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The human microbiome encodes a large repertoire of biochemical enzymes and pathways, most of which remain uncharacterized. Here, using a metagenomics-based search strategy, we discovered that bacterial members of the human gut and oral microbiome encode enzymes that selectively phosphorylate a clinically used antidiabetic drug, acarbose1,2, resulting in its inactivation. Acarbose is an inhibitor of both human and bacterial α-glucosidases3, limiting the ability of the target organism to metabolize complex carbohydrates. Using biochemical assays, X-ray crystallography and metagenomic analyses, we show that microbiome-derived acarbose kinases are specific for acarbose, provide their harbouring organism with a protective advantage against the activity of acarbose, and are widespread in the microbiomes of western and non-western human populations. These results provide an example of widespread microbiome resistance to a non-antibiotic drug, and suggest that acarbose resistance has disseminated in the human microbiome as a defensive strategy against a potential endogenous producer of a closely related molecule. Bacteria in the human gut and oral microbiome encode enzymes that selectively phosphorylate the antidiabetic drug acarbose—an inhibitor of both human and bacterial α-glucosidases—resulting in its inactivation and limiting the drug's effects on the ability of the host to metabolize complex carbohydrates.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes fatal infections. There exists an urgent need for new antimicrobial agents to combat P. aeruginosa. We conducted a screen for molecules that bind the virulence-controlling protein PqsE and characterized hit compounds for inhibition of PqsE enzymatic activity. The binding conformations of two inhibitory molecules, BB391 and BB393, were identified by crystallography, and inhibitor binding was mimicked by the substitution of PqsE residues E182 and S285 with tryptophan. Comparison of the inhibitor-mimetic mutations to the catalytically inactive PqsE D73A protein demonstrated that catalysis is not responsible for the role PqsE plays in driving virulence factor production. Rather, the PqsE E182W protein fails to interact with the quorum-sensing receptor, RhlR, and our results suggest that it is this interaction that is responsible for promoting virulence factor production in P. aeruginosa. These findings provide a new route for drug discovery efforts targeting PqsE.

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation. The co-crystal structure of VSGsur with the trypanocidal compound suramin directly links the binding of the drug to the resistance phenotype displayed by strains of Trypanosoma brucei expressing VSGsur. Therefore, VSGs can have a function beyond that of antigenic variation.

Cryptophyte algae are well known for their ability to survive under low light conditions through the use of their auxiliary light harvesting antennas, phycobiliproteins. Mainly acting to absorb light where chlorophyll cannot (500-650 nm), phycobiliproteins also play an instrumental role in helping cryptophyte algae respond to changes in light intensity through the process of photoacclimation. Until recently, photoacclimation in cryptophyte algae was only observed as a change in the cellular concentration of phycobiliproteins; however, an additional photoacclimation response was recently discovered that causes shifts in the phycobiliprotein absorbance peaks following growth under red, blue, or green light. Here, we reproduce this newly identified photoacclimation response in two other species of cryptophyte algae, P. sulcata and H. pacifica, and elucidate the origin of the response on the protein level. We compare isolated native and photoacclimated phycobiliproteins for these two species using spectroscopy and mass spectrometry, and we report the x-ray structures of the PC577 light harvesting complex and corresponding photoacclimated complex. We find that neither the protein sequences, nor the protein structures are modified by photoacclimation. We conclude that cryptophyte algae change a chromophore in one site of their phycobiliprotein beta-subunits as part of the photoacclimation response to changes in the spectral quality of light. Ultrafast pump-probe spectroscopy shows that the energy transfer is weakly affected by the photoacclimation.

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics (GPAs). The latter are canonical GPAs with broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridium difficile. Here, we investigated the three-dimensional structures and functional consequences for both molecules. Equilibrium binding studies showed tight binding by keratinimicin A, but not keratinicyclin B, to the peptidoglycan terminus. Using protein crystallography methods, we solved the X-ray crystal structures of both GPAs, which, in conjunction with DFT calculations, indicate that the inability of keratinicyclin B to bind the peptidoglycan is governed by steric factors. Keratinicyclin B, therefore, interferes with an alternative target to inhibit C. difficile growth, a conclusion confirmed by checkerboard analysis that revealed synergistic activity with vancomycin. Our results set the stage for identifying the molecular target of keratinicyclins and for exploring their therapeutic utility in combination with vancomycin.

