EvdS6 is a bifunctional decarboxylase from the everninomicin gene cluster.

The everninomicins are bacterially produced antibiotic octasaccharides characterized by the presence of two interglycosidic spirocyclic ortho-δ-lactone (orthoester) moieties. The terminating G- and H-ring sugars, L-lyxose and C-4 branched sugar ß-D-eurekanate, are proposed to be biosynthetically derived from nucleotide diphosphate pentose sugar pyranosides; however, the identity of these precursors and their biosynthetic origin remain to be determined. Herein we identify a new glucuronic acid decarboxylase from Micromonospora belonging to the superfamily of short-chain dehydrogenase/reductase enzymes, EvdS6. Biochemical characterization demonstrated that EvdS6 is an NAD+-dependent bifunctional enzyme that produces a mixture of two products, differing in the sugar C-4 oxidation state. This product distribution is atypical for glucuronic acid decarboxylating enzymes, most of which favor production of the reduced sugar and a minority of which favor release of the oxidized product. Spectroscopic and stereochemical analysis of reaction products revealed that the first product released is the oxidatively produced 4-keto-D-xylose and the second product is the reduced D-xylose. X-ray crystallographic analysis of EvdS6 at 1.51 Å resolution with bound co-factor and TDP demonstrated that the overall geometry of the EvdS6 active site is conserved with other SDR enzymes and enabled studies probing structural determinants for the reductive half of the net neutral catalytic cycle. Critical active site threonine and aspartate residues were unambiguously identified as essential in the reductive step of the reaction and resulted in enzyme variants producing almost exclusively the keto sugar. This work defines potential precursors for the G-ring L-lyxose and resolves likely origins of the H-ring ß-D-eurekanate sugar precursor.

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain.

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-ß polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a KD of 15-47 µM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC50 values in the range of 15-164 µM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.

Atomic View of Aqueous Cyclosporine A: Unpacking a Decades-Old Mystery.

An atomic view of a main aqueous conformation of cyclosporine A (CycA), an important 11-amino-acid macrocyclic immunosuppressant, is reported. For decades, it has been a grand challenge to determine the conformation of free CycA in an aqueous-like solution given its poor water solubility. Using a combination of X-ray and single-crystal neutron diffraction, we unambiguously resolve a unique conformer (A1) with a novel cis-amide between residues 11 and 1 and two water ligands that stabilize hydrogen bond networks. NMR spectroscopy and titration experiments indicate that the novel conformer is as abundant as the closed conformer in 90/10 (v/v) methanol/water and is the main conformer at 10/90 methanol/water. Five other conformers were also detected in 90/10 methanol/water, one in slow exchange with A1, another one in slow exchange with the closed form and three minor ones, one of which contains two cis amides Abu2-Sar3 and MeBmt1-MeVal11. These conformers help better understand the wide spectrum of membrane permeability observed for CycA analogues and, to some extent, the binding of CycA to protein targets.

Structure of a Stable Interstrand DNA Cross-Link Involving a ß-N-Glycosyl Linkage Between an N6-dA Amino Group and an Abasic Site.

Abasic (AP) sites are one of the most common forms of DNA damage. The deoxyribose ring of AP sites undergoes anomerization between α and ß configurations, via an electrophilic aldehyde intermediate. In sequences where an adenine residue is located on the opposing strand and offset 1 nt to the 3' side of the AP site, the nucleophilic N6-dA amino group can react with the AP aldehyde residue to form an interstrand cross-link (ICL). Here, we present an experimentally determined structure of the dA-AP ICL by NMR spectroscopy. The ICL was constructed in the oligodeoxynucleotide 5'-d(T1A2T3G4T5C6T7A8A9G10T11T12C13A14T15C16T17A18)-3':5'-d(T19A20G21A22T23G24A25A26C27X28T29A30G31A32C33A34T35A36)-3' (X=AP site), with the dA-AP ICL forming between A8 and X28. The NMR spectra indicated an ordered structure for the cross-linked DNA duplex and afforded detailed spectroscopic resonance assignments. Structural refinement, using molecular dynamics calculations restrained by NOE data (rMD), revealed the structure of the ICL. In the dA-AP ICL, the 2'-deoxyribosyl ring of the AP site was ring-closed and in the ß configuration. Juxtapositioning the N6-dA amino group and the aldehydic C1 of the AP site within bonding distance while simultaneously maintaining two flanking unpaired A9 and T29 bases stacked within the DNA is accomplished by the unwinding of the DNA at the ICL. The structural data is discussed in the context of recent studies describing the replication-dependent unhooking of the dA-AP ICL by the base excision repair glycosylase NEIL3.

