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PubMed1997

Basal transcription factors TBP and TFIIB and the viral coactivator E1A 13S bind with distinct affinities and kinetics to the transactivation domain of NF-kappaB p65.

Transactivation domains (TADs) are able to contact several components of the basal transcription apparatus and co-activator molecules. In order to study these interactions in biophysical detail, binding of the well-characterized TAD from the human transcription factor NF-kappaB p65 (RelA) to the basal transcription factors TBP and TFIIB and the viral co-activator protein E1A 13S was chosen as a model system to investigate the kinetics and affinities of such protein-protein interactions by surface plasmon resonance analysis. The TAD of NF-kappaB p65 showed remarkably different affinities and kinetics in binding to the various proteins. The real-time kinetic measurements revealed an association rate constant (kass) of 2.3 x 10(6)/M/s for the interaction between the p65 TAD and TBP. The association rate constants of the p65 TAD were much weaker for TFIIB (6.8 x 10(4)/M/s) and for the E1A 13S protein (4.9 x 10(4)/M/s). The dissociation rate constants (kdiss) were determined to be 7.9 x 10(-4)/s for TBP, 1.6 x 10(-3)/s for TFIIB and 1.3 x 10(-3)/s for the E1A protein. Accordingly, the calculated dissociation constants (Kd) differed between 3.4 x 10(-10)M for the strongly binding TBP protein and 2.3 x 10(-8)M and 2.6 x 10(-8)M for the weaker binding TFIIB and E1A 13S proteins respectively. Non-linear analysis of the appropriate part of the sensorgrams revealed monophasic association and dissociation kinetics for binding between the p65 TAD and all three interaction partners. The remarkable differences in protein affinities add another aspect to a more detailed understanding of formation of the transcription preinitiation complex. The co-transfection of TBP and E1A 13S stimulated NF-kappaB p65-dependent gene expression, showing the biological significance of these interactions.

Adenovirus E1A Proteins↗

PubMed1992

A common intermediary factor (p52/54) recognizing "acidic blob"-type domains is required for transcriptional activation by the Jun proteins.

The ability of the c-Jun protein, the main component of the transcription factor AP1, to interact directly or indirectly with the RNA polymerase II-initiation complex to activate transcription was investigated by in vivo transcription interference ("squelching") experiments. Coexpression of a Jun mutant lacking its DNA binding domain strongly represses the activity of wild-type c-Jun. Repression depends on the presence of the transactivation domains (TADs), suggesting that a limiting factor interacting with the TADs is essential to link Jun and the components of the transcriptional machinery. The activity of this intermediary factor(s) is restricted to TADs characterized by an abundance of negatively charged amino acids, as demonstrated by the abilities of the TADs of JunB, GAL4, and VP16 to repress c-Jun activity. Depending on the presence of the TADs of Jun, we found physical interaction between Jun and a cluster of three proteins with molecular masses of 52, 53, and 54 kDa (p52/54). Association between Jun and p52/54 is strongly reduced in the presence of VP16, suggesting that the two proteins compete for binding to p52/54. Transcription factors containing a different type of TAD (e.g., GHF1, estrogen receptor, or serum response factor) fail to inhibit Jun activity, suggesting that these proteins act through a different mechanism. We consider the requirement of Jun to interact with p52/54 utilized by other transcription factors a new mechanism in the regulation of transcription of Jun-dependent target genes.

Base Sequence↗

PubMed2005

Dual regulation of c-Myc by p300 via acetylation-dependent control of Myc protein turnover and coactivation of Myc-induced transcription.

The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. Here, we report a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. We show that p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independently of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene, and p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. Our results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation.

Acetylation↗

PubMed2006

Increased collagen deposition and elevated expression of connective tissue growth factor in human thoracic aortic dissection.

