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1.  Protein Production for Structural Genomics Using E. coli Expression 
The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modified protocol that allows for the production of selenium-labeled proteins in defined media is also offered. Finally, a method for the purification of His6-tagged proteins on immobilized metal affinity chromatography columns that generates high-purity material is described in detail.
doi:10.1007/978-1-4939-0354-2_7
PMCID: PMC4108990  PMID: 24590711
Protein expression; Protein purification; Disposable vessel fermentation; Selenomethionine-labeling; IMAC; His-tag; High-throughput
2.  Salvage of Failed Protein Targets by Reductive Alkylation 
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.
doi:10.1007/978-1-4939-0354-2_15
PMCID: PMC4078742  PMID: 24590719
Chemical modification; Lysine reductive alkylation; Methylation; Ethylation; Isopropylation; Protein crystallization
3.  Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase 
Journal of medicinal chemistry  2013;56(10):4028-4043.
Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition and gastroenteritis as well as posing a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5′-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and > 500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD+. The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An x-ray crystal structure of a representative E•IMP•inhibitor complex is also presented. Overall, the secondary amine derivative 15a (Q67) demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines.
doi:10.1021/jm400241j
PMCID: PMC3756936  PMID: 23668331
4.  Structure and Dimerization Properties of the Aryl Hydrocarbon Receptor PAS-A Domain 
Molecular and Cellular Biology  2013;33(21):4346-4356.
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that binds to xenobiotics and responds by regulating the expression of gene programs required for detoxification and metabolism. AHR and its heterodimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT) belong to the basic helix-loop-helix (bHLH)–PER-ARNT-SIM (PAS) family of transcription factors. Here we report the 2.55-Å-resolution crystal structure of the mouse AHR PAS-A domain, which represents the first AHR-derived protein structure. The AHR PAS-A domain forms a helix-swapped homodimer in the crystal and also in solution. Through a detailed mutational analysis of all interface residues, we identified several hydrophobic residues that are important for AHR dimerization and function. Our crystallographic visualization of AHR PAS-A dimerization leads us to propose a mode of heterodimerization with ARNT that is supported by both biochemical and cell-based data. Our studies also highlight the residues of other mammalian bHLH-PAS proteins that are likely involved in their homo- or heterodimerization.
doi:10.1128/MCB.00698-13
PMCID: PMC3811902  PMID: 24001774
5.  Bacillus anthracis Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion-, Substrate-, and Product-Bound Complexes 
Biochemistry  2012;51(31):10.1021/bi300511w.
Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed “apo”) form and in complex with its substrate, inosine 5′-monophosphate (IMP), and product, xanthosine 5′-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examined for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.
doi:10.1021/bi300511w
PMCID: PMC3836674  PMID: 22788966
6.  Multi-Domain Integration in the Structure of the HNF4α Nuclear Receptor Complex 
Nature  2013;495(7441):394-398.
The hepatocyte nuclear factor 4 alpha (HNF4α, NR2A1) is a member of the nuclear receptor (NR) family of transcription factors that use conserved DNA binding domains (DBDs) and ligand binding domains (LBDs)1,2. HNF4α is the most abundant DNA-binding protein in the liver, where some 40% of the actively transcribed genes have a HNF4α response element 1,3,4. These regulated genes are largely involved in the hepatic gluconeogenic program and lipid metabolism3,5,6. In the pancreas too, HNF4α is a master regulator controlling an estimated 11% of islet genes7. HNF4α protein mutations are linked to Maturity Onset of Diabetes in Young 1 (MODY1) and hyperinsulinemic hypoglycemia (HH)8–11. Prior structural analyses of NRs, while productive with individual domains, have lagged in revealing the connectivity patterns of NR domains. Here, we describe the 2.9 Å crystal structure of the multi-domain HNF4α homodimer bound to its DNA response element and coactivator-derived peptides. A convergence zone connects multiple receptor domains in an asymmetric fashion joining distinct elements from each monomer. An arginine target of PRMT1 methylation protrudes directly into this convergence zone and sustains its integrity. A serine target of protein kinase C is also responsible for maintaining domain-domain interactions. These post-translational modifications manifest into changes in DNA binding by communicating through the tightly connected surfaces of the quaternary fold. We find that some MODY1 mutations, positioned on the LBD and hinge regions of the receptor, compromise DNA binding at a distance by communicating through the inter-junctional surfaces of the complex. The overall domain representation of the HNF4α homodimer is different from that of the PPARγ-RXRα heterodimer, even when both NR complexes are assembled on the same DNA element. Our findings suggest that unique quaternary folds and inter-domain connections in NRs could be exploited by small-molecule allosteric modulators that impact distal functions in these polypeptides.