Failure in repairing ultraviolet radiation-induced DNA damage can lead to mutations and cancer. Among UV-lesions, the pyrimidine–pyrimidone (6-4) photoproduct (6-4PP) is removed from the genome much faster than the cyclobutane pyrimidine dimer (CPD), owing to the more efficient recognition of 6-4PP by XPC-RAD23B, a key initiator of global-genome nucleotide excision repair (NER). Here, we report a crystal structure of a Rad4–Rad23 (yeast XPC-Rad23B ortholog) bound to 6-4PP-containing DNA and 4-μs molecular dynamics (MD) simulations examining the initial binding of Rad4 to 6-4PP or CPD. This first structure of Rad4/XPC bound to a physiological substrate with matched DNA sequence shows that Rad4 flips out both 6-4PP-containing nucleotide pairs, forming an ‘open’ conformation. The MD trajectories detail how Rad4/XPC initiates ‘opening’ 6-4PP: Rad4 initially engages BHD2 to bend/untwist DNA from the minor groove, leading to unstacking and extrusion of the 6-4PP:AA nucleotide pairs towards the major groove. The 5′ partner adenine first flips out and is captured by a BHD2/3 groove, while the 3′ adenine extrudes episodically, facilitating ensuing insertion of the BHD3 β-hairpin to open DNA as in the crystal structure. However, CPD resists such Rad4-induced structural distortions. Untwisting/bending from the minor groove may be a common way to interrogate DNA in NER.

Toxin–antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654–COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis.

Fusion of intracellular trafficking vesicles is mediated by the assembly of soluble N-ethylmaleimide-sensitive fusion protein receptors (SNAREs) to form membrane-bridging complexes. Also required for SNARE-mediated membrane fusion are Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. In the paradigmatic structure of an SM–SNARE complex, Munc18-1 bound to the Qa-SNARE syntaxin 1, the SNARE protein is trapped in an autoinhibited closed conformation that prevents it from entering into SNARE complexes. Here, we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its three-helical Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Tlg2 has a pronounced tendency to self-associate via its SNARE motif, and we demonstrate that Vps45 can rescue Tlg2 oligomers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase-separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein EPYC1 (Essential Pyrenoid Component 1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly-spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of one of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation, and pyrenoid formation. Cryo-electron tomography supports a model where EPYC1 and Rubisco form a co-dependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly one hundred years. Recent studies revealed that trypanosome strains that express the Variant Surface Glycoprotein VSGsur possess heightened resistance to suramin. We show here that VSGsur binds tightly to suramin, other VSGs do not, and that together with VSG13 it defines a structurally divergent subgroup of these coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.

Multisubunit tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for SNARE-mediated membrane fusion in all eukaryotes. MTCs are thought to function as organizers of membrane trafficking, mediating the initial, long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the Dsl1 complex, the simplest known MTC, which is essential for COPI-mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggested how the Dsl1 complex might function to tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other end to ER SNAREs. Here, we use x-ray crystallography to investigate these Dsl1-SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. Our results show that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, depends on the relative orientation of the two Dsl1 legs.

Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1–SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery.

The RicA, RicF, and RicT proteins are widely conserved among the firmicute bacteria and play multiple roles in Bacillus subtilis. Among the phenotypes associated with the inactivation of these proteins are the inability to be genetically transformed or to form biofilms, a decrease in sporulation frequency, and changes in the stability and maturation of multiple RNA species. Despite their importance, the molecular mechanisms of Ric protein activities have not been elucidated and the roles of the two iron-sulfur clusters on the complex of the three proteins are not understood. To unravel the mechanisms of Ric action, molecular characterization of the complex and of its constituent proteins is essential. This report represents a major step toward understanding the structures of the Ric proteins, the arrangement and roles of the Fe-S clusters, and the phenotypes associated with Ric mutations., In Bacillus subtilis, the RicA (YmcA), RicF (YlbF), and RicT (YaaT) proteins accelerate the phosphorylation of the transcription factor Spo0A, contributing to genetic competence, sporulation, and biofilm formation, and are also essential for the correct maturation of several protein-encoding and riboswitch RNAs. These proteins form a stable complex (RicAFT) that carries two [4Fe-4S]+2 clusters. We show here that the complex is a 1:1:1 heterotrimer, and we present the X-ray crystal structures of a RicAF heterotetramer and of a RicA dimer. We also demonstrate that one of the Fe-S clusters (cluster 1) is ligated by cysteine residues donated exclusively by RicT and can be retained when the RicT monomer is purified by itself. Cluster 2 is ligated by C167 from RicT, by C134 and C146 located near the C terminus of RicF, and by C141 at the C terminus of RicA. These findings imply the following novel arrangement: adjacent RicT residues C166 and 167 ligate clusters 1 and 2, respectively, while cluster 2 is ligated by cysteine residues from RicT, RicA, and RicF. Thus, the two clusters must lie close to one another and at the interface of the RicAFT protomers. We also show that the cluster-ligating cysteine residues, and therefore most likely both Fe-S clusters, are essential for cggR-gapA mRNA maturation, for the regulation of ricF transcript stability, and for several Ric-associated developmental phenotypes, including competence for transformation, biofilm formation, and sporulation. Finally, we present evidence that RicAFT, RicAF, and RicA and the RicT monomer may play distinct regulatory roles in vivo.

Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.

Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O-glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1–3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O-glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery.

Many pathogenic bacteria, includingStreptococcus gordonii, possess a pathway for the export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. These adhesins areO-glycosylated by several cytosolic glycosyltransferases and require three accessory Sec proteins (Asp1-3) for export, but how the adhesins are processed for secretion is not well defined. Here, we show thatO-glycosylation ofS. gordoniiadhesin GspB occurs in a sequential manner by three enzymes (GtfA/B, Nss, Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. The modified substrate is subsequently transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures show that both Asp1 and Asp3 are related to carbohydrate binding proteins. Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. These results suggest a mechanism for the modification of adhesin in the cytosol and its subsequent targeting to the export machinery.

The conserved oligomeric Golgi (COG) complex is required, along with SNARE and Sec1/Munc18 (SM) proteins, for vesicle docking and fusion at the Golgi. COG, like other multisubunit tethering complexes (MTCs), is thought to function as a scaffold and/or chaperone to direct the assembly of productive SNARE complexes at the sites of membrane fusion. Reflecting this essential role, mutations in the COG complex can cause congenital disorders of glycosylation. A deeper understanding of COG function and dysfunction will likely depend on elucidating its molecular structure. Despite some progress toward this goal, including EM studies of COG lobe A (subunits 1-4) and higher-resolution structures of portions of Cog2 and Cog4, the structures of COG's eight subunits and the principles governing their assembly are mostly unknown. Here, we report the crystal structure of a complex between two lobe B subunits, Cog5 and Cog7. The structure reveals that Cog5 is a member of the complexes associated with tethering containing helical rods (CATCHR) fold family, with homology to subunits of other MTCs including the Dsl1, exocyst, and Golgi-associated retrograde protein (GARP) complexes. The Cog5-Cog7 interaction is analyzed in relation to the Dsl1 complex, the only other CATCHR-family MTC for which subunit interactions have been characterized in detail. Biochemical and functional studies validate the physiological relevance of the observed Cog5-Cog7 interface, indicate that it is conserved from yeast to humans, and demonstrate that its disruption in human cells causes defects in trafficking and glycosylation.

N-methyl mesoporphyrin IX (NMM) is exceptionally selective for G-quadruplexes (GQ) relative to duplex DNA and, as such, has found a wide range of applications in biology and chemistry. In addition, NMM is selective for parallel versus antiparallel GQ folds, as was recently demonstrated in our laboratory. Here, we present the X-ray crystal structure of a complex between NMM and human telomeric DNA dAGGG(TTAGGG)(3), Tel22, determined in two space groups, P2(1)2(1)2 and P6, at 1.65 and 2.15 Å resolution, respectively. The former is the highest resolution structure of the human telomeric GQ DNA reported to date. The biological unit contains a Tel22 dimer of 5'-5' stacked parallel-stranded quadruplexes capped on both ends with NMM, supporting the spectroscopically determined 1:1 stoichiometry. NMM is capable of adjusting its macrocycle geometry to closely match that of the terminal G-tetrad required for efficient π-π stacking. The out-of-plane N-methyl group of NMM fits perfectly into the center of the parallel GQ core where it aligns with potassium ions. In contrast, the interaction of the N-methyl group with duplex DNA or antiparallel GQ would lead to steric clashes that prevent NMM from binding to these structures, thus explaining its unique selectivity. On the basis of the biochemical data, binding of NMM to Tel22 does not rely on relatively nonspecific electrostatic interactions, which characterize most canonical GQ ligands, but rather it is hydrophobic in nature. The structural features observed in the NMM-Tel22 complex described here will serve as guidelines for developing new quadruplex ligands that have excellent affinity and precisely defined selectivity.

The mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase, a key component of the Carma1/Bcl10/MALT1 signalosome, is critical for NF-κB signaling in multiple contexts. MALT1 is thought to function as a scaffold and protease to promote signaling; however, the biochemical and structural basis of paracaspase action remains largely unknown. Here we report the 1.75-Å resolution crystal structure of the MALT1 paracaspase region, which contains the paracaspase domain and an ensuing Ig-like domain. The paracaspase and the Ig domains appear as a single folding unit and interact with each other through extensive van der Waals contacts and hydrogen bonds. The paracaspase domain adopts a fold that is nearly identical to that of classic caspases and homodimerizes similarly to form an active protease. Unlike caspases, the active and mature form of the paracaspase domain remains a single uncleaved polypeptide and specifically recognizes the bound peptide inhibitor Val-Arg-Pro-Arg. In particular, the carboxyl-terminal amino acid Arg of the inhibitor is coordinated by three highly conserved acidic residues. This structure serves as an important framework for deciphering the function and mechanism of paracaspases exemplified by MALT1.

Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ~60 Å, twice the ~30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multi-domain proteins.

alpha-Hemoglobin (alphaHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with alphaHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting alphaHb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in alphaHb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro(30), in loop 1 of AHSP. Mutation of Pro(30) to a variety of residue types results in reduced ability to convert alphaHb. In complex with alphaHb, AHSP Pro(30) adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in alphaHb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the alphaHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of alphaHb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.

Certain antibodies (Abs) elicited using the cardiac glycoside digoxin (digoxigenin tridigitoxoside) bind preferentially to analogs that differ from digoxin by substitutions on the cardenolide rings, the lactone, or by the presence or absence of attached sugars. Antibody 26-10 binds equally well to digoxin and digitoxin, which differ only by the presence in the former and the absence in the latter of an hydroxyl group at C12. Other antidigoxin Abs, however, can distinguish between these ligands by three orders of magnitude in binding. Inspection of the structure of Fab 26-10 complexed with digoxin shows a gap in complementarity in the region between the digoxin O12 and LCDR3. We proposed that insertions in LCDR3 might result in Abs that bind digitoxin preferentially. We produced libraries of mutants displayed on bacteriophage which were randomized at LCDR3 and contained LCDR3 insertions. Mutants were selected by panning against digoxin and analogs. The mutants bound digitoxin preferentially up to 47-fold greater than digoxin. The mutants that bound well to digitoxin demonstrated a consensus sequence including the substitution of Trp at position L:94. Using site-directed mutagenesis, the binding to digitoxin was shown to be maximized by the combination of an insertion and L:Trp94 mutation, moving the L 94 side chain closer to digoxin. We also selected mutants that bound preferentially to gitoxin, which, like digitoxin, lacks the 12-hydroxyl, increasing relative binding to gitoxin up to 600-fold compared to the unmutated Ab 26-10.

Regulated intramembrane proteolysis by members of the site-2 protease (S2P) family is an important signaling mechanism conserved from bacteria to humans. Here we report the crystal structure of the transmembrane core domain of an S2P metalloprotease from Methanocaldococcus jannaschii . The protease consists of six transmembrane segments, with the catalytic zinc atom coordinated by two histidine residues and one aspartate residue ∼14 angstroms into the lipid membrane surface. The protease exhibits two distinct conformations in the crystals. In the closed conformation, the active site is surrounded by transmembrane helices and is impermeable to substrate peptide; water molecules gain access to zinc through a polar, central channel that opens to the cytosolic side. In the open conformation, transmembrane helices α1 and α6 separate from each other by 10 to 12 angstroms, exposing the active site to substrate entry. The structure reveals how zinc embedded in an integral membrane protein can catalyze peptide cleavage.

The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3-6, which overlaps with the binding site for the B' (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B' for the PP2A core enzyme. Consequently, ST does not efficiently displace B' from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes.

SummaryThe serine/threonine phosphatase protein phosphatase 2A (PP2A) plays an essential role in many aspects of cellular functions and has been shown to be an important tumor suppressor. The core enzyme of PP2A comprises a 65 kDa scaffolding subunit and a 36 kDa catalytic subunit. Here we report the crystal structures of the PP2A core enzyme bound to two of its inhibitors, the tumor-inducing agents okadaic acid and microcystin-LR, at 2.6 and 2.8 Å resolution, respectively. The catalytic subunit recognizes one end of the elongated scaffolding subunit by interacting with the conserved ridges of HEAT repeats 11–15. Formation of the core enzyme forces the scaffolding subunit to undergo pronounced structural rearrangement. The scaffolding subunit exhibits considerable conformational flexibility, which is proposed to play an essential role in PP2A function. These structures, together with biochemical analyses, reveal significant insights into PP2A function and serve as a framework for deciphering the diverse roles of PP2A in cellular physiology.