BACKGROUND: Thoracic aortic dissection (TAD) is characterized by dysregulated extracellular matrix. Little is known about the alterations of collagen and stimulators of collagen synthesis, eg, connective tissue growth factor (CTGF), in patients with TAD. In this study, we examined their roles in TAD. METHODS AND RESULTS: Surgical specimens of the aortic wall of TAD patients (n=10) and controls (n=10) were tested for collagen types I and III and CTGF expression. When compared with controls, protein levels of type I and III collagen and CTGF were significantly increased by 3.2-, 3.7-, and 5.3-fold, respectively (P<0.05 for all). Similar patterns were shown in mRNA levels of type Ialpha and Ialpha2 collagen and CTGF. Using immunohistochemistry and trichrome staining, we also observed elevated levels of collagen in the aortic media and adventitia. Treatment with recombinant human CTGF increased collagen synthesis in cultured aortic smooth muscle cells in a dose- and time-dependent fashion, in which expression of collagens increased from 506+/-108 counts per minute to 2764+/-240 cpm by 50 ng/mL CTGF, and from 30+/-43 cpm to 429+/-102 cpm at 48 hours. CONCLUSIONS: TAD patients exhibited significantly increased expression of aortic collagen types I and III as well as CTGF, which is likely to be responsible for the compromised aortic distensibility and systemic compliance. Because CTGF can increase collagen expression, CTGF may be a new target molecule in the pathogenesis and progression of TAD.

Adult↗

PubMed1999

Multiple NF-ATc isoforms with individual transcriptional properties are synthesized in T lymphocytes.

The transcription factor NF-ATc that controls gene expression in T lymphocytes and embryonic cardiac cells is expressed in three prominent isoforms. This is due to alternative splice/polyadenylation events that lead to the predominant synthesis of two long isoforms in naive T cells and a shorter NF-ATc isoform in effector T cells. Whereas the previously described isoform NF-ATc/A contains a relatively short C terminus, the longer isoforms, B and C, span extra C-terminal peptides of 128 and 246 aa, respectively. We show here that in addition to the strong N-terminal trans-activation domain, TAD-A, which is common to all three NF-ATc isoforms, NF-ATc/C contains a second trans-activation domain, TAD-B, in its C-terminal peptide. Various stimuli of T cells that induce the activity of TAD-A also enhance the activity of TAD-B, but, unlike TAD-A, TAD-B remains unphosphorylated by protein from 12-O-tetradecanoyl 12-phorbol 13-acetate-stimulated T cells. The shorter C-terminal peptide of isoform NF-ATc/B exerts a suppressive transcriptional effect. These properties of NF-ATc/B and -C might be of importance for gene regulation in naive T lymphocytes in which NF-ATc/B and -C are predominantly synthesized.

Amino Acid Sequence↗

PubMed1999

Incidence of transient acantholytic dermatosis (Grover's disease) in a hospital setting.

BACKGROUND: Grovers's disease may be triggered by bed rest. METHODS: We have prospectively analyzed, over a 30-month period, all cases of transient acantholytic dermatosis (TAD) diagnosed in the context of a dermatological consultation for inpatients of a community hospital. RESULTS: A total of 28 cases of TAD were diagnosed within a total of 3,750 patients examined (0.8%). The mean age of patients with TAD was 66.7 years, and the male-to-female ratio was 1.8. In 83% of cases the length of hospitalization preceding TAD was 66.7 years, and the male-to-female ratio was 1.8. In 83% of cases the length of hospitalization preceding TAD exceeded 2 weeks, and in all cases there was an association with strict bed rest. No association with malignant disease or other specific pathologies was observed. CONCLUSION: Our results suggest that TAD, which is frequent in elderly patients within a hospital setting, is not paraneoplastic and favor the hypothesis of a sweat-related pathogenesis.

Acantholysis↗

PubMed1991

Wound complications after modified radical mastectomy compared with tylectomy with axillary lymph node dissection.

Tylectomy with axillary lymph node dissection and radiotherapy (TAD) has become an accepted treatment for early breast cancer and has been shown to result in equal 5- and 8-year survival when compared with modified radical mastectomy (MRM). In order to determine the safety of TAD with respect to wound complications and to identify potential risk factors, we reviewed the charts of 560 patients undergoing MRM (n = 387) and TAD (n = 173) at Virginia Mason Medical Center from 1983 through 1989. The incidence of infection, seroma, hematoma, and epidermolysis were compared, and obesity, age 60 years or older, smoking, antibiotics, and wound drainage were examined as possible risk factors. There were more wound complications in the MRM group versus the TAD group (49% versus 35%; p less than 0.01), specifically more seromas (29% versus 18%; p less than 0.01) and epidermolysis (18% versus 0%). In the MRM group, age 60 years or older was associated with seroma (p less than 0.01) and smoking was associated with epidermolysis (p less than 0.01). In the TAD group, obesity was associated with infection. In both groups, volume of drainage from closed suction wound drains greater than 30 mL in the 24 hours prior to removal of the last drain was associated with seroma (p less than 0.05).