doi:10.1038/nature11966
PMCID: PMC3606643  PMID: 23485969
7.  High-throughput Protein Purification and Quality Assessment for Crystallization 
Methods (San Diego, Calif.)  2011;55(1):12-28.
The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. 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. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; [1] the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are discussed in this chapter.
doi:10.1016/j.ymeth.2011.07.010
PMCID: PMC3690762  PMID: 21907284
domain design; expression vectors; gene cloning; protein purification; crystallization screening; quality assessment
8.  A Structural Basis for IκB Kinase 2 Activation Via Oligomerization-Dependent Trans Auto-Phosphorylation 
PLoS Biology  2013;11(6):e1001581.
Conformational change in human IKK2 permits dimers to form higher-order oligomers that support interaction between kinase domains and promote activation through trans auto-phosphorylation.
Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.
Author Summary
IκB kinase (IKK) is an enzyme that quickly becomes active in response to diverse stresses on a cell. Once activated, IKK promotes an array of cellular defense processes by phosphorylating IκB, thereby promoting its degradation and liberating its partner, the pro-survival transcription factor NF-κB; NF-κB is then free to relocate to the nucleus where it can modulate gene expression. Our X-ray crystallographic studies on an active version of the human IKK2 isoform reveal that the enzyme adopts a unique open conformation that permits pairs of IKK2 enzymes to form higher order assemblies in which their catalytic domains are in close proximity. Disruption of IKK2's ability to form these assemblies, by introducing changes that interfere with the surfaces that mediate oligomerization, results in IKK2 enzymes that are greatly impaired in their ability to become activated in cells. We propose that after oligomerization the neighboring catalytic domains then phosphorylate each other as part of the activation process. Our findings also suggest that targeted small molecules might disrupt cell survival by blocking IKK2 assembly in cells.
doi:10.1371/journal.pbio.1001581
PMCID: PMC3678999  PMID: 23776406
9.  Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria 
The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS.
Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3′,5′-adenosine diphosphate (3′,5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′,5′-ADP is bound as the 3′,5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′,5′-ADP–AcpS binary complexes. The position of the second 3′,5′-ADP has never been described before. It is in close proximity to the first 3′,5′-­ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.
doi:10.1107/S0907444912029101
PMCID: PMC3447402  PMID: 22993090
acyl-carrier-protein synthase; acyl carrier protein; type II fatty-acid synthesis; inhibition; 3′,5′-adenosine diphosphate; coenzyme A
10.  HIGH-THROUGHPUT PROTEIN PURIFICATION FOR X-RAY CRYSTALLOGRAPHY AND NMR 
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.
doi:10.1016/S0065-3233(07)75003-9
PMCID: PMC3366499  PMID: 20731990
11.  High-Resolution Structure of the Nitrile Reductase QueF Combined with Molecular Simulations Provide Insight into Enzyme Mechanism 
Journal of Molecular Biology  2010;404(1):127-137.
Here, we report the 1.53-Å crystal structure of the enzyme 7-cyano-7-deazaguanine reductase (QueF) from Vibrio cholerae, which is responsible for the complete reduction of a nitrile (C≡N) bond to a primary amine (H2C–NH2). At present, this is the only example of a biological pathway that includes reduction of a nitrile bond, establishing QueF as particularly noteworthy. The structure of the QueF monomer resembles two connected ferrodoxin-like domains that assemble into dimers. Ligands identified in the crystal structure suggest the likely binding conformation of the native substrates NADPH and 7-cyano-7-deazaguanine. We also report on a series of numerical simulations that have shed light on the mechanism by which this enzyme affects the transfer of four protons (and electrons) to the 7-cyano-7-deazaguanine substrate. In particular, the simulations suggest that the initial step of the catalytic process is the formation of a covalent adduct with the residue Cys194, in agreement with previous studies. The crystal structure also suggests that two conserved residues (His233 and Asp102) play an important role in the delivery of a fourth proton to the substrate.
doi:10.1016/j.jmb.2010.09.042
PMCID: PMC3366508  PMID: 20875425
queuosine; oxidoreductase; QueF; nitrile reduction
12.  Structure of Apo- and Monometalated Forms of NDM-1—A Highly Potent Carbapenem-Hydrolyzing Metallo-β-Lactamase 
PLoS ONE  2011;6(9):e24621.
