The structural basis of macrolide-ribosome binding assessed using mutagenesis of 23S rRNA positions 2058 and 2059

Macrolides are a diverse group of antibiotics that inhibit bacterial growth by binding within the peptide tunnel of the 50S ribosomal subunit. There is good agreement about the architecture of the macrolide site from different crystallography studies of bacterial and archaeal 50S subunits. These structures show plainly that 23S rRNA nucleotides A2058 and A2059 are located accessibly on the surface of the tunnel wall where they act as key contact sites for macrolide binding. However, the molecular details of how macrolides fit into this site remain a matter of contention. Here, we have generated an isogenic set of single and dual substitutions at A2058 and A2059 in Mycobacterium smegmatis to investigate the effects of the rRNA mutations on macrolide binding. Resistances conferred to a comprehensive array of 11 macrolide compounds are used to assess models of macrolide binding predicted from the crystal structures. The data indicate that all macrolides and their derivatives bind at the same site in the tunnel with their C5 amino sugar in a similar orientation. Our data are compatible with the lactone rings of 14-membered and 16-membered macrolides adopting different conformations, enabling the latter compounds to avoid a steric clash with 2058G. This difference, together with interactions conveyed via substituents that are specific to certain ketolide and macrolide sub-classes, influences the binding to the large ribosomal subunit. Our genetic data show no support for a derivatized-macrolide binding site that has been proposed to be located further down the tunnel.     

Unique capping activity of the recombinant RNA polymerase (L) of vesicular stomatitis virus: association of cellular capping enzyme with the L protein

Vesicular stomatitis virus (VSV), a prototype of non-segmented negative strand RNA viruses, packages an RNA-dependent RNA polymerase (L) which, together with an associated phosphoprotein (P), transcribes the genome RNA, in vitro and in vivo, into mRNAs that are capped at the 5'-ends. However, unlike cellular guanlylyltransferase (GT), the RNA polymerase incorporates GDP in the capped structure, as Gp(alpha)p(beta)-p(alpha)A. In an effort to characterize the capping activity of the RNA polymerase, we have purified recombinant L (rL) protein expressed in insect cells. The rL, like the virion L polymerase, also caps transcribed mRNAs with identical unique cap structure. Interestingly, the purified rL is found to be tightly bound to the GT of the insect cell during all stages of purification. VSV grown in baby hamster kidney cells also packages cellular GT of the murine cell, suggesting that VSV L protein or its associated proteins may have a strong affinity for the cellular GT. The GT bound to rL, however, formed E-GMP complex, whereas no such complex was detected with the rL protein. It appears that the L protein may contain the putative active site for the unique capping reaction or the tightly bound cellular GT may by some unknown mechanism participate in the unique capping reaction.     

Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 Å. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant’s catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.     

A novel electron transfer mechanism suggested by crystallographic studies of mitochondrial cytochrome bc1 complex.

The crystal structure of bovine mitochondrial cytochrome bc1 complex, an integral membrane protein complex of 11 different subunits with a total molecular mass of 242 kDa, demonstrated a tightly associated dimer consisting of three major regions: a matrix region primarily made of subunits core1, core2, 6, and 9; a transmembrane-helix region of 26 helices in the dimer contributed by cytochrome b, cytochrome c1, the Rieske iron-sulfur protein (ISP), subunits 7, 10, and 11; and an intermembrane-space region composed of extramembrane domains of ISP, cytochrome c1, and subunit 8. The structure also revealed the positions of and distances between irons of prosthetic groups, and two symmetry related cavities in the transmembrane-helix region upon dimerization of the bc1 complex. Extensive crystallographic studies on crystals of bc1 complexed with inhibitors of electron transfer identified binding pockets for both Qo and Qi site inhibitors. Discrete binding sites for subtypes of Qo site inhibitors have been mapped onto the Qo binding pocket, and bindings of different subtypes of Qo site inhibitors are capable of inducing dramatic conformational changes in the extramembrane domain of ISP. A novel electron transfer mechanism for the bc1 complex consistent with crystallographic observations is discussed.     

Crystal structure of botulinum neurotoxin type A in complex with the cell surface co-receptor GT1b-insight into the toxin-neuron interaction

Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.     

