Precision medicine integrating whole-genome sequencing, comprehensive metabolomics, and advanced imaging.

Genome sequencing has established clinical utility for rare disease diagnosis. While increasing numbers of individuals have undergone elective genome sequencing, a comprehensive study surveying genome-wide disease-associated genes in adults with deep phenotyping has not been reported. Here we report the results of a 3-y precision medicine study with a goal to integrate whole-genome sequencing with deep phenotyping. A cohort of 1,190 adult participants (402 female [33.8%]; mean age, 54 y [range 20 to 89+]; 70.6% European) had whole-genome sequencing, and were deeply phenotyped using metabolomics, advanced imaging, and clinical laboratory tests in addition to family/medical history. Of 1,190 adults, 206 (17.3%) had at least 1 genetic variant with pathogenic (P) or likely pathogenic (LP) assessment that suggests a predisposition of genetic risk. A multidisciplinary clinical team reviewed all reportable findings for the assessment of genotype and phenotype associations, and 137 (11.5%) had genotype and phenotype associations. A high percentage of genotype and phenotype associations (>75%) was observed for dyslipidemia (n = 24), cardiomyopathy, arrhythmia, and other cardiac diseases (n = 42), and diabetes and endocrine diseases (n = 17). A lack of genotype and phenotype associations, a potential burden for patient care, was observed in 69 (5.8%) individuals with P/LP variants. Genomics and metabolomics associations identified 61 (5.1%) heterozygotes with phenotype manifestations affecting serum metabolite levels in amino acid, lipid and cofactor, and vitamin pathways. Our descriptive analysis provides results on the integration of whole-genome sequencing and deep phenotyping for clinical assessments in adults.

Precision medicine screening using whole-genome sequencing and advanced imaging to identify disease risk in adults.

Reducing premature mortality associated with age-related chronic diseases, such as cancer and cardiovascular disease, is an urgent priority. We report early results using genomics in combination with advanced imaging and other clinical testing to proactively screen for age-related chronic disease risk among adults. We enrolled active, symptom-free adults in a study of screening for age-related chronic diseases associated with premature mortality. In addition to personal and family medical history and other clinical testing, we obtained whole-genome sequencing (WGS), noncontrast whole-body MRI, dual-energy X-ray absorptiometry (DXA), global metabolomics, a new blood test for prediabetes (Quantose IR), echocardiography (ECHO), ECG, and cardiac rhythm monitoring to identify age-related chronic disease risks. Precision medicine screening using WGS and advanced imaging along with other testing among active, symptom-free adults identified a broad set of complementary age-related chronic disease risks associated with premature mortality and strengthened WGS variant interpretation. This and other similarly designed screening approaches anchored by WGS and advanced imaging may have the potential to extend healthy life among active adults through improved prevention and early detection of age-related chronic diseases (and their risk factors) associated with premature mortality.

Cognitive enhancement in old dogs from dietary supplementation with a nutrient blend containing arginine, antioxidants, B vitamins and fish oil.

This study focused on the hypothesis that cognitive decline in aged dogs could be attenuated by dietary supplementation with a nutrient blend consisting of antioxidants, B vitamins, fish oil and l-arginine, referred to hereafter as the Brain Protection Blend (BPB). Baseline cognitive assessment before the start of treatment was used to establish cognitively equivalent control (10·464+2·33 kg) and treatment (12·118+3·386 kg) groups of aged dogs between 9·1 and 11·5 years of age and with body condition score of 5. After an initial wash-in period, all dogs were tested over a 6-month period on cognitive test protocols that assessed four phases of a landmark discrimination learning protocol, which assessed a spatial learning skill based on utilisation of external cues, and egocentric discrimination task, which assessed spatial learning based on internal body-centred cues. The BPB-supplemented group showed significantly better performance than the controls on the landmark 1 (P=0·0446) discrimination learning tasks, and on two egocentric discrimination reversal learning tasks (P=0·005 and P=0·01, respectively). The groups did not differ significantly (P>0·10) on the landmark zero discrimination task and the egocentric discrimination learning task. These results suggest beneficial effects are positively linked to task complexity. Many of the nutrients supplemented in the BPB diet were significantly higher in plasma, including arginine, α-tocopherol, DHA and EPA. These results indicate that long-term supplementation with the BPB can have cognition-improving effects and support the use of nutritional strategies in targeting brain ageing-associated risk factors as an intervention to delay cognitive ageing.

Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria.

The primary hyperoxalurias (PH), types 1-3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157-515 contain the catalytic core with one FAD molecule. The 12-fold higher k(cat)/K(m) value of 0.93 M⁻¹·s⁻¹ for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a K(d) value of 125 µM for Hyp, a value ~1600-fold lower than the K(m) value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ1 reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ10 as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH.

Structure of the sulphiredoxin-peroxiredoxin complex reveals an essential repair embrace.

Typical 2-Cys peroxiredoxins (Prxs) have an important role in regulating hydrogen peroxide-mediated cell signalling. In this process, Prxs can become inactivated through the hyperoxidation of an active site Cys residue to Cys sulphinic acid. The unique repair of this moiety by sulphiredoxin (Srx) restores peroxidase activity and terminates the signal. The hyperoxidized form of Prx exists as a stable decameric structure with each active site buried. Therefore, it is unclear how Srx can access the sulphinic acid moiety. Here we present the 2.6 A crystal structure of the human Srx-PrxI complex. This complex reveals the complete unfolding of the carboxy terminus of Prx, and its unexpected packing onto the backside of Srx away from the Srx active site. Binding studies and activity analyses of site-directed mutants at this interface show that the interaction is required for repair to occur. Moreover, rearrangements in the Prx active site lead to a juxtaposition of the Prx Gly-Gly-Leu-Gly and Srx ATP-binding motifs, providing a structural basis for the first step of the catalytic mechanism. The results also suggest that the observed interactions may represent a common mode for other proteins to bind to Prxs.

Oxidation state governs structural transitions in peroxiredoxin II that correlate with cell cycle arrest and recovery.

Inactivation of eukaryotic 2-Cys peroxiredoxins (Prxs) by hyperoxidation has been proposed to promote accumulation of hydrogen peroxide (H2O2) for redox-dependent signaling events. We examined the oxidation and oligomeric states of PrxI and -II in epithelial cells during mitogenic signaling and in response to fluxes of H2O2. During normal mitogenic signaling, hyperoxidation of PrxI and -II was not detected. In contrast, H2O2-dependent cell cycle arrest was correlated with hyperoxidation of PrxII, which resulted in quantitative recruitment of approximately 66- and approximately 140-kD PrxII complexes into large filamentous oligomers. Expression of cyclin D1 and cell proliferation did not resume until PrxII-SO2H was reduced and native PrxII complexes were regenerated. Ectopic expression of PrxI or -II increased Prx-SO2H levels in response to oxidant exposure and failed to protect cells from arrest. We propose a model in which Prxs function as peroxide dosimeters in subcellular processes that involve redox cycling, with hyperoxidation controlling structural transitions that alert cells of perturbations in peroxide homeostasis.

Specificity of Human Sulfiredoxin for Reductant and Peroxiredoxin Oligomeric State.

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., H2O2), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SO2H) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (H2S) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and H2S in supporting Srx activity.

Protein engineering of the quaternary sulfiredoxin.peroxiredoxin enzyme.substrate complex reveals the molecular basis for cysteine sulfinic acid phosphorylation.