The New Delhi Metallo-β-lactamase (NDM-1) gene makes multiple pathogenic microorganisms resistant to all known β-lactam antibiotics. The rapid emergence of NDM-1 has been linked to mobile plasmids that move between different strains resulting in world-wide dissemination. Biochemical studies revealed that NDM-1 is capable of efficiently hydrolyzing a wide range of β-lactams, including many carbapenems considered as “last resort” antibiotics. The crystal structures of metal-free apo- and monozinc forms of NDM-1 presented here revealed an enlarged and flexible active site of class B1 metallo-β-lactamase. This site is capable of accommodating many β-lactam substrates by having many of the catalytic residues on flexible loops, which explains the observed extended spectrum activity of this zinc dependent β-lactamase. Indeed, five loops contribute “keg” residues in the active site including side chains involved in metal binding. Loop 1 in particular, shows conformational flexibility, apparently related to the acceptance and positioning of substrates for cleavage by a zinc-activated water molecule.
doi:10.1371/journal.pone.0024621
PMCID: PMC3169612  PMID: 21931780
13.  Cleavable C-terminal His-tag vectors for structure determination 
High-throughput structural genomics projects seek to delineate protein structure space by determining the structure of representatives of all major protein families. Generally this is accomplished by processing numerous proteins through standardized protocols, for the most part involving purification of N-terminally His-tagged proteins. Often proteins that fail this approach are abandoned, but in many cases further effort is warranted because of a protein’s intrinsic value. In addition, failure often occurs relatively far into the path to structure determination, and many failed proteins passed the first critical step, expression as a soluble protein. Salvage pathways seek to recoup the investment in this subset of failed proteins through alternative cloning, nested truncations, chemical modification, mutagenesis, screening buffers, ligands and modifying processing steps. To this end we have developed a series of ligation-independent cloning expression vectors that append various cleavable C-terminal tags instead of the conventional N-terminal tags. In an initial set of 16 proteins that failed with an N-terminal appendage, structures were obtained for C-terminally tagged derivatives of five proteins, including an example for which several alternative salvaging steps had failed. The new vectors allow appending C-terminal His6-tag and His6- and MBP-tags, and are cleavable with TEV or with both TEV and TVMV proteases.
doi:10.1007/s10969-010-9082-y
PMCID: PMC2885959  PMID: 20213425
LIC; TEV; C-terminal; His-tag; High-throughput; Structural genomics
14.  The Structural Basis for Substrate Anchoring, Active Site Selectivity, and Product Formation by P450 PikC from Streptomyces venezuelae 
The Journal of biological chemistry  2006;281(36):26289-26297.
The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae represents a valuable system for dissecting the fundamental mechanisms of modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and hydroxylation leading to the production of a series of macrolide antibiotics, including the natural ketolides narbomycin and pikromycin. In this study, we describe four x-ray crystal structures and allied functional studies for PikC, the remarkable P450 monooxygenase responsible for production of a number of related macrolide products from the Pik pathway. The results provide important new insights into the structural basis for the C10/C12, and C12/C14 hydroxylation patterns for the 12- (YC-17) and 14-membered ring (narbomycin) macrolides, respectively. This includes two different ligand-free structures in an asymmetric unit (resolution 2.1 Å) and two co-crystal structures with bound endogenous substrates YC-17 (resolution 2.35 Å) or narbomycin (resolution 1.7 Å). A central feature of the enzyme-substrate interaction involves anchoring of the desosamine residue in two alternative binding pockets based on a series of distinct amino acid residues that form a salt bridge and a hydrogen bonding network with the deoxysugar C3′ dimethylamino group. Functional significance of the salt bridge was corroborated by site-directed mutagenesis that revealed a key role for E94 in YC-17 binding, and E85 for narbomycin binding. Taken together, the x-ray structure analysis, site-directed mutagenesis and corresponding product distribution studies reveal that PikC substrate tolerance, and product diversity result from a combination of alternative anchoring modes, rather than an induced fit mechanism.
doi:10.1074/jbc.M605478200
PMCID: PMC2939096  PMID: 16825192
15.  Structure- and Function-based Characterization of a New Phosphoglycolate Phosphatase from Thermoplasma acidophilum* 
The Journal of biological chemistry  2003;279(1):517-526.
