than to the isolated heme domain (Kd = 0.2 µM for Manduca ß H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ∼ 1 µM). These data indicate the α subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the α subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

Per-ARNT-Sim Domains in Nitric Oxide Signaling by Soluble Guanylyl Cyclase.

Nitric oxide (NO) regulates large swaths of animal physiology including wound healing, vasodilation, memory formation, odor detection, sexual function, and response to infectious disease. The primary NO receptor is soluble guanyly/guanylate cyclase (sGC), a dimeric protein of ∼150 kDa that detects NO through a ferrous heme, leading to a large change in conformation and enhanced production of cGMP from GTP. In humans, loss of sGC function contributes to multiple disease states, including cardiovascular disease and cancer, and is the target of a new class of drugs, sGC stimulators, now in clinical use. sGC evolved through the fusion of four ancient domains, a heme nitric oxide / oxygen (H-NOX) domain, a Per-ARNT-Sim (PAS) domain, a coiled coil, and a cyclase domain, with catalysis occurring at the interface of the two cyclase domains. In animals, the predominant dimer is the α1ß1 heterodimer, with the α1 subunit formed through gene duplication of the ß1 subunit. The PAS domain provides an extensive dimer interface that remains unchanged during sGC activation, acting as a core anchor. A large cleft formed at the PAS-PAS dimer interface tightly binds the N-terminal end of the coiled coil, keeping this region intact and unchanged while the rest of the coiled coil repacks, and the other domains reposition. This interface buries ∼3000 Å2 of monomer surface and includes highly conserved apolar and hydrogen bonding residues. Herein, we discuss the evolutionary history of sGC, describe the role of PAS domains in sGC function, and explore the regulatory factors affecting sGC function.

Health Behavior Change Intervention Preferences Expressed by American Indian Cancer Survivors From a Southwest Tribal Community: Semistructured Interview Study.

BACKGROUND: While many factors, including social determinants of health, affect cancer mortality, one modifiable risk factor that may contribute to cancer disparities is obesity. The prevalence of obesity in the American Indian/Alaska Native population is 48.1% per the Centers for Disease Control and Prevention. The overall cancer mortality for the American Indian/Alaska Native population is 18% higher than the White population as reported by the American Cancer Society. Interventions tailored to American Indian/Alaska Native communities that promote healthy lifestyle behaviors after cancer diagnosis and prior to cancer surgery (prehab) might improve cancer outcomes for this population. OBJECTIVE: The aim of the study is to characterize the lifestyle behaviors of San Carlos Apache cancer survivors and identify preferences for the adaption of a prehab intervention. METHODS: Semistructured interviews and validated questionnaires were completed with San Carlos Apache cancer survivors (N=4), exploring their viewpoints on healthy lifestyle and cancer risk and preferences for program development. A thematic content analysis was conducted. RESULTS: Participants had an average BMI of 31 kg/m2 and walked 53 minutes daily. The majority of participants reported a high willingness to change eating habits (n=3, 75%). All 4 reported willingness to participate in a diet and exercise program. Important themes and subthemes were identified: (1) cancer is perceived as a serious health condition in the community (N=4, 100%); (2) environmental exposures are perceived as cancer-causing threats (n=3, 75%); (3) healthy diet, exercise, and avoiding harmful substances are perceived as mitigating cancer risk (n=3, 75%); (4) barriers to healthy habits include distance to affordable groceries (n=3, 75%) and lack of transportation (n=2, 50%); (5) there is high interest in a prehab program geared toward patients with cancer (N=4, 100%); and (6) standard monitoring practiced in published prehab programs showed early acceptability with participants (N=4, 100%). CONCLUSIONS: Collaboration with tribal partners provided important insight that can help inform the adaptation of a culturally appropriate prehab program for San Carlos Apache patients diagnosed with cancer.

Extracellular signals induce dynamic ER remodeling through αTAT1-dependent microtubule acetylation.

Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to various growth factors and cytokines including TGF-ß and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT1-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT1 pathway may be a key target in ER stress and dysfunction.

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 ß1 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.

Nitric oxide delivery and heme-assisted S-nitrosation by the bedbug nitrophorin.

Nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect's salivary glands, NO binds tightly to cNP. During a blood meal, cNP-NO is delivered to the feeding site where dilution and increased pH lead to NO release. In a previous study, cNP was shown to not only bind heme, but to also nitrosate the proximal cysteine, leading to Cys-NO (SNO) formation. SNO formation requires oxidation of the proximal cysteine, which was proposed to be metal-assisted through accompanying reduction of ferric heme and formation of Fe(II)-NO. Here, we report the 1.6 Å crystal structure of cNP first chemically reduced and then exposed to NO, and show that Fe(II)-NO is formed but SNO is not, supporting a metal-assisted SNO formation mechanism. Crystallographic and spectroscopic studies of mutated cNP show that steric crowding of the proximal site inhibits SNO formation while a sterically relaxed proximal site enhances SNO formation, providing insight into specificity for this poorly understood modification. Experiments examining the pH dependence for NO implicate direct protonation of the proximal cysteine as the underlying mechanism. At lower pH, thiol heme ligation predominates, leading to a smaller trans effect and 60-fold enhanced NO affinity (Kd = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.

Sorting nexin-dependent therapeutic targeting of oncogenic epidermal growth factor receptor.