Oxidative stress can damage the active site cysteine of the antioxidant enzyme peroxiredoxin (Prx) to the sulfinic acid form, Prx-SO(2)(-). This modification leads to inactivation. Sulfiredoxin (Srx) utilizes a unique ATP-Mg(2+)-dependent mechanism to repair the Prx molecule. Using selective protein engineering that involves disulfide bond formation and site-directed mutagenesis, a mimic of the enzyme.substrate complex has been trapped. Here, we present the 2.1 A crystal structure of human Srx in complex with PrxI, ATP, and Mg(2+). The Cys(52) sulfinic acid moiety was substituted by mutating this residue to Asp, leading to a replacement of the sulfur atom with a carbon atom. Because the Srx reaction cannot occur, the structural changes in the Prx active site that lead to the attack on ATP may be visualized. The local unfolding of the helix containing C52D resulted in the packing of Phe(50) in PrxI within a hydrophobic pocket of Srx. Importantly, this structural rearrangement positioned one of the oxygen atoms of Asp(52) within 4.3 A of the gamma-phosphate of ATP bound to Srx. These observations support a mechanism where phosphorylation of Prx-SO(2)(-) is the first chemical step.

Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins.

The Prxs (peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 microM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation.

The peroxiredoxin repair proteins.

Sulfiredoxin and sestrin are cysteine sulfinic acid reductases that selectively reduce or repair the hyperoxidized forms of typical 2-Cys peroxiredoxins within eukaryotes. As such these enzymes play key roles in the modulation of peroxide-mediated cell signaling and cellular defense mechanisms. The unique structure of sulfiredoxin facilitates access to the peroxiredoxin active site and novel sulfur chemistry.

A metabolome-wide characterization of the diabetic phenotype in ZDF rats and its reversal by pioglitazone.

Type 2 diabetes (T2D) is a complex metabolic disease associated with alterations in glucose, lipid and protein metabolism. In order to characterize the biochemical phenotype of the Zucker diabetic fatty (ZDF) rat, the most common animal model for the study of T2D, and the impact of the insulin sensitizer pioglitazone, a global, mass spectrometry-based analysis of the metabolome was conducted. Overall, 420 metabolites in serum, 443 in the liver and 603 in the intestine were identified at study end. In comparison to two control groups, obese diabetic ZDF rats showed characteristic metabolic signatures that included hyperglycemia, elevated ß-oxidation, dyslipidemia-featured by an increase in saturated and monounsaturated fatty acids and a decrease of medium chain and of polyunsaturated fatty acids in serum-and decreased amino acid levels, consistent with their utilization in hepatic gluconeogenesis. A 13-week treatment with the PPARγ agonist pioglitazone reversed most of these signatures: Pioglitazone improved glycemic control and the fatty acid profile, elevated amino acid levels in the liver, but decreased branched chain amino acids in serum. The hitherto most comprehensive metabolic profiling study identified a biochemical blueprint for the ZDF diabetic model and captured the impact of genetic, nutritional and pharmacological perturbations. The in-depth characterization on the molecular level deepens the understanding and further validates the ZDF rat as a suitable preclinical model of diabetes in humans.

Acetaminophen (Paracetamol) Use Modifies the Sulfation of Sex Hormones.

BACKGROUND: Acetaminophen (paracetamol) is one of the most common medications used for management of pain in the world. There is lack of consensus about the mechanism of action, and concern about the possibility of adverse effects on reproductive health. METHODS: We first established the metabolome profile that characterizes use of acetaminophen, and we subsequently trained and tested a model that identified metabolomic differences across samples from 455 individuals with and without acetaminophen use. We validated the findings in a European ancestry adult twin cohort of 1880 individuals (TwinsUK), and in a study of 1235 individuals of African American and Hispanic ancestry. We used genomics to elucidate the mechanisms targeted by acetaminophen. FINDINGS: We identified a distinctive pattern of depletion of sulfated sex hormones with use of acetaminophen across all populations. We used a Mendelian randomization approach to characterize the role of Sulfotransferase Family 2A Member 1 (SULT2A1) as the site of the interaction. Although CYP3A7-CYP3A51P variants also modified levels of some sulfated sex hormones, only acetaminophen use phenocopied the effect of genetic variants of SULT2A1. Overall, acetaminophen use, age, gender and SULT2A1 and CYP3A7-CYP3A51P genetic variants are key determinants of variation in levels of sulfated sex hormones in blood. The effect of taking acetaminophen on sulfated sex hormones was roughly equivalent to the effect of 35years of aging. INTERPRETATION: These findings raise concerns of the impact of acetaminophen use on hormonal homeostasis. In addition, it modifies views on the mechanism of action of acetaminophen in pain management as sulfated sex hormones can function as neurosteroids and modify nociceptive thresholds.

