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The structure of Streptococcus pneumoniae TrpA, which is the α subunit of tryptophan synthase, reveals 3D domain swapping, which has never before been observed for a TIM-barrel protein., Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and β, encoded by trpA and trpB genes, that function as an αββα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0–S1–H1–S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2′. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α–β interface of the tryptophan synthase αββα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the β subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.

The tryptophan synthases from three human pathogens show remarkable structural conservation, but at the same time display local differences in both their catalytic and allosteric sites that may be responsible for the observed differences in catalysis and inhibitor binding. This functional dissimilarity may be exploited in the design of species-specific enzyme inhibitors., Tryptophan biosynthesis is one of the most characterized processes in bacteria, in which the enzymes from Salmonella typhimurium and Escherichia coli serve as model systems. Tryptophan synthase (TrpAB) catalyzes the final two steps of tryptophan biosynthesis in plants, fungi and bacteria. This pyridoxal 5′-phosphate (PLP)-dependent enzyme consists of two protein chains, α (TrpA) and β (TrpB), functioning as a linear αββα heterotetrameric complex containing two TrpAB units. The reaction has a complicated, multistep mechanism resulting in the β-replacement of the hydroxyl group of l-serine with an indole moiety. Recent studies have shown that functional TrpAB is required for the survival of pathogenic bacteria in macrophages and for evading host defense. Therefore, TrpAB is a promising target for drug discovery, as its orthologs include enzymes from the important human pathogens Streptococcus pneumoniae, Legionella pneumophila and Francisella tularensis, the causative agents of pneumonia, legionnaires’ disease and tularemia, respectively. However, specific biochemical and structural properties of the TrpABs from these organisms have not been investigated. To fill the important phylogenetic gaps in the understanding of TrpABs and to uncover unique features of TrpAB orthologs to spearhead future drug-discovery efforts, the TrpABs from L. pneumophila, F. tularensis and S. pneumoniae have been characterized. In addition to kinetic properties and inhibitor-sensitivity data, structural information gathered using X-ray crystallo­graphy is presented. The enzymes show remarkable structural conservation, but at the same time display local differences in both their catalytic and allosteric sites that may be responsible for the observed differences in catalysis and inhibitor binding. This functional dissimilarity may be exploited in the design of species-specific enzyme inhibitors.

The Gram-negative cell envelope is a remarkable structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. Multiple molecular systems function to maintain integrity of this essential barrier between the interior of the cell and its surrounding environment. We show that a conserved DUF1849 family protein, EipB, is secreted to the periplasmic space of Brucella species, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual 14-stranded β-spiral structure that resembles the LolA and LolB lipoprotein delivery system, though the overall fold of EipB is distinct from LolA/LolB. Deletion of eipB results in defects in Brucella cell envelope integrity in vitro and in maintenance of spleen colonization in a mouse model of Brucella abortus infection. Transposon disruption of ttpA, which encodes a periplasmic protein containing tetratricopeptide repeats, is synthetically lethal with eipB deletion. ttpA is a reported virulence determinant in Brucella, and our studies of ttpA deletion and overexpression strains provide evidence that this gene also contributes to cell envelope function. We conclude that eipB and ttpA function in the Brucella periplasmic space to maintain cell envelope integrity, which facilitates survival in a mammalian host. IMPORTANCEBrucella species cause brucellosis, a global zoonosis. A gene encoding a conserved DUF1849-family protein, which we have named EipB, is present in all sequenced Brucella and several other genera in the class Alphaproteobacteria. The manuscript provides the first functional and structural characterization of a DUF1849 protein. We show that EipB is secreted to the periplasm where it forms a spiral-shaped antiparallel β protein that is a determinant of cell envelope integrity in vitro and virulence in an animal model of disease. eipB genetically interacts with ttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in select Alphaproteobacteria.