Phosphotyrosyl phosphatase activator (PTPA), also known as PP2A phosphatase activator, is a conserved protein from yeast to human. Here we report the 1.9 A crystal structure of human PTPA, which reveals a previously unreported fold consisting of three subdomains: core, lid, and linker. Structural analysis uncovers a highly conserved surface patch, which borders the three subdomains, and an associated deep pocket located between the core and the linker subdomains. The conserved surface patch and the deep pocket are responsible for binding to PP2A and ATP, respectively. PTPA and PP2A A-C dimer together constitute a composite ATPase. PTPA binding to PP2A results in a dramatic alteration of substrate specificity, with enhanced phosphotyrosine phosphatase activity and decreased phosphoserine phosphatase activity. This function of PTPA strictly depends on the composite ATPase activity. These observations reveal significant insights into the function and mechanism of PTPA and have important ramifications for understanding PP2A function.

Recent structural studies of epidermal growth factor receptor (EGFR) family extracellular regions have identified an unexpected mechanism for ligand-induced receptor dimerization that has important implications for activation and inhibition of these receptors. Here we describe the 2.8 angstroms resolution X-ray crystal structure of the antigen binding (Fab) fragment from cetuximab (Erbitux), an inhibitory anti-EGFR antibody, in complex with the soluble extracellular region of EGFR (sEGFR). The sEGFR is in the characteristic "autoinhibited" or "tethered" inactive configuration. Cetuximab interacts exclusively with domain III of sEGFR, partially occluding the ligand binding region on this domain and sterically preventing the receptor from adopting the extended conformation required for dimerization. We suggest that both these effects contribute to potent inhibition of EGFR activation.

Mutations in the BRCA2 (breast cancer susceptibility gene 2) tumor suppressor lead to chromosomal instability due to defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, but BRCA2's role in this process has been unclear. Here, we present the 3.1 angstrom crystal structure of a ∼90-kilodalton BRCA2 domain bound to DSS1, which reveals three oligonucleotide-binding (OB) folds and a helix-turn-helix (HTH) motif. We also (i) demonstrate that this BRCA2 domain binds single-stranded DNA, (ii) present its 3.5 angstrom structure bound to oligo(dT) 9 , (iii) provide data that implicate the HTH motif in dsDNA binding, and (iv) show that BRCA2 stimulates RAD51-mediated recombination in vitro. These findings establish that BRCA2 functions directly in homologous recombination and provide a structural and biochemical basis for understanding the loss of recombination-mediated DSB repair in BRCA2-associated cancers.

The Chfr mitotic checkpoint protein is frequently inactivated in human cancer. We determined the three-dimensional structure of its FHA domain in its native form and in complex with tungstate, an analog of phosphate. The structures revealed a β sandwich fold similar to the previously determined folds of the Rad53 N- and C-terminal FHA domains, except that the Rad53 domains were monomeric, whereas the Chfr FHA domain crystallized as a segment-swapped dimer. The ability of the Chfr FHA domain to recognize tungstate suggests that it shares the ability with other FHA domains to bind phosphoproteins. Nevertheless, differences in the sequence and structure of the Chfr and Rad53 FHA domains suggest that FHA domains can be divided into families with distinct binding properties.

Brca1 C-terminal (BRCT) domains are a common protein–protein interaction motif in proteins involved in the DNA damage response and DNA repair. The DNA-damage response protein 53BP1 has two BRCT domains that bind to the DNA-binding domain of p53. The 53BP1 tandem-BRCT region is homologous to the tandem-BRCT region of Brca1, which is involved in double-strand break repair and homologous recombination and which binds BACH1, a member of the DEAH helicase family. Here we report the structures of a human 53BP1–p53 complex and of the rat Brca1 BRCT repeats. The 53BP1–p53 structure shows that the two BRCT repeats are arranged tandemly and pack extensively through an interface that also involves the inter-repeat linker. The first BRCT repeat and the linker together bind p53 on a region that overlaps with the DNA-binding surface of p53 and involves p53 residues that are mutated in cancer and are important for DNA binding. Comparison with the structure of the tandem-BRCT region of Brca1 shows a remarkable conservation of the repeat arrangement and of the inter-BRCT repeat interface. Analysis of human BRCA1 tumor-derived mutations and conservation identifies a potential protein-binding site that we show through mutagenesis is involved in BACH1 binding. The BACH1-binding region of Brca1 consists of a unique insertion in the first BRCT repeat and the inter-repeat linker and is analogous to the region of 53BP1 that binds p53.