Metabolic Profiling Reveals Effects of Age, Sexual Development and Neutering in Plasma of Young Male Cats.

Neutering is a significant risk factor for obesity in cats. The mechanisms that promote neuter-associated weight gain are not well understood but following neutering, acute changes in energy expenditure and energy consumption have been observed. Metabolic profiling (GC-MS and UHPLC-MS-MS) was used in a longitudinal study to identify changes associated with age, sexual development and neutering in male cats fed a nutritionally-complete dry diet to maintain an ideal body condition score. At eight time points, between 19 and 52 weeks of age, fasted blood samples were taken from kittens neutered at either 19 weeks of age (Early Neuter (EN), n = 8) or at 31 weeks of age (Conventional Neuter (CN), n = 7). Univariate and multivariate analyses were used to compare plasma metabolites (n = 370) from EN and CN cats. Age was the primary driver of variance in the plasma metabolome, including a developmental change independent of neuter group between 19 and 21 weeks in lysolipids and fatty acid amides. Changes associated with sexual development and its subsequent loss were also observed, with differences at some time points observed between EN and CN cats for 45 metabolites (FDR p<0.05). Pathway Enrichment Analysis also identified significant effects in 20 pathways, dominated by amino acid, sterol and fatty acid metabolism. Most changes were interpretable within the context of male sexual development, and changed following neutering in the CN group. Felinine metabolism in CN cats was the most significantly altered pathway, increasing during sexual development and decreasing acutely following neutering. Felinine is a testosterone-regulated, felid-specific glutathione derivative secreted in urine. Alterations in tryptophan, histidine and tocopherol metabolism observed in peripubertal cats may be to support physiological functions of glutathione following diversion of S-amino acids for urinary felinine secretion.

Oxidized and synchrotron cleaved structures of the disulfide redox center in the N-terminal domain of Salmonella typhimurium AhpF.

The flavoprotein component (AhpF) of Salmonella typhimurium alkyl hydroperoxide reductase contains an N-terminal domain (NTD) with two contiguous thioredoxin folds but only one redox-active disulfide (within the sequence -Cys129-His-Asn-Cys132-). This active site is responsible for mediating the transfer of electrons from the thioredoxin reductase-like segment of AhpF to AhpC, the peroxiredoxin component of the two-protein peroxidase system. The previously reported crystal structure of AhpF possessed a reduced NTD active site, although fully oxidized protein was used for crystallization. To further investigate this active site, we crystallized an isolated recombinant NTD (rNTD); using diffraction data sets collected first at our in-house X-ray source and subsequently at a synchrotron, we showed that the active site disulfide bond (Cys129-Cys132) is oxidized in the native crystals but becomes reduced during synchrotron data collection. The NTD disulfide bond is apparently particularly sensitive to radiation cleavage compared with other protein disulfides. The two data sets provide the first view of an oxidized (disulfide) form of NTD and show that the changes in conformation upon reduction of the disulfide are localized and small. Furthermore, we report the apparent pKa of the active site thiol to be approximately 5.1, a relatively low pKa given its redox potential (approximately 265 mV) compared with most members of the thioredoxin family.

Metabolite profiles correlate closely with neurobehavioral function in experimental spinal cord injury in rats.