Axilla↗

PubMed1984

Studies on the mechanism of action of 2-beta-D-ribofuranosylthiazole-4-carboxamide--V. Factors governing the response of murine tumors to tiazofurin.

The pharmacological effects and metabolism of tiazofurin have been compared in the six transplantable tumors comprising the NCI rodent tumor panel, viz. the P388 leukemia (S); the L1210 leukemia (S); the Lewis lung carcinoma (S); the B16 melanoma (R); the colon 38 carcinoma (R); and the M5076 sarcoma (R), where (S) denotes sensitivity and (R) resistance to tiazofurin. In addition, a variant of the P388 leukemia rendered resistant to the drug in vitro, and maintaining stable resistance in vivo, P388/TR, was also studied. Intraperitoneal administration of tiazofurin (100 mg/kg) resulted in a 3- to 30-fold greater accumulation of thiazole-4-carboxamide adenine dinucleotide (TAD), the proposed active metabolite of the drug in S versus R lines. In general, levels of TAD, percent inhibition of IMP dehydrogenase (mean 40% in S versus 10% in R), depression in the concentration of guanosine nucleotides, (50% in S versus 20% in R) and percent elevation of levels of IMP (500% in S versus 60% in R) correlated well with sensitivity or resistance. However, the B16 melanoma, although resistant to tiazofurin treatment, showed certain biochemical features characteristic of an S line. The sensitive and resistant tumors displayed comparable abilities to phosphorylate tiazofurin, but there was significant depression only in the R lines of the pyrophosphorylase which converts tiazofurin-5'-monophosphate to TAD (mean 78 nmoles/mg protein/hr in S versus 22 nmoles/mg protein/hr in R). The naturally resistant tumors were also found to exhibit a greater ability to degrade synthetic TAD than the sensitive lines (mean 102 nmoles/mg protein/hr in R versus 29 nmoles/mg protein/hr in S lines). The state of sensitivity or resistance could not be attributed to the basal levels of IMP dehydrogenase, to the specific activities of the enzymes of purine salvage, or to the basal concentration of purine and pyrimidine nucleotides. Moreover, treatment with tiazofurin did not influence the enzymes of TAD synthesis or of purine salvage.

Adenine Nucleotides↗

PubMed1982

Failure of physostigmine in intoxications with tricyclic antidepressants in rats.

In experiments on rats there is a moderate antagonistic effect of physostigmine against intoxications with the tricyclic antidepressants (TAD) clomipramine, desipramine and imipramine, respectively. During the first hours after TAD intoxication the survival rate is higher in physostigmine treated rats. Especially after relatively low doses of TAD the lethality seems to be reduced by physostigmine treatment. However, at the end of the observation period (96 h) the lethality after TAD is equal with and without physostigmine treatment. The effectivity of physostigmine does not depend on the mode of administration: repeated administration and intravenous infusion are not more effective than a single injection of physostigmine. The influence of TAD on heart rate and respiratory rate was not abolished by physostigmine salicylate. Intoxications with high doses of physostigmine were antagonized by atropine; on the other hand there are no signs for an antagonistic effect of desipramine against physostigmine intoxication, that means their anticipated anticholinergic properties could not be proved.

Animals↗

PubMed1995

Comparative effectiveness of hypothermia rewarming techniques: radio frequency energy vs. warm water.