Metabolite glycosylation is affected by three classes of enzymes: nucleotidylyltransferases, which activate sugars as nucleotide diphospho-derivatives, intermediate sugar-modifying enzymes and glycosyltransferases, which transfer the final derivatized activated sugars to aglycon substrates. One of the first crystal structures of an enzyme responsible for the first step in this cascade, alpha-D-glucopyranosyl phosphate thymidylyltransferase (Ep) from Salmonella, in complex with product (UDP-Glc) and substrate (dTTP) is reported at 2.0 A and 2.1 A resolution, respectively. These structures, in conjunction with the kinetic characterization of Ep, clarify the catalytic mechanism of this important enzyme class. Structure-based engineering of Ep produced modified enzymes capable of utilizing 'unnatural' sugar phosphates not accepted by wild type Ep. The demonstrated ability to alter nucleotidylyltransferase specificity by design is an integral component of in vitro glycosylation systems developed for the production of diverse glycorandomized libraries.

The HyHEL-5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg-white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL-5, an anti-hen egg-white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side-chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL-5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side-chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL-5 is similar to that in previously determined lysozyme-HyHEL-5 complexes. © 1996 Wiley-Liss, Inc.

The X-ray structure of the uncomplexed human chimeric Fab′ of the anti-tumor antibody BR96 has been determined at 2.6 A resolution. The structure has been compared with Lewis Y antigen-complexed structures of BR96 which were determined previously. The comparison reveals segmental motions and/or conformational rearrangements of three CDR loops (L1, L3, and H2), whereas CDR H3 does not undergo changes upon complexation despite its significant main-chain contacts to the carbo hydrate antigen. In light of the uncomplexed chimeric Fab′ structure reported here, the previously observed high mobility of the CL:CH1 domains of the complexed chimeric BR96 Fab is rationalized as a “swinging” motion approximately about the axis of the elbow bend.

We constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 of the anti-digoxin antibody 26-10 Fab to investigate sequence constraints necessary for high affinity binding in an antibody of known crystal structure. Phage were selected by panning against digoxin and three C-16-substituted analogues. All antigen-positive mutants selected using other analogues also bound digoxin. Among 73 antigen-positive clones, 26 different nucleotide sequences were found. The majority of Fabs had high affinity for digoxin (Ka 3.4 x 10(9) M-1) despite wide sequence diversity. Two mutants displayed affinities 2- and 4-fold higher than the parental antibody. Analysis of the statistical distribution of sequences showed that highest affinity binding occurred with a restricted set of amino acid substitutions at positions H33-35. All clones save two retained the parental Asn-H35, which contacts hapten and hydrogen bonds to other binding site residues in the parental structure. Positions H30-32 display remarkable diversity, with 10-14 different substitutions for each residue, consistent with high affinity binding. Thus complementarity can be retained and even improved despite diversity in the conformation of the N-terminal portion of the H-CDR1 loop.

The crystal structure of the human cyclinA-cyclin-dependent kinase2 (CDK2)-ATP complex has been determined at 2.3 A resolution. CyclinA binds to one side of CDK2's catalytic cleft, inducing large conformational changes in its PSTAIRE helix and T-loop. These changes activate the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft.

We determined the sequence, specificity for structurally related cardenolides, and three-dimensional structure of the anti-digoxin antibody 40-50 Fab in complex with ouabain. The 40-50 antibody does not share close sequence homology with other high-affinity anti-digoxin antibodies. Measurement of the binding constants of structurally distinct digoxin analogs indicated a well-defined specificity pattern also distinct from other anti-digoxin antibodies. The 40-50-ouabain Fab complex crystallizes in space group C2 with cell dimensions of a = 93.7 A, b = 84.8 A, c = 70.1 A, beta = 128.0 degrees. The structure of the complex was determined by X-ray crystallography and refined at a resolution of 2.7 A. The hapten is bound in a pocket extending as a groove from the center of the combining site across the light chain variable domain, with five of the six complementarity-determining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; two hydrogen bonds are formed between the antibody and hapten. The surface area of the antibody combining site buried by ouabain is contributed equally by the light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chains. The surface complementarity between hapten and antibody is sufficient to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40-50-ouabain complex allows qualitative explanation of the observed fine specificities of 40-50, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40-50 complexed with ouabain and the previously determined 26-10 anti-digoxin Fab complexed with digoxin, demonstrates that the antibodies bind these structurally related haptens in different orientations, consistent with their different fine specificities. These results demonstrate that the immune system can generate antibodies that provide diverse structural solutions to the binding of even small molecules.