DNA Sequence Modulates the Efficiency of NEIL1-Catalyzed Excision of the Aflatoxin B1-Induced Formamidopyrimidine Guanine Adduct.

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B1 (AFB1) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB1-FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB1-FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB1-FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB1-FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB1-FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB1-FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

Bicelles Rich in both Sphingolipids and Cholesterol and Their Use in Studies of Membrane Proteins.

How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be cosolubilized by n-dodecyl-ß-melibioside to form bicelles. Small-angle X-ray and neutron scattering, as well as cryo-electron microscopy, demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low-order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.

Configurational and Conformational Equilibria of N6-(2-Deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-methylformamidopyrimidine (MeFapy-dG) Lesion in DNA.

The most common lesion in DNA occurring due to clinical treatment with Temozolomide or cellular exposures to other methylating agents is 7-methylguanine (N7-Me-dG). It can undergo a secondary reaction to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-methylformamidopyrimidine (MeFapy-dG). MeFapy-dG undergoes epimerization in DNA to produce either α or ß deoxyribose anomers. Additionally, conformational rotation around the formyl bond, C5- N5 bond, and glycosidic bond may occur. To characterize and quantitate the mixture of these isomers in DNA, a 13C-MeFapy-dG lesion, in which the CH3 group of the MeFapy-dG was isotopically labeled, was incorporated into the trimer 5'-TXT-3' and the dodecamer 5'-CATXATGACGCT-3' (X = 13C-MeFapy-dG). NMR spectroscopy of both the trimer and dodecamer revealed that the MeFapy-dG lesion exists in single strand DNA as ten configurationally and conformationally discrete species, eight of which may be unequivocally assigned. In the duplex dodecamer, the MeFapy-dG lesion exists as six configurationally and conformationally discrete species. Analyses of NMR data in the single strand trimer confirm that for each deoxyribose anomer, atropisomerism occurs around the C5- N5 bond to produce R a and S a atropisomers. Each atropisomer exhibits geometrical isomerism about the formyl bond yielding E and Z conformations. 1H NMR experiments allow the relative abundances of the species to be determined. For the single strand trimer, the α and ß anomers exist in a 3:7 ratio, favoring the ß anomer. For the ß anomer, with respect to the C5- N5 bond, the R a and S a atropisomers are equally populated. However, the Z geometrical isomer of the formyl moiety is preferred. For the α anomer, the E- S a isomer is present at 12%, whereas all other isomers are present at 5-7%. DNA processing enzymes may differentially recognize different isomers of the MeFapy-dG lesion. Moreover, DNA sequence-specific differences in the populations of configurational and conformational species may modulate biological responses to the MeFapy-dG lesion.

Blood Lipids, Lipoproteins, and Apolipoproteins With Risk of Coronary Heart Disease: A Prospective Study Among Racially Diverse Populations.