The purpose of this study was to compare the rewarming effectiveness of a radio frequency coil (13.56 MHz) at a specific absorption rate (SAR) of 2.5 W/kg (RF) with warm water immersion (40 degrees C) (WW) and an insulated mummy-type insulating sack (IS) under simulated field conditions. Four male subjects, ages 24-35, were immersed in 10 degrees C water for up to 90 min or until their rectal temperatures (Tre) decreased to 35 degrees C. Each subject had 3 trials in which they were immersed. After each immersion, rewarming was accomplished with either RF, WW, or IS, so that each subject was rewarmed once with each method. Comparisons of the 3 rewarming methods were based on the rate of increase of Tre during rewarming (Tre/t), Tre 60 min after the start of rewarming (Tre60), the time-interval measured from extraction from the water to the end of afterdrop (tad), and the magnitude of any observed Tre afterdrop (Tad). WW had significantly greater Tre/t and Tre60 than either RF or IS (P < 0.03) and a smaller tad than IS (P < 0.05). IS had significantly greater Tad than either WW or RF (P < 0.05). No significant differences in Tre/t, Tre60, or tad were observed between IS and RF. The results of this study indicate that for mildly hypothermic individuals, active rewarming with RF at a SAR of 2.5 W/kg is less effective than WW and roughly equivalent to passive rewarming with IS.

Adult↗

PubMed2005

The effect of tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs) and newer antidepressant drugs on the activity and level of rat CYP3A.

The aim of the present study was to investigate the influence of tricyclic antidepressants (TADs: imipramine, amitriptyline, clomipramine, and desipramine), selective serotonin reuptake inhibitors (SSRIs: fluoxetine and sertraline) and novel antidepressant drugs (mirtazapine and nefazodone) on the activity of CYP3A measured as a rate of testosterone 2beta- and 6beta-hydroxylation. The reaction was studied in control liver microsomes in the presence of the antidepressants, as well as in microsomes of rats treated intraperitoneally (i.p.) for 1 day or 2 weeks with pharmacological doses of the drugs (imipramine, amitriptyline, clomipramine, nefazodone 10 mg kg(-1) i.p.; desipramine, fluoxetine, sertraline 5 mg kg(-1) i.p.; mirtazapine 3 mg kg(-1) i.p.), in the absence of the antidepressants in vitro. The investigated antidepressants added to control liver microsomes produced some inhibitory effects on CYP3A activity, which were very weak (most of TADs, K(i)=145-212 microM), modest (clomipramine and sertraline, K(i)=67.5 and 62 microM, respectively) or moderate (nefazodone and fluoxetine, K(i)=42 and 43 microM, respectively). Mirtazapine did not display this kind of properties. One-day exposure of rats to TADs substantially decreased the activity of CYP3A in liver microsomes, which was maintained during chronic treatment. The observed decreases in the enzyme activity were in contrast to the increased CYP3A protein level found after chronic treatment with TADs. On the other hand, sertraline increased the activity of the enzyme after its prolonged administration and its effect correlated positively with the observed elevation in CYP3A protein level. Fluoxetine, mirtazapine and nefazodone did not change the activity of CYP3A in liver microsomes after their administration to rats. Three different mechanisms of the antidepressants-CYP3A interaction are postulated: 1) a direct inhibition of CYP3A by nefazodone, SSRIs and clomipramine, shown in vitro, with the inhibitory effect of nefazodone being the strongest, but weaker than the effects of this drug on human CYP3A4; 2) in vivo inhibition of CYP3A produced by 1 day and maintained during chronic treatment with TADs, which suggests inactivation of the enzyme by reactive metabolites; 3) in vivo induction by sertraline of CYP3A produced only by chronic treatment with the antidepressant, which suggests its influence on the enzyme regulation.

Animals↗

PubMed2005

Decreased expression of fibulin-5 correlates with reduced elastin in thoracic aortic dissection.

BACKGROUND: Thoracic aortic aneurysmal diseases are characterized by degeneration of elastin within the aortic wall. Although proteinases, such as matrix metalloproteinase, appear to contribute to elastin degradation, little is known about the role of elastic fiber assembly in such diseases. Fibulin-5 is an extracellular protein that is expressed in the vascular basement membrane and regulates elastic fiber assembly by microfibril machinery. In this study, we examined whether thoracic aortic dissection (TAD) is associated with abnormal fibulin-5 expression. METHODS: Intraoperative aortic samples were obtained from 21 patients with proximal aortic dissection. Control aortic tissue was obtained from 11 organ donors, heart transplant recipients, and patients undergoing coronary artery bypass. An in vitro culture of vascular smooth muscle cells was obtained from 2 TAD patients and 1 control subject. To evaluate elastin expression, we stained tissue sections with Verhoeff-Van Gieson stain. Fibulin-5 messenger RNA (mRNA) expression was determined by quantitative real-time reverse-transcriptase-polymerase chain reaction. RESULTS: Aortic fibulin-5 mRNA and elastin content were decreased in TAD patients, compared with controls (P=.001 and P=.02, respectively). Decreased fibulin-5 expression strongly correlated with decreased amounts and fragmentation of elastin in aortic samples from patients with TAD (r=0.83, P < .0001 and F=20.7, P < .0001 respectively). The fibulin-5 mRNA in aortic vascular smooth muscle cells collected from TAD demonstrated a 38% decrease in expression, compared with the control. CONCLUSIONS: Patients with proximal aortic dissection exhibited significantly decreased expression of aortic fibulin-5. Decreased fibulin-5 may contribute to the pathogenesis of aortic dissection by impairing elastic fiber assembly.