Overexpression and/or overactivation of the Epidermal Growth Factor Receptor (EGFR) is oncogenic in several tumor types yet targeting the kinase domain of wildtype EGFR has had limited success. EGFR has numerous kinase-independent roles, one of which is accomplished through the Sorting Nexin-dependent retrotranslocation of EGFR to the nucleus, which is observed in some metastatic cancers and therapeutically resistant disease. Here, we have utilized the BAR domain of Sorting Nexin 1 to create a peptide-based therapeutic (cSNX1.3) that promotes cell death in EGFR-expressing cancer. We evaluated the efficacy of cSNX1.3 in tumor-bearing WAP-TGFα transgenic mice (an EGFR-dependent model of breast cancer), where cSNX1.3 treatment resulted in significant tumor regression without observable toxicity. Evaluation of remaining tumor tissues found evidence of increased PARP cleavage, suggesting apoptotic tumor cell death. To evaluate the mechanism of action for cSNX1.3, we found that cSNX1.3 binds the C-terminus of the EGFR kinase domain at an interface site opposite the ATP binding domain with a Kd of ~4.0 µM. In vitro analysis found that cSNX1.3 inhibits the nuclear localization of EGFR. To determine specificity, we evaluated cancer cell lines expressing wildtype EGFR (MDA-MB-468, BT20 and A549), mutant EGFR (H1975) and non-transformed lines (CHO and MCF10A). Only transformed lines expressing wildtype EGFR responded to cSNX1.3, while mutant EGFR and normal cells responded better to an EGFR kinase inhibitor. Phenotypically, cSNX1.3 inhibits EGF-, NRG-, and HGF-dependent migration, but not HA-dependent migration. Together, these data indicate that targeting retrotranslocation of EGFR may be a potent therapeutic for RTK-active cancer.

Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence.

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 Šwith 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 k(cat) 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.

Thrombospondin-1 and angiotensin II inhibit soluble guanylyl cyclase through an increase in intracellular calcium concentration.

Nitric oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca(2+)](i)), and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells and that inhibition requires an increase in [Ca(2+)](i). Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca(2+)](i), up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca(2+)](i), also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca(2+) remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in K(m) for GTP, which rises to 834 µM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca(2+)](i) in response to NO, inducing vasodilation, but also is inhibited by high [Ca(2+)](i), providing a fine balance between signals for vasodilation and vasoconstriction.

Oxidation and loss of heme in soluble guanylyl cyclase from Manduca sexta.

Oxidation and loss of heme in soluble guanylyl/guanylate cyclase (sGC), the nitric oxide receptor, is thought to be a major contributor to cardiovascular disease and is the target of compounds BAY 58-2667 and HMR1766. Using spectroelectrochemical titration, we found a truncated sGC to be highly stable in the ferrous state (234 mV) and to bind ferrous heme tightly even in the presence of NO, despite the NO-induced release of the proximal histidine. In contrast, oxidized sGC readily loses ferric heme to myoglobin (0.47 ± 0.02 h(-1)). Peroxynitrite, the presumed cellular oxidant, readily oxidizes sGC in 5 mM glutathione.

Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds.

Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a "stepping-stone" method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and lambda. We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and lambda. The domains show 40% sequence identity but differ by switching of alpha-helix to beta-sheet in a C-terminal region spanning approximately 25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization.

Solution structures of the Shewanella woodyi H-NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP-051.

Heme-nitric oxide/oxygen binding (H-NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H-NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H-NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H-NOX and coiled-coil domains. Here, we report the full NMR structure for CO-ligated Sw H-NOX in the presence and absence of stimulator compound IWP-051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi-empirical quantum potential energy approach. Although IWP-051 binding is weak, a single binding conformation was found at the interface of the two H-NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix-connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure-based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H-NOX proteins. The largest dynamical loop lies at the interface between Sw H-NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.

Ultrafast dynamics of diatomic ligand binding to nitrophorin 4.

Nitrophorin 4 (NP4) is a heme protein that stores and delivers nitric oxide (NO) through pH-sensitive conformational change. This protein uses the ferric state of a highly ruffled heme to bind NO tightly at low pH and release it at high pH. In this work, the rebinding kinetics of NO and CO to NP4 are investigated as a function of iron oxidation state and the acidity of the environment. The geminate recombination process of NO to ferrous NP4 at both pH 5 and pH 7 is dominated by a single approximately 7 ps kinetic phase that we attribute to the rebinding of NO directly from the distal pocket. The lack of pH dependence explains in part why NP4 cannot use the ferrous state to fulfill its function. The kinetic response of ferric NP4NO shows two distinct phases. The relative geminate amplitude of the slower phase increases dramatically as the pH is raised from 5 to 8. We assign the fast phase of NO rebinding to a conformation of the ferric protein with a closed hydrophobic pocket. The slow phase is assigned to the protein in an open conformation with a more hydrophilic heme pocket environment. Analysis of the ultrafast kinetics finds the equilibrium off-rate of NO to be proportional to the open state population as well as the pH-dependent amplitude of escape from the open pocket. When both factors are considered, the off-rate increases by more than an order of magnitude as the pH changes from 5 to 8. The recombination of CO to ferrous NP4 is observed to have a large nonexponential geminate amplitude with rebinding time scales of approximately 10(-11)-10(-9) s at pH 5 and approximately 10(-10)-10(-8) s at pH 7. The nonexponential CO rebinding kinetics at both pH 5 and pH 7 are accounted for using a simple model that has proven effective for understanding CO binding in a variety of other heme systems (Ye, X.; et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14682).

Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta 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.