BACKGROUND: Comprehensive blood lipoprotein profiles and their association with incident coronary heart disease (CHD) among racially and geographically diverse populations remain understudied. METHODS AND RESULTS: We conducted nested case-control studies of CHD among 3438 individuals (1719 pairs), including 1084 White Americans (542 pairs), 1244 Black Americans (622 pairs), and 1110 Chinese adults (555 pairs). We examined 36 plasma lipids, lipoproteins, and apolipoproteins, measured by nuclear magnetic resonance spectroscopy, with incident CHD among all participants and subgroups by demographics, lifestyle, and metabolic health status using conditional or unconditional logistic regression adjusted for potential confounders. Conventionally measured blood lipids, that is, total cholesterol, triglycerides, low-density lipoprotein-cholesterol, and high-density lipoprotein-cholesterol, were each associated with incident CHD, with odds ratios (ORs) being 1.33, 1.32, 1.24, and 0.79 per 1-SD increase among all participants. Seventeen lipoprotein biomarkers showed numerically stronger associations than conventional lipids, with ORs per 1-SD among all participants ranging from 1.35 to 1.57 and a negative OR of 0.78 (all false discovery rate <0.05), including apolipoprotein B100 to apolipoprotein A1 ratio (OR, 1.57 [95% CI, 1.45-1.7]), low-density lipoprotein-triglycerides (OR, 1.55 [95% CI, 1.43-1.69]), and apolipoprotein B (OR, 1.49 [95% CI, 1.37-1.62]). All these associations were significant and consistent across racial groups and other subgroups defined by age, sex, smoking, obesity, and metabolic health status, including individuals with normal levels of conventionally measured lipids. CONCLUSIONS: Our study highlighted several lipoprotein biomarkers, including apolipoprotein B/ apolipoprotein A1 ratio, apolipoprotein B, and low-density lipoprotein-triglycerides, strongly and consistently associated with incident CHD. Our results suggest that comprehensive lipoprotein measures may complement the standard lipid panel to inform CHD risk among diverse populations.

Site-specific stabilization of DNA by a tethered major groove amine, 7-aminomethyl-7-deaza-2'-deoxyguanosine.

A cationic 7-aminomethyl-7-deaza-2'-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d(G¹A²G³A4X5C6G7C8T9C¹°T¹¹C¹²)2 (X = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X5 imino resonance remained sharp at 55 °C. Additionally, two 5'-neighboring base pairs, A4:T9 and G³:C¹°, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A4:T9 base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A4:T9 base pair in the unmodified duplex, which confirmed that the overall fraction of the A4:T9 base pair in the open state of the modified duplex decreased. This was also observed for the G³:C¹° base pair, where αK(op) for the G³:C¹° base pair in the modified duplex was 3.0 × 106 versus 4.1 × 106 for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X5:C8 and C6:G7 base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation.

Observation of two modes of inhibition of human microsomal prostaglandin E synthase 1 by the cyclopentenone 15-deoxy-Δ(12,14)-prostaglandin J(2).

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E(2) (PGE(2)). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a naturally occurring degradation product of prostaglandin D(2), is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ(2) can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH(2)) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ(2) were determined. The question of whether the anti-inflammatory properties of 15d-PGJ(2) are due to inhibition of MPGES1 is discussed.

γ-Hydroxy-1,N2-propano-2'-deoxyguanosine DNA adduct conjugates the N-terminal amine of the KWKK peptide via a carbinolamine linkage.

The γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine adduct (γ-OH-PdG) was introduced into 5'-d(GCTAGCXAGTCC)-3'·5'-d(GGACTCGCTAGC)-3' (X = γ-OH-PdG). In the presence of excess peptide KWKK, (13)C isotope-edited NMR revealed the formation of two spectroscopically distinct DNA-KWKK conjugates. These involved the reaction of the KWKK N-terminal amino group with the N(2)-dG propylaldehyde tautomer of the γ-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited (15)N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH(3), suggesting that they existed, in part, as imine (Schiff base) linkages. However, (13)C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNA-KWKK conjugates were predicted from potential energy minimization of model structures derived from the refined structure of the fully reduced cross-link [ Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. ( 2010 ) Biochemistry , 49 , 6155 -6164 ]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the S-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNA-KWKK conjugates thermally stabilized duplex DNA. However, the DNA-KWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 5'-CpX-3' sequence, the DNA-KWKK conjugates slowly converted to interstrand N(2)-dG:N(2)-dG DNA cross-links and ring-opened γ-OH-PdG derivatives over a period of weeks.

Modulation of the structure, catalytic activity, and fidelity of African swine fever virus DNA polymerase X by a reversible disulfide switch.