Conformational Changes in the tryptophan synthase from a hyperthermophile upon alpha2beta2 complex formation: crystal structure of the complex

The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.     

Structural basis of UDP-galactose binding by alpha-1,3-galactosyltransferase (alpha3GT): role of negative charge on aspartic acid 316 in structure and activity

alpha-1,3-Galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-galactose to form an alpha 1-3 link with beta-linked galactosides; it is part of a family of homologous retaining glycosyltransferases that includes the histo-blood group A and B glycosyltransferases, Forssman glycolipid synthase, iGb3 synthase, and some uncharacterized prokaryotic glycosyltransferases. In mammals, the presence or absence of active forms of these enzymes results in antigenic differences between individuals and species that modulate the interplay between the immune system and pathogens. The catalytic mechanism of alpha3GT is controversial, but the structure of an enzyme complex with the donor substrate could illuminate both this and the basis of donor substrate specificity. We report here the structure of the complex of a low-activity mutant alpha3GT with UDP-galactose (UDP-gal) exhibiting a bent configuration stabilized by interactions of the galactose with multiple residues in the enzyme including those in a highly conserved region (His315 to Ser318). Analysis of the properties of mutants containing substitutions for these residues shows that catalytic activity is strongly affected by His315 and Asp316. The negative charge of Asp316 is crucial for catalytic activity, and structural studies of two mutants show that its interaction with Arg202 is needed for an active site structure that facilitates the binding of UDP-gal in a catalytically competent conformation.     

Conformational analysis of GT1B ganglioside and its interaction with botulinum neurotoxin type B: a study by molecular modeling and molecular dynamics

The conformational property of oligosaccharide GT1B in aqueous environment was studied by molecular dynamics (MD) simulation using all-atom model. Based on the trajectory analysis, three prominent conformational models were proposed for GT1B. Direct and water-mediated hydrogen bonding interactions stabilize these structures. The molecular modeling and 15 ns MD simulation of the Botulinum Neuro Toxin/B (BoNT/B) - GT1B complex revealed that BoNT/B can accommodate the GT1B in the single binding mode. Least mobility was seen for oligo-GT1B in the binding pocket. The bound conformation of GT1B obtained from the MD simulation of the BoNT/B-GT1B complex bear a close conformational similarity with the crystal structure of BoNT/A-GT1B complex. The mobility noticed for Arg 1268 in the dynamics was accounted for its favorable interaction with terminal NeuNAc. The internal NeuNAc1 tends to form 10 hydrogen bonds with BoNT/B, hence specifying this particular site as a crucial space for the therapeutic design that can restrict the pathogenic activity of BoNT/B.     

Binding of cations in Bacillus subtilis phosphoribosyldiphosphate synthetase and their role in catalysis.

The binding sites for the two cations essential for the catalytic function of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthases have been identified from the structure of the Bacillus subtilis phosphoribosyldiphosphate synthetase (PRPPsase) with bound Cd(2+). The structure determined from X-ray diffraction data to 2.8-A resolution reveals the same hexameric arrangement of the subunits that was observed in the complexes of the enzyme with the activator sulfate and the allosteric inhibitor ADP. Two cation binding sites were localized in each of the two domains of the subunits that compose the hexamer; each domain of the subunit has an associated cation. In addition to the bound Cd(2+), the Cd(2+)-PRPPsase structure contains a sulfate ion in the regulatory site, a sulfate ion at the ribose-5-phosphate binding site, and an AMP moiety at the ATP binding site. Comparison of the Cd(2+)-PRPPsase to the structures of the PRPPsase complexed with sulfate and mADP reveals the structural rearrangement induced by the binding of the free cation, which is essential for the initiation of the reaction. The comparison to the cPRPP complex of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli, a type I phosphoribosyltransferase, provided information about the binding of PRPP. This strongly indicates that the binding of both substrates must lead to a stabilized conformation of the loop region, which remains unresolved in the known PRPPsase complex structures.     