SummaryThe Gram-negative cell envelope is a remarkably diverse structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. We show that a conserved DUF1849-family protein, EipB, is secreted to the periplasmic space ofBrucella, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual fourteen-stranded β-spiral structure that contains a conserved disulfide bond. EipB has structural features that resemble the LolA and LolB lipoprotein delivery system, though the overall topology and fold of EipB is distinct from LolA/LolB. Deletion ofeipBresults in defects in both cell envelope integrityin vitroand in maintenance of spleen colonization in a mouse model ofB. abortusinfection. Transposon disruption ofttpA, which encodes a periplasmic tetratricopeptide repeat (TPR) protein, is synthetically lethal witheipBdeletion inB. abortus.ttpAis a known virulence determinant inB. melitensis, and our studies ofttpAdeletion and overexpression strains provide evidence thatttpA, likeeipB, contributes to cell envelope function inBrucella. We conclude thateipBandttpAfunction in theBrucellaperiplasmic space to maintain cell envelope integrity and to facilitate survival in a mammalian host.ImportanceBrucellaspecies cause brucellosis, a global zoonosis. A gene encoding a conserved uncharacterized protein, EipB, is present in all sequencedBrucellaand several other genera in the classAlphaproteobacteria.To our knowledge, this study presents the first functional and structural characterization of a protein from the DUF1849 family, to which EipB belongs. EipB is secreted to the periplasm where it forms a spiral-like anti-parallel β structure. Deletion ofBrucella eipBresults in defects of the cell envelope and in reduced virulence in an animal model of disease.eipBgenetically interacts withttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in selectAlphaproteobacteria.

Summary Hsp104 is an AAA+ protein disaggregase with powerful amyloid-remodeling activity. All nonmetazoan eukaryotes express Hsp104 while eubacteria express an Hsp104 ortholog, ClpB. However, most studies have focused on Hsp104 from Saccharomyces cerevisiae and ClpB orthologs from two eubacterial species. Thus, the natural spectrum of Hsp104/ClpB molecular architectures and protein-remodeling activities remains largely unexplored. Here, we report two structures of Hsp104 from the thermophilic fungus Calcarisporiella thermophila (CtHsp104), a 2.70A crystal structure and 4.0A cryo-electron microscopy structure. Both structures reveal left-handed, helical assemblies with all domains clearly resolved. We thus provide the highest resolution and most complete view of Hsp104 hexamers to date. We also establish that CtHsp104 antagonizes several toxic protein-misfolding events in vivo where S. cerevisiae Hsp104 is ineffective, including rescue of TDP-43, polyglutamine, and α-synuclein toxicity. We suggest that natural Hsp104 variation is an invaluable, untapped resource for illuminating therapeutic disaggregases for fatal neurodegenerative diseases.

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-β-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 Å resolution, respectively. SgcE6 shares a similar β-barrel fold with the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few other known FADH2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. The crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.

SummaryMolecular components of theBrucella abortuscell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function inB. abortusenvelope stress resistance and infection. Expression of this gene, which we nameeipA,is directly activated by the essential cell cycle regulator, CtrA.eipAencodes a soluble periplasmic protein that adopts an unusual eight-stranded β-barrel fold. Deletion ofeipAattenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the integrity of the cell envelope. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal witheipAdeletion. This genetic connection between O-polysaccharide andeipAis corroborated by our discovery thateipAis essential inBrucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion ofeipAexpression inB. ovisresults in a cell chaining phenotype, providing evidence thateipAdirectly or indirectly influences cell division inBrucella. We conclude that EipA is a molecular determinant ofBrucellavirulence that functions to maintain cell envelope integrity and influences cell division.