Traumatic spinal cord injury (SCI) results in direct physical damage and the generation of local factors contributing to secondary pathogenesis. Untargeted metabolomic profiling was used to uncover metabolic changes and to identify relationships between metabolites and neurobehavioral functions in the spinal cord after injury in rats. In the early metabolic phase, neuronal signaling, stress, and inflammation-associated metabolites were strongly altered. A dynamic inflammatory response consisting of elevated levels of prostaglandin E2 and palmitoyl ethanolamide as well as pro- and anti-inflammatory polyunsaturated fatty acids was observed. N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) were significantly decreased possibly reflecting neuronal cell death. A second metabolic phase was also seen, consistent with membrane remodeling and antioxidant defense response. These metabolomic changes were consistent with the pathology and progression of SCI. Several metabolites, including NAA, NAAG, and the ω-3 fatty acids docosapentaenoate and docosahexaenoate correlated greatly with the established Basso, Beattie and Bresnahan locomotive score (BBB score). Our findings suggest the possibility of a biochemical basis for BBB score and illustrate that metabolites may correlate with neurobehavior. In particular the NAA level in the spinal cord might provide a meaningful biomarker that could help to determine the degree of injury severity and prognosticate neurologic recovery.

Reduction of cysteine sulfinic acid in peroxiredoxin by sulfiredoxin proceeds directly through a sulfinic phosphoryl ester intermediate.

Sulfiredoxin (Srx) catalyzes a novel enzymatic reaction, the reduction of protein cysteine sulfinic acid, Cys-SO(2)(-). This reaction is unique to the typical 2-Cys peroxiredoxins (Prx) and plays a role in peroxide-mediated signaling by regulating the activity of Prxs. Two mechanistic schemes have been proposed that differ regarding the first step of the reaction. This step involves either the direct transfer of the gamma-phosphate of ATP to the Prx molecule or through Srx acting as a phosphorylated intermediary. In an effort to clarify this step of the Srx reaction, we have determined the 1.8A resolution crystal structure of Srx in complex with ATP and Mg(2+). This structure reveals the role of the Mg(2+) ion to position the gamma-phosphate toward solvent, thus preventing an in-line attack by the catalytic residue Cys-99 of Srx. A model of the quaternary complex is consistent with this proposal. Furthermore, phosphorylation studies on several site-directed mutants of Srx and Prx, including the Prx-Asp mimic of the Prx-SO(2)(-) species, support a mechanism where phosphorylation of Prx-SO(2)(-) is the first chemical step.

Identification of intact protein thiosulfinate intermediate in the reduction of cysteine sulfinic acid in peroxiredoxin by human sulfiredoxin.

The reversible oxidation of the active site cysteine in typical 2-Cys peroxiredoxins (Prx) to sulfinic acid during oxidative stress plays an important role in peroxide-mediated cell signaling. The catalytic retroreduction of Prx-SO(2)(-) by sulfiredoxin (Srx) has been proposed to proceed through two novel reaction intermediates, a sulfinic phosphoryl ester and protein-based thiosulfinate. Two scenarios for the repair mechanism have been suggested that differ in the second step of the reaction. The attack of Srx or GSH on the Prx-SO(2)PO(3)(2-) intermediate would result in either the formation of Prx-Cys-S(=O)-S-Cys-Srx or the formation of Prx-Cys-S(=O)-S-G thiosulfinates, respectively. To elucidate the mechanism of Prx repair, we monitored the reduction of human PrxII-SO(2)(-) using rapid chemical quench methodology and electrospray ionization time-of-flight mass spectrometry. An (18)O exchange study revealed that the Prx sulfinic acid phosphoryl ester is rapidly formed and hydrolyzed (k = 0.35 min(-1)). Furthermore, we observed the exclusive formation of a thiosulfinate linkage between Prx and Srx (k = 1.4 min(-1)) that collapses to the disulfide-bonded Srx-Prx species (k = 0.14 min(-1)). Thus, the kinetic and chemical competences of the first two steps in the Srx reaction have been demonstrated. It is clear, however, that GSH may influence thiosulfinate formation and that GSH and Srx may play additional roles in the resolution of the thiosulfinate intermediate.

Mutant AhpC peroxiredoxins suppress thiol-disulfide redox deficiencies and acquire deglutathionylating activity.