Aged↗

PubMed2006

Amino acid substitutions that specifically impair the transcriptional activity of papillomavirus E2 affect binding to the long isoform of Brd4.

The E2 protein of papillomaviruses binds to specific sites in the viral genome to regulate its transcription, replication and segregation in mitosis. Amino acid substitutions in the transactivation domain (TAD) of E2, of Arg37 and Ile73, have been shown previously to impair the transcriptional activity of the protein but not its ability to support viral DNA replication. To understand the biochemical basis of this defect, we have used the TADs of a low-risk (HPV11) and a high-risk (HPV31) human papillomavirus (HPV) as affinity ligands to capture proteins from whole cell extracts that can associate with these domains. The major TAD-binding protein was identified by mass spectrometry and western blotting as the long isoform of Brd4. Binding to Brd4 was also demonstrated for the E2 TADs of other papillomaviruses including cutaneous and animal types. For HPV11, HPV31 and CRPV E2, we found that binding to Brd4 is significantly reduced by substitutions of Arg37 and Ile73. Since these amino acids are located near each other in the 3-dimensional structure of the TAD, we suggest that they define a conserved surface involved in binding Brd4 to regulate viral gene transcription.

Amino Acid Substitution↗

PubMed2002

The subdomains of the transactivation domain of the aryl hydrocarbon receptor (AhR) inhibit AhR and estrogen receptor transcriptional activity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) activates the aryl hydrocarbon receptor (AhR) to mediate transcriptional activity of dioxin-responsive genes. The transactivation domain (TAD) of human AhR (hAhR) has potentially distinct acidic, glutamine-rich, and proline/serine/threonine-rich subdomains. Cotransfection of exogenous hAhR into BP8 cells with isolated subdomains of hAhR TAD fused to glutathione S-transferase exhibited squelching of TCDD-dependent dioxin-response element (DRE)-driven luciferase reporter-gene activity with each subdomain. To study the potential cross talk between AhR- and estrogen receptor (ER)-mediated activities, BP8 cells were cotransfected with hAhR TAD subdomain constructs and ERalpha. The three hAhR TAD subdomains inhibited the 17beta-estradiol-induced estrogen-response element-mediated reporter-gene transactivation. Cotransfection of hAhR with the ligand-binding domain (LBD) of ERalpha also squelched TCDD-dependent DRE-driven reporter-gene activity in the presence of 17beta-estradiol. Similar results were observed in T47D cells that express functional AhR and ERalpha. These results indicate that the isolated subdomains of hAhR's TAD and LBD of ERalpha are capable of squelching ligand-dependent transactivation of either the AhR or the ER, by titrating crucial proteins from an existing common pool of cofactors.

Amino Acid Sequence↗

PubMed1984

The association of transient acantholytic dermatosis with other skin diseases.

A retrospective study of 375 patients with transient acantholytic dermatosis (TAD) showed significant statistical associations between TAD and asteatotic eczema, allergic contact dermatitis, atopic dermatitis, and irritation from adhesive tape, and these associations were believed to be clinically significant. A similar statistical correlation between TAD and the presence of skin cancer was not deemed significant on clinical grounds. There was a clinical suggestion of an etiologic relationship between TAD and bullous pemphigoid, but this was not demonstrated statistically. Most of these associations seem explicable in terms of the Koebner phenomenon, and it is suggested that prevention and treatment of eczematous inflammation and irritation of the skin may be helpful in the management of TAD.