African swine fever virus polymerase X (pol X) is the smallest DNA polymerase known (174 amino acids), and its tertiary structure resembles the C-terminal half of prototypical X-family pol beta, which includes a catalytic dNTP-binding site (palm domain) and a finger domain. This structural similarity and the presence of viral genes coding for other base excision repair proteins suggest that pol X functions in a manner similar to pol beta, but inconsistencies concerning pol X catalysis have been reported. We examined the structural and functional properties of two forms of pol X using spectroscopic and kinetic analysis. Using (1)H-(15)N correlated NMR, we unambiguously demonstrated the slow interconversion of pol X between a reduced (pol X(red)) and an oxidized form (pol X(ox)), confirmed by mass spectrometry. Steady-state kinetic analysis revealed that pol X(ox), with a disulfide bond between Cys-81 and Cys-86, has approximately 10-fold lower fidelity than pol X(red) during dNTP insertion opposite a template G. The disulfide linkage is located between two beta-strands in the palm domain, near the putative dNTP-binding site. Structural alignment of pol X with a pol beta ternary structure suggests that the disulfide switch may modulate fidelity by altering the ability of the palm domain to align and stabilize the primer terminus and catalytic metal ion for deprotonation of the 3'-OH group and subsequent phosphoryl transfer. Thus, DNA polymerase fidelity is altered by the redox state of the enzyme and its related conformational changes.

Structural perturbations induced by the alpha-anomer of the aflatoxin B(1) formamidopyrimidine adduct in duplex and single-strand DNA.

The guanine N7 adduct of aflatoxin B(1) exo-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between alpha and beta anomers. The beta anomer is highly mutagenic in Escherichia coli, producing G --> T transversions; it thermally stabilizes the DNA duplex. The AFB-alpha-FAPY adduct blocks replication; it destabilizes the DNA duplex. Herein, the structure of the AFB-alpha-FAPY adduct has been elucidated in 5'-d(C(1)T(2)A(3)T(4)X(5)A(6)T(7)T(8)C(9)A(10))-3'.5'-d(T(11)G(12)A(13)A(14)T(15)C(16)A(17)T(18)A(19)G(20))-3' (X = AFB-alpha-FAPY) using molecular dynamics calculations restrained by NMR-derived distances and torsion angles. The AFB moiety intercalates on the 5' face of the pyrimidine moiety at the damaged nucleotide between base pairs T(4).A(17) and X(5).C(16), placing the FAPY C5-N(5) bond in the R(a) axial conformation. Large perturbations of the epsilon and zeta backbone torsion angles are observed, and the base stacking register of the duplex is perturbed. The deoxyribose orientation shifts to become parallel to the FAPY base and displaced toward the minor groove. Intrastrand stacking between the AFB moiety and the 5' neighbor thymine remains, but strong interstrand stacking is not observed. A hydrogen bond between the formyl group and the exocyclic amine of the 3'-neighbor adenine stabilizes the E conformation of the formamide moiety. NMR studies reveal a similar 5'-intercalation of the AFB moiety for the AFB-alpha-FAPY adduct in the tetramer 5'-d(C(1)T(2)X(3)A(4))-3', involving the R(a) axial conformation of the FAPY C5-N(5) bond and the E conformation of the formamide moiety. Since in duplex DNA the AFB moiety of the AFB-beta-FAPY adduct also intercalates on the 5' side of the pyrimidine moiety at the damaged nucleotide, we conclude that favorable 5'-stacking leads to the R(a) conformational preference about the C5-N(5) bond; the same conformational preference about this bond is also observed at the nucleoside and base levels. The structural distortions and the less favorable stacking interactions induced by the AFB-alpha-FAPY adduct explain its lower stability as compared to the AFB-beta-FAPY adduct in duplex DNA. In this DNA sequence, hydrogen bonding between the formyl oxygen and the exocyclic amine of the 3'-neighboring adenine stabilizing the E configuration of the formamide moiety is also observed for the AFB-beta-FAPY adduct, and suggests that the identity of the 3'-neighbor nucleotide modulates the stability and biological processing of AFB adducts.

NMR resonance assignments of RNase P protein from Thermotoga maritima.