C-1027 is a chromoprotein enediyne antitumor antibiotic, consisting of the CagA apoprotein and the C-1027 chromophore. The C-1027 chromophore features a nine-membered enediyne core appended with three peripheral moieties, including an ( S)-3-chloro-5-hydroxy-β-tyrosine. In a convergent biosynthesis of the C-1027 chromophore, the ( S)-3-chloro-5-hydroxy-β-tyrosine moiety is appended to the enediyne core by the free-standing condensation enzyme SgcC5. Unlike canonical condensation domains from the modular nonribosomal peptide synthetases that catalyze amide-bond formation, SgcC5 catalyzes ester-bond formation, as demonstrated in vitro, between SgcC2-tethered ( S)-3-chloro-5-hydroxy-β-tyrosine and ( R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core as an acceptor substrate. Here, we report that (i) genes encoding SgcC5 homologues are widespread among both experimentally confirmed and bioinformatically predicted enediyne biosynthetic gene clusters, forming a new clade of condensation enzymes, (ii) SgcC5 shares a similar overall structure with the canonical condensation domains but forms a homodimer in solution, the active site of which is located in a cavity rather than a tunnel typically seen in condensation domains, and (iii) the catalytic histidine of SgcC5 activates the 2-hydroxyl group, while a hydrogen-bond network in SgcC5 prefers the R-enantiomer of the acceptor substrate, accounting for the regio- and stereospecific ester-bond formation between SgcC2-tethered ( S)-3-chloro-5-hydroxy-β-tyrosine and ( R)-1-phenyl-1,2-ethanediol upon acid-base catalysis. These findings expand the catalytic repertoire and reveal new insights into the structure and mechanism of condensation enzymes.

AT2433 from Actinomadura melliaura is an indolocarbazole antitumor antibiotic structurally distinguished by its unique aminodideoxypentose-containing disaccharide moiety. The corresponding sugar nucleotide-based biosynthetic pathway for this unusual sugar derives from comparative genomics where AtmS13 has been suggested as the contributing sugar aminotransferase (SAT). Determination of the AtmS13 X-ray structure at 1.50-A resolution reveals it as a member of the aspartate aminotransferase fold type I (AAT-I). Structural comparisons of AtmS13 with homologous SATs that act upon similar substrates implicate potential active site residues that contribute to distinctions in sugar C5 (hexose vs. pentose) and/or sugar C2 (deoxy vs. hydroxyl) substrate specificity.

Salmonella enterica serovar Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded β-barrel transmembrane domain and a C-terminal domain (OmpACTD ). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpACTD from two pathogens: S. typhimurium (STOmpACTD ) in open and closed forms and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpACTD ), in closed form. In the open form of STOmpACTD , an aspartate residue from a long β2-α3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD , a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpACTD , suggest a large conformational change that includes an extension of α3 helix by ordering a part of β2-α3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD suggesting PG-anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD , or possibly that of full length STOmpA.

Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs.

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe–S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1–Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70's ATPase activity. However, in apparent contradiction, we previously reported that an 8-fold decrease in Jac1's affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here, we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, we determined that a total of eight surface-exposed residues play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype, and a reduction in the activity of Fe–S cluster-containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensable role in the essential process of mitochondrial Fe–S cluster biogenesis.

The crystal structure of the uncharacterized protein SO2946 from Shewanella oneidensis MR-1 was determined with single-wavelength anomalous diffraction (SAD) and refined to 2.0 Å resolution. The SO2946 protein consists of a short helical N-terminal domain and a large C-terminal domain with the “jelly-roll” topology. The protein assembles into a propeller consisting of three C-terminal blades arranged around a central core formed by the N-terminal domains. The function of SO2946 could not be inferred from the sequence since the protein represents an orphan with no sequence homologs, but the protein’s structure bears a fold similar to that of proteins containing carbohydrate-binding modules. Features such as fold conservation, the presence of a conserved groove and a metal binding region are indicative that SO2946 may be an enzyme and could be involved in binding carbohydrate molecules.

The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at the high resolutions of 1.90 Å and 1.65 Å, respectively. Comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein-ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM-family of antitumor antibiotics.