Arc repressor is a small, dimeric DNA-binding protein that belongs to the ribbon-helix-helix family of transcription factors. Replacing Pro8 at the N-terminal end of the beta-sheet with leucine increases the stability of the mutant protein by 2.5 kcal/mol of dimer. However, this enhanced stability is achieved at the expense of significantly reduced DNA binding affinity. The structure of the PL8 mutant dimer has been determined to 2.4-A resolution by X-ray crystallography. The overall structure of the mutant is very similar to wild type, but Leu8 makes an additional interstrand hydrogen bond at each end of the beta-sheet of the mutant, increasing the total number of beta-sheet hydrogen bonds from six to eight. Comparison of the refolding and unfolding kinetics of the PL8 mutant and wild-type Arc shows that the enhanced stability of the mutant is accounted for by a decrease in the rate of protein unfolding, suggesting that the mutation acts to stabilize the native state and that the beta-sheet forms after the rate-limiting step in folding. The reduced operator affinity of the PL8 dimer appears to arise because the mutant cannot make the new interstrand hydrogen bonds and simultaneously make the wild-type set of contacts with operator DNA.

Mutations in the p53 tumor suppressor are the most frequently observed genetic alterations in human cancer. The majority of the mutations occur in the core domain which contains the sequence-specific DNA binding activity of the p53 protein (residues 102-292), and they result in loss of DNA binding. The crystal structure of a complex containing the core domain of human p53 and a DNA binding site has been determined at 2.2 angstroms resolution and refined to a crystallographic R factor of 20.5 percent. The core domain structure consists of a beta sandwich that serves as a scaffold for two large loops and a loop-sheet-helix motif. The two loops, which are held together in part by a tetrahedrally coordinated zinc atom, and the loop-sheet-helix motif form the DNA binding surface of p53. Residues from the loop-sheet-helix motif interact in the major groove of the DNA, while an arginine from one of the two large loops interacts in the minor groove. The loops and the loop-sheet-helix motif consist of the conserved regions of the core domain and contain the majority of the p53 mutations identified in tumors. The structure supports the hypothesis that DNA binding is critical for the biological activity of p53, and provides a framework for understanding how mutations inactivate it.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.

We have determined the three-dimensional structures of the antigen-binding fragment of the anti-digoxin monoclonal antibody 26-10 in the uncomplexed state at 2.7 A resolution and as a complex with digoxin at 2.5 A resolution. Neither the antibody nor digoxin undergoes any significant conformational changes upon forming the complex. Digoxin interacts primarily with the antibody heavy chain and is oriented such that the carbohydrate groups are exposed to solvent and the lactone ring is buried in a deep pocket at the bottom of the combining site. Despite extensive interactions between antibody and antigen, no hydrogen bonds or salt links are formed between 26-10 and digoxin. Thus the 26-10-digoxin complex is unique among the known three-dimensional structures of antibody-antigen complexes in that specificity and high affinity arise primarily from shape complementarity.

The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody VL domain has been characterized by measurements of 15N T1 and T2 relaxation times, 1H–15N NOE values, and 1H–2H exchange rates. These data were acquired with 2D inverse detected heteronuclear 1H–15N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R2 (= 1/T2). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R2 exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R2 exchange contributions. The measured 1H–2H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the VL domain, including the three hypervariable loops, undergo dynamic changes upon VL:VH association and/ or complexation with digoxin. Overall, the 26–10 VL domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc.