O-linked glycosylation of rat renal gamma-glutamyltranspeptidase adjacent to its membrane anchor domain

Large domains rich in serine and threonine, that are likely to exhibit clusters of O-linked oligosaccharides, have been reported adjacent to the anchor of several cell surface proteins. No such domain is evident in the primary sequence of rat renal gamma-glutamyltranspeptidase. However, papain treatment of the amphipathic enzyme (Triton-purified gamma-glutamyltranspeptidase, T gamma GT), pretreated with galactose oxidase and NaB3H4 (Frielle, T., and Curthoys, N. P. (1983) Biochemistry 22, 5709-5714), yields the hydrophilic enzyme (papain-treated Triton-purified gamma-glutamyltranspeptidase, PT gamma GT) and a labeled peptide which contains both the amino-terminal membrane anchor and the sequence Pro27-Thr28-Thr29-Ser30. Since [3H]galactose was identified in this peptide, the presence of O-linked oligosaccharides was investigated. Carbohydrate analysis is consistent with the presence of two simple O-linked oligosaccharides on T gamma GT and one on PT gamma GT. Lectin blot analysis of T gamma GT and PT gamma GT was carried out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The small subunits of both T gamma GT and PT gamma GT and the large amphipathic subunit of T gamma GT all react with the peanut agglutinin lectin, but the large subunit of PT gamma GT exhibits no such reactivity. The reactivity with PNA is consistent with the presence of one oligosaccharide with the structure galactose beta 1-3N-acetylgalactosamine alpha 1-Ser/Thr attached to each subunit of T gamma GT. The papain-sensitivity of the oligosaccharide from the larger subunit is consistent with O-glycosylation at the Thr28-Thr29-Ser30 sequence. The results of lectin blot analysis with wheat germ agglutinin imply that the content of N-linked oligosaccharides is unaffected by papain treatment of the transpeptidase. These data represent the first direct evidence for O-glycosylation of a microvillar hydrolase at a site immediately adjacent to the membrane anchor and indicates that even small clusters of Thr and Ser can be O-glycosylated. Isolated O-linked oligosaccharides may have functional significance since single Ser and Thr residues are consistently found near the membrane anchor of many cell surface proteins.     

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 ? resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ～392 residues and two L subunits of ～150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ～100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 ? away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.     

Conformational changes induced by binding UDP-2F-galactose to alpha-1,3 galactosyltransferase- implications for catalysis

Alpha-1,3 galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-galactose to beta-linked galactosides with retention of its alpha configuration. Although several complexes of alpha3GT with inhibitors and substrates have been reported, no structure has been determined of a complex containing intact UDP-galactose. We describe the structure of a complex containing an inhibitory analogue of UDP-galactose, UDP-2F-galactose, in a complex with the Arg365Lys mutant of alpha3GT. The inhibitor is bound in a distorted, bent configuration and comparison with the structure of the apo form of this mutant shows that the interaction induces structural changes in the enzyme, implying a role for ground state destabilization in catalysis. In addition to a general reduction in flexibility in the enzyme indicated by a large reduction in crystallographic B-factors, two loops, one centred around Trp195 and one encompassing the C-terminal 11 residues undergo large structural changes in complexes with UDP and UDP derivatives. The distorted configuration of the bound UDP-2F-galactose in its complex is stabilized, in part, by interactions with residues that are part of or near the flexible loops. Mutagenesis and truncation studies indicate that two highly conserved basic amino acid residues in the C-terminal region, Lys359 and Arg365 are important for catalysis, probably reflecting their roles in these ligand-mediated conformational changes. A second Mn(2+) cofactor has been identified in the catalytic site of a complex of the Arg365Lys with UDP, in a location that suggests it could play a role in facilitating UDP release, consistent with kinetic studies that show alpha3GT activity depends on the binding of two manganese ions. Conformational changes in the C-terminal 11 residues require an initial reorganization of the Trp195 loop and are linked to enzyme progress through the catalytic cycle, including donor substrate distortion, cleavage of the UDP-galactose bond, galactose transfer, and UDP release.     

X-ray scattering study of activated Arp2/3 complex with bound actin-WCA

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.     

The archaeal exosome core is a hexameric ring structure with three catalytic subunits

The exosome is a 3' --> 5' exoribonuclease complex involved in RNA processing. We report the crystal structure of the RNase PH core complex of the Sulfolobus solfataricus exosome determined at a resolution of 2.8 A. The structure reveals a hexameric ring-like arrangement of three Rrp41-Rrp42 heterodimers, where both subunits adopt the RNase PH fold common to phosphorolytic exoribonucleases. Structure-guided mutagenesis reveals that the activity of the complex resides within the active sites of the Rrp41 subunits, all three of which face the same side of the hexameric structure. The Rrp42 subunit is inactive but contributes to the structuring of the Rrp41 active site. The high sequence similarity of this archaeal exosome to eukaryotic exosomes and its high structural similarity to the bacterial mRNA-degrading PNPase support a common basis for RNA-degrading machineries in all three domains of life.     

Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling.

NADH:ubiquinone oxidoreductase (complex I) is the first, largest and most complicated enzyme of the mitochondrial electron transport chain. Photoaffinity labeling with the highly potent and specific inhibitor trifluoromethyldiazirinyl-[(3)H]pyridaben ([(3)H]TDP) labels only the PSST and ND1 subunits of complex I in electron transport particles. PSST is labeled at a high-affinity site responsible for inhibition of enzymatic activity while ND1 is labeled at a low-affinity site not related to enzyme inhibition. In this study we found, as expected, that 13 complex I inhibitors decreased labeling at the PSST site without effect on ND1 labeling. However, there were striking exceptions where an apparent interaction was found between the PSST and ND1 subunits: preincubation with NADH increases PSST labeling and decreases ND1 labeling; the very weak complex I inhibitor 1-methyl-4-phenylpyridinium ion (MPP(+)) and the semiquinone analogue stigmatellin show the opposite effect with increased labeling at ND1 coupled to decreased labeling at PSST in a concentration- and time-dependent manner. MPP(+), stigmatellin and ubisemiquinone have similarly positioned centers of highly negative and positive electrostatic potential surfaces. Perhaps the common action of MPP(+) and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I.     

The domain structure of tryptophan synthase. A neutron scattering study

Tryptophan synthase from Escherichia coli is a complex of two alpha subunits and two beta subunits. Small-angle neutron scattering involving deuterium-labelled isomers revealed the quaternary structure of the enzyme at the level of the beta 2 subunit and the two structural domains P1 and P2 which constitute the alpha subunits. Within the alpha 2 beta 2 complex, the two alpha subunits are completely separated. They are situated on opposite sides of the beta 2 subunit. The most probable distance between the two alpha protomers is 10.5 +/- 1 nm; the nearest distance is 5.8 +/- 0.5 nm, and the largest distance is 13.5 +/- 0.5 nm. The two domains of the same alpha subunit are intimately juxtaposed. The distances between two like or unlike domains belonging to opposite alpha subunits are roughly equal. All domains exhibit about equal distances to the beta 2 subunit which is situated in the centre of the complex. Thus the cleft between P1 and P2, which probably contains the active site of the alpha subunit, makes intimate contact with the beta 2 subunit. Neutron scattering allows us to determine the shape of the beta 2 subunit within the complex. Comparison with the free dimer suggests a conformational change, upon assembly, from an elongated into a more compact form.     

Structure of UDP complex of UDP-galactose:beta-galactoside-alpha -1,3-galactosyltransferase at 1.53-A resolution reveals a conformational change in the catalytically important C terminus

UDP-galactose:beta-galactosyl alpha-1,3-galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-alpha-d-galactose into an alpha-1,3 linkage with beta-galactosyl groups in glycoconjugates. The enzyme is expressed in many mammalian species but is absent from humans, apes, and old world monkeys as a result of the mutational inactivation of the gene; in humans, a large fraction of natural antibodies are directed against its product, the alpha-galactose epitope. alpha3GT is a member of a family of metal-dependent retaining glycosyltransferases including the histo-blood group A and B synthases. A crystal structure of the catalytic domain of alpha3GT was recently reported (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638-649). However, because of the limited resolution (2.3 A) and high mobility of the atoms (as indicated by high B-factors) this structure (form I) does not provide a clear depiction of the catalytic site of the enzyme. Here we report a new, highly ordered structure for the catalytic domain of alpha3GT at 1.53-A resolution (form II). This provides a more accurate picture of the details of the catalytic site that includes a bound UDP molecule and a Mn(2+) cofactor. Significantly, in the new structure, the C-terminal segment (residues 358-368) adopts a very different, highly structured conformation and appears to form part of the active site. The properties of an Arg-365 to Lys mutant indicate that this region is important for catalysis, possibly reflecting its role in a donor substrate-induced conformational change.