Classical UDP-glucose 6-dehydrogenases (UGDH; EC 1.1.1.22) catalyze the conversion of UDP-α-D-glucose (UDP-Glc) to the key metabolic precursor UDP-alpha-D-glucuronic acid (UDP-GlcA) and display specificity for UDP-Glc. The fundamental biochemical and structural study of the UGDH homolog CalS8 encoded by the calicheamicin biosynthetic gene is reported and represents one of the first studies of a UGDH homolog involved in secondary metabolism. The corresponding biochemical characterization of CalS8 reveals CalS8 as one of the first characterized base permissive UGDH homologs with a >15-fold preference for TDP-Glc over UDP-Glc. The corresponding structure elucidation of apo-CalS8 and the CalS8/substrate/cofactor ternary complex (at 2.47 angstrom and 1.95 angstrom resolution, respectively) highlight a notably high degree of conservation between CalS8 and classical UGDHs where structural divergence within the intersubunit loop structure likely contributes to the CalS8 base permissivity. As such, this study begins to provide a putative blueprint for base specificity among sugar nucleotide-dependent dehydrogenases and, in conjunction with prior studies on the base specificity of the calicheamicin aminopentosyltransferase CalG4, provides growing support for the calicheamicin aminopentose pathway as a TDP-sugar-dependent process.

Bacterial catabolism of aromatic compounds from various sources including phenylpropanoids and flavonoids that are abundant in soil plays an important role in the recycling of carbon in the ecosystem. We have determined the crystal structures of apo-HcaR from Acinetobacter sp. ADP1, a MarR/SlyA transcription factor, in complexes with hydroxycinnamates and a specific DNA operator. The protein regulates the expression of the hca catabolic operon in Acinetobacter and related bacterial strains, allowing utilization of hydroxycinnamates as sole sources of carbon. HcaR binds multiple ligands, and as a result the transcription of genes encoding several catabolic enzymes is increased. The 1.9-2.4 A resolution structures presented here explain how HcaR recognizes four ligands (ferulate, 3,4-dihydroxybenzoate, p-coumarate, and vanillin) using the same binding site. The ligand promiscuity appears to be an adaptation to match a broad specificity of hydroxycinnamate catabolic enzymes while responding to toxic thioester intermediates. Structures of apo-HcaR and in complex with a specific DNA hca operator when combined with binding studies of hydroxycinnamates show how aromatic ligands render HcaR unproductive in recognizing a specific DNA target. The current study contributes to a better understanding of the hca catabolic operon regulation mechanism by the transcription factor HcaR.

AT2433 from Actinomadura melliaura is an indolocarbazole antitumor antibiotic structurally distinguished by its unique aminodideoxypentose-containing disaccharide moiety. The corresponding sugar nucleotide-based biosynthetic pathway for this unusual sugar derives from comparative genomics where AtmS13 has been suggested as the contributing sugar aminotransferase (SAT). Determination of the AtmS13 X-ray structure at 1.50 Å resolution reveals it as a member of the aspartate aminotransferase fold type I (AAT-I). Structural comparisons of AtmS13 with homologous SATs that act upon similar substrates implicate potential active site residues that contribute to distinctions in sugar C5 (hexose versus pentose) and/or sugar C2 (deoxy versus hydroxyl) substrate specificity.

Tryptophan synthase is an enzyme participating in the last two steps of tryptophan synthesis in plants, fungi and bacteria. It consists of two protein chains, α and β, encoded by trpA and trpB gene...

Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10°C compared to those grown at 37°C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses ( flaA and flp ), regulatory adaptive responses ( rpoN , lhkA , yycJ , bglG , adaB , and psr ), general microbial stress responses ( groEL , clpP , clpB , flp , and trxB ), amino acid metabolism ( hisJ , trpG , cysS , and aroA ), cell surface alterations ( fbp , psr , and flaA ), and degradative metabolism ( eutB , celD , and mleA ). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10°C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.

Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs.

The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins.

CalE6 from Micromonospora echinospora is a (pyridoxal 5′ phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. We report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structural analysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis and holoenzyme maturation.