Acantholysis↗

PubMed2004

Characterization of the binding site for inhibitors of the HPV11 E1-E2 protein interaction on the E2 transactivation domain by photoaffinity labeling and mass spectrometry.

An indandione-containing class of inhibitors abrogates DNA replication of human papillomavirus (HPV) types 6 and 11 by binding reversibly to the transactivation domain (TAD) of the viral E2 protein and inhibiting its interaction with the viral E1 helicase. To locate the binding site of this class of protein-protein interaction inhibitors, a benzophenone derivative was used to generate an irreversibly labeled E2-TAD polypeptide. The single site of covalent modification of the E2-TAD was identified by proteolytic digestions using trypsin, LysC, and V8 proteases and characterization of the resulting peptides by LC-MS procedures. Through this methodology, the benzophenone attachment point was located at the terminal methyl of residue Met101. Evidence further pinpointed the site of photoaffinity attachment to the terminal carbon atom, which is significant in providing a definitive example of the ability to locate photoinduced cross-linking to a polypeptide with atomic resolution using solely mass spectrometric detection. The location of the inhibitor binding site vis-à-vis the Glu39 and Glu100 residues sensitive to mutation for HPV 11 E2-TAD is discussed in relation to the crystal structure of the E2-TAD from the related HPV type 16.

Antiviral Agents↗

PubMed1990

Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with the conversion of NAD to NADH. An ordered sequential mechanism where IMP is the first substrate bound and XMP is the last product released was proposed for Tritrichomonas foetus IMPDH on the basis of product inhibition studies. Thiazole adenine dinucleotide (TAD) is an uncompetitive inhibitor versus IMP and a noncompetitive inhibitor versus NAD, which suggests that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive inhibitor versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and mycophenolic acid are mutually exclusive with each other and with NADH. Therefore, mycophenolic acid most probably binds to the dinucleotide site of T. foetus IMPDH. The mycophenolic acid binding site was further localized to the nicotinamide subsite within the dinucleotide site: mycophenolic acid was mutually exclusive with tiazofurin, but could form ternary enzyme complexes with ADP or adenosine diphosphate ribose. NAD inhibits the IMPDH reaction at concentrations greater than 3 mM. NAD substrate inhibition is uncompetitive versus IMP, which suggests that NAD inhibits by binding to E-XMP. TAD is mutually exclusive with both NAD and NADH in multiple-inhibitor experiments, which suggests that there is one dinucleotide binding site. The ordered mechanism predicts that multiple-inhibitor experiments with XMP and TAD, mycophenolic acid, or NAD should have an interaction constant (alpha) between 0 and 1. However, alpha was greater than 1 in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗

PubMed2002

Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: NAD+ orients the active site loop for catalysis.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP with the reduction of NAD(+), which is the rate-limiting step in the biosynthesis of guanine nucleotides. IMPDH is a promising target for chemotherapy. Microbial IMPDHs differ from mammalian enzymes in their lower affinity for inhibitors such as mycophenolic acid (MPA) and thiazole-4-carboxamide adenine dinucleotide (TAD). Part of this resistance is determined by the coupling between nicotinamide and adenosine subsites in the NAD(+) binding site that is postulated to involve an active site flap. To understand the structural basis of the drug selectivity, we solved the X-ray crystal structure of the catalytic core domain of Tritrichomonas foetus IMPDH in complex with IMP and beta-methylene-TAD at 2.2 A resolution. Unlike previous structures of this enzyme, the active site loop is ordered in this complex, and the catalytic Cys319 is 3.6 A from IMP, in the same plane as the hypoxanthine ring. The active site loop forms hydrogen bonds to the carboxamide of beta-Me-TAD which suggests that NAD(+) promotes the nucleophillic attack of Cys319 on IMP. The interactions of the adenosine end of TAD are very different from those in the human enzyme, suggesting the NAD(+) site may be an exploitable target for the design of antimicrobial drugs. In addition, a new K(+) site is observed at the subunit interface. This site is adjacent to beta-Me-TAD, consistent with the link between the K(+) activation and NAD(+). However, contrary to the coupling model, the flap does not cover the adenosine subsite and remains largely disordered.

Adenosine Diphosphate↗