Antibody LB4, produced by a spontaneous variant of the murine anti-digoxin monoclonal antibody 26-10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640-4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26-10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26-10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are algal toxins that can accumulate in shellfish and cause diarrhetic shellfish poisoning. Recent studies indicate that DTX-2 is about half as toxic and has about half the affinity for protein phosphatase 2A (PP2A) as OA. NMR structural studies showed that DTX-1 possessed an equatorial 35-methyl group but that DTX-2 had an axial 35-methyl group. Molecular modeling studies indicated that an axial 35-methyl could exhibit unfavorable interactions in the PP2A binding site, and this has been proposed as the reason for the reduced toxicity of DTX-2. Statistical analyses of published data indicate that the affinity of PP2A for DTX-1 is 1.6-fold higher, and for DTX-2 is 2-fold lower, than for OA. We obtained X-ray crystal structures of DTX-1 and DTX-2 bound to PP2A. The crystal structures independently confirm the C-35 stereochemistries determined in the earlier NMR study. The structure for the DTX-1 complex was virtually identical to that of the OA-PP2A complex, except for the presence of the equatorial 35-methyl on the ligand. The favorable placement of the equatorial 35-methyl group of DTX-1 against the aromatic {pi}-bonds of His191 may account for the increased affinity of PP2A toward DTX-1. In contrast, themore » axial 35-methyl of DTX-2 caused the side chain of His191 to rotate 140{sup o} so that it pointed toward the solvent, thereby opening one end of the hydrophobic binding cage. This rearrangement to accommodate the unfavorable interaction from the axial 35-methyl of DTX-2 reduces the binding energy and appears to be responsible for the reduced affinity of PP2A for DTX-2. These results highlight the potential of molecular modeling studies for understanding the relative toxicity of analogues once the binding site at the molecular target has been properly characterized.« less

The proper glycosylation of proteins trafficking through the Golgi apparatus depends upon the conserved oligomeric Golgi (COG) complex. Defects in COG can cause fatal congenital disorders of glycosylation (CDGs) in humans. The recent discovery of a form of CDG, caused in part by a COG4 missense mutation changing Arg 729 to Trp, prompted us to determine the 1.9 Å crystal structure of a Cog4 C-terminal fragment. Arg 729 is found to occupy a key position at the center of a salt bridge network, thereby stabilizing Cog4's small C-terminal domain. Studies in HeLa cells reveal that this C-terminal domain, while not needed for the incorporation of Cog4 into COG complexes, is essential for the proper glycosylation of cell surface proteins. We also find that Cog4 bears a strong structural resemblance to exocyst and Dsl1p complex subunits. These complexes and others have been proposed to function by mediating the initial tethering between transport vesicles and their membrane targets; the emerging structural similarities provide strong evidence of a common evolutionary origin and may reflect shared mechanisms of action.

Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate (PLP)-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this, we find cells release ≥100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anti-cholera treatment.

This study was designed to investigate whether brain unbound concentration (C u,brain ) is a better predictor of dopamine D 2 receptor occupancy than total brain concentration, cerebrospinal fluid concentration (C CSF ), or blood unbound concentration (C u,blood ). The ex vivo D 2 receptor occupancy and concentration-time profiles in cerebrospinal fluid, blood, and brain of six marketed antipsychotic drugs were determined after oral administration in rats at a range of dose levels. The C u,brain was estimated from the product of total brain concentration and unbound fraction, which was determined using a brain homogenate method. In conclusion, the C u,brain of selected antipsychotic agents is a good predictor of D 2 receptor occupancy in rats. Furthermore, C u,brain seems to provide a better prediction of D 2 receptor occupancy than C CSF or C u,blood for those compounds whose mechanism of entry into brain tissue is influenced by factors other than simple passive diffusion.

Bacteria can communicate via diffusible signal molecules they generate and release to coordinate their behavior in response to the environment. Signal molecule concentration is often proportional to bacterial population density, and when this reaches a critical concentration, reflecting a bacterial quorum, specific behaviors including virulence, symbiosis, and horizontal gene transfer are activated. Quorum-sensing regulation in many Gram-negative bacteria involves acylated homoserine lactone signals that are perceived through binding to LuxR-type, acylated-homoserine-lactone-responsive transcription factors. Bacteria of the rhizobial group employ the LuxR-type transcriptional activator TraR in quorum sensing, and its activity is further regulated through interactions with the TraM antiactivator. In this study, we have crystallographically determined the 3D structure of the TraR–TraM antiactivation complex from Rhizobium sp. strain NGR234. Unexpectedly, the antiactivator TraM binds to TraR at a site distinct from its DNA-binding motif and induces an allosteric conformational change in the protein, thereby preventing DNA binding. Structural analysis reveals a highly conserved TraR–TraM interface and suggests a mechanism for antiactivation complex formation. This structure may inform alternative strategies to control quorum-sensing-regulated microbial activity including amelioration of infectious disease and antibiotic resistance. In addition, the structural basis of antiactivation presents a regulatory interaction that provides general insights relevant to the field of transcription regulation and signal transduction.