Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors

Cytochrome P450 14α-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14α-methyl group from sterol precursors such as lanosterol, obtusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydrolanosterol. Inhibitors of CYP51 include triazole antifungal agents fluconazole and itraconazole, drugs used in treatment of topical and systemic mycoses. The 2.1- and 2.2-Å crystal structures reported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) are the first structures of an authentic P450 drug target. MTCYP51 exhibits the P450 fold with the exception of two striking differences—a bent I helix and an open conformation of BC loop—that define an active site-access channel running along the heme plane perpendicular to the direction observed for the substrate entry in P450BM3. Although a channel analogous to that in P450BM3 is evident also in MTCYP51, it is not open at the surface. The presence of two different channels, with one being open to the surface, suggests the possibility of conformationally regulated substrate-in/product-out openings in CYP51. Mapping mutations identified in Candida albicans azole-resistant isolates indicates that azole resistance in fungi develops in protein regions involved in orchestrating passage of CYP51 through different conformational stages along the catalytic cycle rather than in residues directly contacting fluconazole. These new structures provide a basis for rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.

Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2α

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1–181) of SYT-SSX1 and 50 amino acids (aa 156–205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription–PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.

Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii1

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii.

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity in vivo

The integrin αLβ2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the αM and α2 subunits has been crystallized in both open and closed conformations; however, the αL I domain has been crystallized in only the closed conformation. We hypothesized that the αL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the αL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg2+. Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The kon, koff, and KD values for the locked open I domain were within 1.5-fold of values previously determined for the αLβ2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized αLβ2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

Mutant G protein α subunit activated by Gβγ: A model for receptor activation?

How receptors catalyze exchange of GTP for GDP bound to the Gα subunit of trimeric G proteins is not known. One proposal is that the receptor uses the G protein's βγ heterodimer as a lever, tilting it to pull open the guanine nucleotide binding pocket of Gα. To test this possibility, we designed a mutant Gα that would bind to βγ in the tilted conformation. To do so, we excised a helical turn (four residues) from the N-terminal region of αs, the α subunit of GS, the stimulatory regulator of adenylyl cyclase. In the presence, but not in the absence, of transiently expressed β1 and γ2, this mutant (αsΔ), markedly stimulated cAMP accumulation. This effect depended on the ability of the coexpressed β protein to interact normally with the lip of the nucleotide binding pocket of αsΔ. We substituted alanine for an aspartate in β1 that binds to a lysine (K206) in the lip of the α subunit's nucleotide binding pocket. Coexpressed with αsΔ and γ2, this mutant, β1-D228A, elevated cAMP much less than did β1-wild type; it did bind to αsΔ normally, however, as indicated by its unimpaired ability to target αsΔ to the plasma membrane. We conclude that βγ can activate αs and that this effect probably involves both a tilt of βγ relative to αs and interaction of β with the lip of the nucleotide binding pocket. We speculate that receptors use a similar mechanism to activate trimeric G proteins.

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

Molecular mechanisms of translation initiation in eukaryotes

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5′ end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III–box I hairpin with a 3′ single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III–box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III–box I hairpin in partially unwound DNA.

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat1

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.

Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean1

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.

Biochemical Characterization of Stromal and Thylakoid-Bound Isoforms of Isoprene Synthase in Willow Leaves1

Isoprene synthase is the enzyme responsible for the foliar emission of the hydrocarbon isoprene (2-methyl-1,3-butadiene) from many C3 plants. Previously, thylakoid-bound and soluble forms of isoprene synthase had been isolated separately, each from different plant species using different procedures. Here we describe the isolation of thylakoid-bound and soluble isoprene synthases from a single willow (Salix discolor L.) leaf-fractionation protocol. Willow leaf isoprene synthase appears to be plastidic, with whole-leaf and intact chloroplast fractionations yielding approximately equal soluble (i.e. stromal) and thylakoid-bound isoprene synthase activities. Although thylakoid-bound isoprene synthase is tightly bound to the thylakoid membrane (M.C. Wildermuth, R. Fall [1996] Plant Physiol 112: 171–182), it can be solubilized by pH 10.0 treatment. The solubilized thylakoid-bound and stromal isoprene synthases exhibit similar catalytic properties, and contain essential cysteine, histidine, and arginine residues, as do other isoprenoid synthases. In addition, two regulators of foliar isoprene emission, leaf age and light, do not alter the percentage of isoprene synthase activity in the bound or soluble form. The relationship between the isoprene synthase isoforms and the implications for function and regulation of isoprene production are discussed.

Synthesis and spectroscopic characterization of site-specific 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine oligodeoxyribonucleotide adducts

The aim of the present study is to determine the chemical structure and conformation of DNA adducts formed by incubation of the bioactive form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-acetoxy-PhIP, with a single-stranded 11mer oligodeoxyribonucleotide. Using conditions optimized to give the C8-dG-PhIP adduct as the major product, sufficient material was synthesized for NMR solution structure determination. The NMR data indicate that in duplex DNA this adduct exists in equilibrium between two different conformational states. In the main conformer, the covalently bound PhIP molecule intercalates in the helix, whilst in the minor conformation the PhIP ligand is probably solvent exposed. In addition to the C8-dG-PhIP adduct, at least eight polar adducts are found after reaction of N-acetoxy-PhIP with the oligonucleotide. Three of these were purified for further characterization and shown to exhibit lowest energy UV absorption bands in the range 342–347 nm, confirming the presence of PhIP or PhIP derivative. Accurate mass determination of two of the polar adducts by negative ion MALDI-TOF MS revealed ions consistent with a spirobisguanidino-PhIP derivative and a ring-opened adduct. The third adduct, which has the same mass as the C8-dG-PhIP oligonucleotide adduct, may contain PhIP bound to the N2 position of guanine.

ATP bound to the origin recognition complex is important for preRC formation

The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.

BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} acts as a phosphate analog in proteins phosphorylated on aspartate: Structure of a BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} complex with phosphoserine phosphatase

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.