The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel β-sheet with strand order S10, S9, S8, S1, S2 and a small β-hairpin, strands S3 and S4. This central sheet is flanked by a set of three α-helices on one side and two helices on the other. The smaller domain is composed of an open faced β-sandwich represented by three antiparallel β-strands, S5, S6, and S7, flanked by two oppositely oriented α-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl− ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase.
doi:10.1074/jbc.M306054200
PMCID: PMC2795321  PMID: 14555659
17.  The Crystal Structure of Aq_328 from the Hyperthermophilic Bacteria Aquifex aeolicus Shows an Ancestral Histone Fold 
Proteins  2006;62(1):8-16.
The structure of Aq_328, an uncharacterized protein from hyperthermophilic bacteria Aquifex aeolicus, has been determined to 1.9 Å by using multi-wavelength anomalous diffraction (MAD) phasing. Although the amino acid sequence analysis shows that Aq_328 has no significant similarity to proteins with a known structure and function, the structure comparison by using the Dali server reveals that it: (1) assumes a histone-like fold, and (2) is similar to an ancestral nuclear histone protein (PDB code 1F1E) with z-score 8.1 and RMSD 3.6 Å over 124 residues. A sedimentation equilibrium experiment indicates that Aq_328 is a monomer in solution, with an average sedimentation coefficient of 2.4 and an apparent molecular weight of about 20 kDa. The overall architecture of Aq_328 consists of two noncanonical histone domains in tandem repeat within a single chain, and is similar to eukaryotic heterodimer (H2A/H2B and H3/H4) and an archaeal histone heterodimer (HMfA/HMfB). The sequence comparisons between the two histone domains of Aq_328 and six eukaryotic/archaeal histones demonstrate that most of the conserved residues that underlie the Aq_328 architecture are used to build and stabilize the two cross-shaped antiparallel histone domains. The high percentage of salt bridges in the structure could be a factor in the protein’s thermostability. The structural similarities to other histone-like proteins, molecular properties, and potential function of Aq_328 are discussed in this paper.
doi:10.1002/prot.20590
PMCID: PMC2792020  PMID: 16287087
structural genomics; MAD phasing; synchrotron radiation; histone fold; thermostability
18.  Novel hexamerization motif is discovered in a conserved cytoplasmic protein from Salmonella typhimurium 
The cytoplasmic protein Stm3548 of unknown function obtained from a strain of Salmonella typhimurium was determined by X-ray crystallography at a resolution of 2.25 Å. The asymmetric unit contains a hexamer of structurally identical monomers. The monomer is a globular domain with a long β-hairpin protrusion that distinguishes this structure. This β-hairpin occupies a central position in the hexamer, and its residues participate in the majority of interactions between subunits of the hexamer. We suggest that the structure of Stm3548 presents a new hexamerization motif. Because the residues participating in interdomain interactions are highly conserved among close members of protein family DUF1355 and buried solvent accessible area for the hexamer is significant, the hexamer is most likely conserved as well. A light scattering experiment confirmed the presence of hexamer in solution.
doi:10.1007/s10969-007-9028-1
PMCID: PMC2792014  PMID: 17968677
β-hairpin; Protein family DUF1355; Salmonella typhimurium
20.  Crystal Structure of Thermotoga maritima 0065, a Member of the IclR Transcriptional Factor Family* 
The Journal of biological chemistry  2002;277(21):19183-19190.