In structural biology, the most critical issue is the availability of high-quality samples. “Structural-biology-grade” proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. Where the ultimate goal of structural biology is the same—to understand the structural basis of proteins in cellular processes, the structural genomics approach is different in that the functional aspects of individual protein or family are not ignored, however, emphasis here is on the number of unique structures, covering most of the protein folding space and developing new technologies with high efficiency. At the Mid-west Center Structural Genomics (MCSG), we have developed semiautomated protocols for high-throughput parallel protein purification. In brief, a protein, expressed as a fusion with a cleavable affinity tag, is purified in two immobilized metal affinity chromatography (IMAC) steps: (i) first IMAC coupled with buffer-exchange step, and after tag cleavage using TEV protease, (ii) second IMAC and buffer exchange to clean up cleaved tags and tagged TEV protease. Size exclusion chromatography is also applied as needed. These protocols have been implemented on multidimensional chromatography workstations AKTAexplorer and AKTAxpress (GE Healthcare). All methods and protocols used for purification, some developed in MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Disease (CSGID) purification pipeline, are discussed in this chapter.

In structural biology, the most critical issue is the availability of high-quality samples. "Structural-biology-grade" proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. Where the ultimate goal of structural biology is the same-to understand the structural basis of proteins in cellular processes, the structural genomics approach is different in that the functional aspects of individual protein or family are not ignored, however, emphasis here is on the number of unique structures, covering most of the protein folding space and developing new technologies with high efficiency. At the Midwest Center Structural Genomics (MCSG), we have developed semiautomated protocols for high-throughput parallel protein purification. In brief, a protein, expressed as a fusion with a cleavable affinity tag, is purified in two immobilized metal affinity chromatography (IMAC) steps: (i) first IMAC coupled with buffer-exchange step, and after tag cleavage using TEV protease, (ii) second IMAC and buffer exchange to clean up cleaved tags and tagged TEV protease. Size exclusion chromatography is also applied as needed. These protocols have been implemented on multidimensional chromatography workstations AKTAexplorer and AKTAxpress (GE Healthcare). All methods and protocols used for purification, some developed in MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Disease (CSGID) purification pipeline, are discussed in this chapter.

Precise tuning of gene expression by transcriptional regulators determines the response to internal and external chemical signals and adjusts the metabolic machinery for many cellular processes. As a part of ongoing efforts by the Midwest Center for Structural Genomics, a number of transcription factors were selected to study protein-ligand and protein-DNA interactions. HcaR, a new member of the MarR/SlyA family of transcription regulators from soil bacteria Acinetobacter sp. ADP1, is an evolutionarily atypical regulator and represses hydroxycinnamate (hca) catabolic genes. Hydroxycinnamates containing an aromatic ring play diverse, critical roles in plant architecture and defense. HcaR regulates the expression of the hca catabolic operon, allowing this and related bacterial strains to utilize hydroxycinnamates: ferulate, p-coumarate, and caffeate as sole sources of carbon and energy. HcaR appears to be capable of responding to multiple aromatic ligands. These aromatic compounds bind to HcaR and reduce its affinity to the specific DNA sites. As a result, the transcription of genes encoding several catabolic enzymes is up-regulated. The HcaR structures of the apo-form and in a complex with several ligands: ferulic acid, 3,4 dihydroxybenzoic acid, vanillin and p-coumaric acid have been determined to understand how HcaR accommodates various aromatic compounds using the same binding pocket. We also have identified a potential DNA site for HcaR in the regulatory region upstream of the genes of the hca catabolic operon in Acinetobacter sp. ADP1 and have confirmed DNA binding by EMSA. The co-crystal structure of HcaR and palindromic 24-mer DNA has been determined for this DNA site. The crystal structures of HcaR, the apo-form, ligand-bound forms, and the specific DNA-bound form provide critical structural basis of protein-ligand (substrates or product) and protein-DNA interactions to understand the regulation of the expression of hydroxycinnamate (hca) catabolic genes. Our studies allow for better understanding of DNA-binding and regulation by this important group of transcription factors belonging to the MarR/SlyA families. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

Large-scale evaluation of protein reductive methylation for improving protein crystallization