The bacterial peroxiredoxin AhpC, a cysteine-dependent peroxidase, can be converted through a single amino acid insertion to a disulfide reductase, AhpC*, active in the glutathione and glutaredoxin pathway. Here we show that, whereas AhpC* is inactive as a peroxidase, other point mutants in AhpC can confer the in vivo disulfide reductase activity without abrogating peroxidase activity. Moreover, AhpC* and several point mutants tested in vitro exhibit an enhanced reductase activity toward mixed disulfides between glutathione and glutaredoxin (Grx-S-SG), consistent with the in vivo requirements for these components. Remarkably, this Grx-S-SG reductase activity relies not on the peroxidatic cysteine but rather on the resolving cysteine that plays only a secondary role in the peroxidase mechanism. Furthermore, putative conformational changes, which impart this unusual Grx-S-SG reductase activity, are transmissible across subunits. Thus, AhpC and potentially other peroxiredoxins in this widespread family can elaborate a new reductase function that alleviates disulfide stress.

Cysteine reactivity and thiol-disulfide interchange pathways in AhpF and AhpC of the bacterial alkyl hydroperoxide reductase system.

AhpC and AhpF from Salmonella typhimurium undergo a series of electron transfers to catalyze the pyridine nucleotide-dependent reduction of hydroperoxide substrates. AhpC, the peroxide-reducing (peroxiredoxin) component of this alkyl hydroperoxidase system, is an important scavenger of endogenous hydrogen peroxide in bacteria and acts through a reactive, peroxidatic cysteine, Cys46, and a second cysteine, Cys165, that forms an active site disulfide bond. AhpF, a separate disulfide reductase protein, regenerates AhpC every catalytic cycle via electrons from NADH which are transferred to AhpC through a tightly bound flavin and two disulfide centers, Cys345-Cys348 and Cys129-Cys132, through putative large domain movements. In order to assess cysteine reactivity and interdomain interactions in both proteins, a comprehensive set of single and double cysteine mutants (replacing cysteine with serine) of both proteins were prepared. Based on 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and AhpC reactivity with multiple mutants of AhpF, the thiolate of Cys129 in the N-terminal domain of AhpF initiates attack on Cys165 of the intersubunit disulfide bond within AhpC for electron transfer between proteins. Cys348 of AhpF has also been identified as the nucleophile attacking the Cys129 sulfur of the N-terminal disulfide bond to initiate electron transfer between these two redox centers. These findings support the modular architecture of AhpF and its need for domain rotations for function, and emphasize the importance of Cys165 in the reductive reactivation of AhpC. In addition, two new constructs have been generated, an AhpF-AhpC complex and a "twisted" form of AhpF, in which redox centers are locked together by stable disulfide bonds which mimic catalytic intermediates.

The thioredoxin domain of Neisseria gonorrhoeae PilB can use electrons from DsbD to reduce downstream methionine sulfoxide reductases.

The PilB protein from Neisseria gonorrhoeae is located in the periplasm and made up of three domains. The N-terminal, thioredoxin-like domain (NT domain) is fused to tandem methionine sulfoxide reductase A and B domains (MsrA/B). We show that the alpha domain of Escherichia coli DsbD is able to reduce the oxidized NT domain, which suggests that DsbD in Neisseria can transfer electrons from the cytoplasmic thioredoxin to the periplasm for the reduction of the MsrA/B domains. An analysis of the available complete genomes provides further evidence for this proposition in other bacteria where DsbD/CcdA, Trx, MsrA, and MsrB gene homologs are all located in a gene cluster with a common transcriptional direction. An examination of wild-type PilB and a panel of Cys to Ser mutants of the full-length protein and the individually expressed domains have also shown that the NT domain more efficiently reduces the MsrA/B domains when in the polyprotein context. Within this frame-work there does not appear to be a preference for the NT domain to reduce the proximal MsrA domain over MsrB domain. Finally, we report the 1.6A crystal structure of the NT domain. This structure confirms the presence of a surface loop that makes it different from other membrane-tethered, Trx-like molecules, including TlpA, CcmG, and ResA. Subtle differences are observed in this loop when compared with the Neisseria meningitidis NT domain structure. The data taken together supports the formation of specific NT domain interactions with the MsrA/B domains and its in vivo recycling partner, DsbD.