Members of the IclR family of transcription regulators modulate signal-dependent expression of genes involved in carbon metabolism in bacteria and archaea. The Thermotoga maritima TM0065 gene codes for a protein (TM-IclR) that is homologous to the IclR family. We have determined the crystal structure of TM-IclR at 2.2 Å resolution using MAD phasing and synchrotron radiation. The protein is composed of two domains: the N-terminal DNA-binding domain contains the winged helix-turn-helix motif, and the C-terminal presumed regulatory domain is involved in binding signal molecule. In a proposed signal-binding site, a bound Zn2+ ion was found. In the crystal, TM-IclR forms a dimer through interactions between DNA-binding domains. In the dimer, the DNA-binding domains are 2-fold related, but the dimer is asymmetric with respect to the orientation of signal-binding domains. Crystal packing analysis showed that TM-IclR dimers form a tetramer through interactions exclusively by signal-binding domains. A model is proposed for binding of IclR-like factors to DNA, and it suggests that signal-dependent transcription regulation is accomplished by affecting an oligomerization state of IclR and therefore its affinity for DNA target.
doi:10.1074/jbc.M112171200
PMCID: PMC2792004  PMID: 11877432
21.  Automation of protein purification for structural genomics 
A critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. ‘Structural-biology-grade’ proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.
doi:10.1023/B:JSFG.0000029206.07778.fc
PMCID: PMC2778303  PMID: 15263850
affinity chromatography; automation; protein purification; structural genomics
22.  Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †  
Molecular and Cellular Biology  2008;29(5):1306-1320.
Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.
doi:10.1128/MCB.01271-08
PMCID: PMC2643833  PMID: 19103740
23.  Functional and Structural Characterization of Four Glutaminases from Escherichia coli and Bacillus subtilis† 
Biochemistry  2008;47(21):5724-5735.
Glutaminases belong to the large superfamily of serine-dependent β-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of l-glutamine to l-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to l-glutamine and did not hydrolyze d-glutamine or l-asparagine. In each organism, one glutaminase showed higher affinity to glutamine (E. coli YbaS and B. subtilis YlaM; Km 7.3 and 7.6 mM, respectively) than the second glutaminase (E. coli YneH and B. subtilis YbgJ; Km 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical β-lactamase-like fold and conservation of several key catalytic residues of β-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme–glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.
doi:10.1021/bi800097h
PMCID: PMC2735108  PMID: 18459799
25.  Small-Molecule Scaffolds for CYP51 Inhibitors Identified by High-Throughput Screening and Defined by X-Ray Crystallography▿  
Antimicrobial Agents and Chemotherapy  2007;51(11):3915-3923.
Sterol 14α-demethylase (CYP51), a major checkpoint in membrane sterol biosynthesis, is a key target for fungal antibiotic therapy. We sought small organic molecules for lead candidate CYP51 inhibitors. The changes in CYP51 spectral properties following ligand binding make CYP51 a convenient target for high-throughput screening technologies. These changes are characteristic of either substrate binding (type I) or inhibitor binding (type II) in the active site. We screened a library of 20,000 organic molecules against Mycobacterium tuberculosis CYP51 (CYP51Mt), examined the top type I and type II binding hits for their inhibitory effects on M. tuberculosis in broth culture, and analyzed them spectrally for their ability to discriminate between CYP51Mt and two reference M. tuberculosis CYP proteins, CYP130 and CYP125. We determined the binding mode for one of the top type II hits, α-ethyl-N-4-pyridinyl-benzeneacetamide (EPBA), by solving the X-ray structure of the CYP51Mt-EPBA complex to a resolution of 1.53 Å. EPBA binds coordinately to the heme iron in the CYP51Mt active site through a lone pair of nitrogen electrons and also through hydrogen bonds with residues H259 and Y76, which are invariable in the CYP51 family, and hydrophobic interactions in a phylum- and/or substrate-specific cavity of CYP51. We also identified a second compound with structural and binding properties similar to those of EPBA, 2-(benzo[d]-2,1,3-thiadiazole-4-sulfonyl)-2-amino-2-phenyl-N-(pyridinyl-4)-acetamide (BSPPA). The congruence between the geometries of EPBA and BSPPA and the CYP51 binding site singles out EPBA and BSPPA as lead candidate CYP51 inhibitors with optimization potential for efficient discrimination between host and pathogen enzymes.
doi:10.1128/AAC.00311-07
PMCID: PMC2151439  PMID: 17846131

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