Ribonuclase P (RNase P) is an essential metallo-endonuclease that catalyzes 5' precursor-tRNA (ptRNA) processing and exists as an RNA-based enzyme in bacteria, archaea, and eukaryotes. In bacteria, a large catalytic RNA and a small protein component assemble to recognize and accurately cleave ptRNA and tRNA-like molecular scaffolds. Substrate recognition of ptRNA by bacterial RNase P requires RNA-RNA shape complementarity, intermolecular base pairing, and a dynamic protein-ptRNA binding interface. To gain insight into the binding specificity and dynamics of the bacterial protein-ptRNA interface, we report the backbone and side chain 1H, 13C, and 15N resonance assignments of the hyperthermophilic Thermatoga maritima RNase P protein in solution at 318 K. Our data confirm the formation of a stable RNA recognition motif (RRM) with intrinsic heterogeneity at both the N- and C-terminus of the protein, consistent with available structural information. Comprehensive resonance assignments of the bacterial RNase P protein serve as an important first step in understanding how coupled RNA binding and protein-RNA conformational changes give rise to ribonucleoprotein function.

A Mechanism of Calmodulin Modulation of the Human Cardiac Sodium Channel.

The function of the human cardiac sodium channel (NaV1.5) is modulated by the Ca2+ sensor calmodulin (CaM), but the underlying mechanism(s) are controversial and poorly defined. CaM has been reported to bind in a Ca2+-dependent manner to two sites in the intracellular loop that is critical for inactivation of NaV1.5 (inactivation gate [IG]). The affinity of CaM for the complete IG was significantly stronger than that of fragments that lacked both complete binding sites. Structural analysis by nuclear magnetic resonance, crystallographic, and scattering approaches revealed that CaM simultaneously engages both IG sites using an extended configuration. Patch-clamp recordings for wild-type and mutant channels with an impaired CaM-IG interaction revealed CaM binding to the IG promotes recovery from inactivation while impeding the kinetics of inactivation. Models of full-length NaV1.5 suggest that CaM binding to the IG directly modulates channel function by destabilizing the inactivated state, which would promote resetting of the IG after channels close.

Structural and biochemical differences between the Notch and the amyloid precursor protein transmembrane domains.

γ-Secretase cleavage of the Notch receptor transmembrane domain is a critical signaling event for various cellular processes. Efforts to develop inhibitors of γ-secretase cleavage of the amyloid-ß precursor C99 protein as potential Alzheimer's disease therapeutics have been confounded by toxicity resulting from the inhibition of normal cleavage of Notch. We present biochemical and structural data for the combined transmembrane and juxtamembrane Notch domains (Notch-TMD) that illuminate Notch signaling and that can be compared and contrasted with the corresponding traits of C99. The Notch-TMD and C99 have very different conformations, adapt differently to changes in model membrane hydrophobic span, and exhibit different cholesterol-binding properties. These differences may be exploited in the design of agents that inhibit cleavage of C99 while allowing Notch cleavage.

Performance of cryogenic probes as a function of ionic strength and sample tube geometry.

The pursuit for more sensitive NMR probes culminated with development of the cryogenic cooled NMR probe. A key factor for the sensitivity is the overall resistance of RF circuitry and sample. Lowering the coil temperature to approximately 25 K and the use of superconducting coil material has greatly reduced the resistance contribution of the hardware. However, the resistance of a salty sample remains the same and evolves as the major factor determining the signal-to-noise ratio. Several approaches have been proposed to reduce the resistance contribution of the sample. These range from encapsulating proteins in a water cavity formed by reverse micelles in low viscosity fluids to the optimal selection of low mobility, low conductivity buffer ions. Here we demonstrate that changing the sample diameter has a pronounced effect on the sample resistance and this results in dramatic improvements of the signal-to-noise ratio and shorter pi/2 pulses. We determined these parameters for common 5 mm NMR tubes under different experimental conditions and compared them to the 2, 3 and 4 mm tubes, in addition, 5mm Shigemi tubes were included since these are widely used. We demonstrate benefits and applicability of studying NMR samples with up to 4M salt concentrations in cryogenic probes. Under high salt conditions, best results in terms of short pi/2 pulses and high signal-to-noise ratios are obtained using 2 or 3mm NMR tubes, especially when limited sample is available. The 4 mm tube is preferred when sample amounts are abundant at intermediate salt conditions.

Unraveling the aflatoxin-FAPY conundrum: structural basis for differential replicative processing of isomeric forms of the formamidopyrimidine-type DNA adduct of aflatoxin B1.

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.