High-resolution structure of an engineered Cro monomer shows changes in conformation relative to the native dimer

A rationally designed, genetically engineered, monomeric form of the Cro protein from bacteriophage lambda has been crystallized and its structure determined by isomorphous replacement and refined to a resolution of 1.54 A. The structure confirms the rationale of the design but, at the same time, reveals 1-2 A shifts throughout the monomer structure relative to the previously determined structure of the dimeric wild-type protein. These changes include a 1.6 A main-chain shift in part of the beta-sheet region of the molecule relative to the alpha-helical region and a 1.1 A shift of a buried phenylalanine within the core as well as a correlated 2.2 A shift in a solvent-exposed beta-hairpin. The conformational adjustments appear to reflect an inherent flexibility of the protein that is associated with its DNA-binding function.     

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.     

Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence

The crystal structure of the type II restriction endonuclease BglI bound to DNA containing its specific recognition sequence has been determined at 2.2 A resolution. This is the first structure of a restriction endonuclease that recognizes and cleaves an interrupted DNA sequence, producing 3' overhanging ends. BglI is a homodimer that binds its specific DNA sequence with the minor groove facing the protein. Parts of the enzyme reach into both the major and minor grooves to contact the edges of the bases within the recognition half-sites. The arrangement of active site residues is strikingly similar to other restriction endonucleases, but the co-ordination of two calcium ions at the active site gives new insight into the catalytic mechanism. Surprisingly, the core of a BglI subunit displays a striking similarity to subunits of EcoRV and PvuII, but the dimer structure is dramatically different. The BglI-DNA complex demonstrates, for the first time, that a conserved subunit fold can dimerize in more than one way, resulting in different DNA cleavage patterns.     

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.     

The crystal structure of the hyperthermophile chromosomal protein Sso7d bound to DNA

BACKGROUND: Whether herd immunity will occur with widespread Haemophilus influenzae type b (Hib) vaccination in developing countries is dependent on whether the vaccines are capable of reducing carriage in these settings. However, few population-based studies of Hib carriage in developing countries exist. METHODS: To study Hib carriage in the Dominican Republic, we collected nasopharyngeal swab specimens from a population-based sample of 983 children 0 to 47 months old in a periurban area of Santo Domingo. RESULTS: Nasopharyngeal swabs of 76 (7.7%) children were positive for Hib. Hib carriage varied by age group with a low of 1.5% among 0 to 5 month olds, a peak of 12.5% in 6 to 11 month olds and prevalence rates of 6.0, 7.9 and 9.8% among 1-, 2- and 3-year-olds, respectively. Hib carriage was 51% lower among currently breast-fed 6 to 11 month olds than among those not currently breast-fed (18.2% vs. 9.0%; P=0.08). CONCLUSIONS: Infants and young children in Santo Domingo have high rates of Hib carriage, characterized by an early peak in carriage that corresponds with the peak of risk for Hib meningitis. The ability of Hib vaccines to diminish carriage to levels that will effectively reduce transmission and lead to herd immunity in this setting needs to be determined.     

Three-dimensional structure of AmpC beta-lactamase from Escherichia coli bound to a transition-state analogue: possible implications for the oxyanion hypothesis and for inhibitor design

The structures of AmpC beta-lactamase from Escherichia coli, alone and in complex with a transition-state analogue, have been determined by X-ray crystallography. The native enzyme was determined to 2.0 A resolution, and the structure with the transition-state analogue m-aminophenylboronic acid was determined to 2.3 A resolution. The structure of AmpC from E. coli resembles those previously determined for the class C enzymes from Enterobacter cloacae and Citrobacter freundii. The transition-state analogue, m-aminophenylboronic acid, makes several interactions with AmpC that were unexpected. Perhaps most surprisingly, the putative "oxyanion" of the boronic acid forms what appears to be a hydrogen bond with the backbone carbonyl oxygen of Ala318, suggesting that this atom is protonated. Although this interaction has not previously been discussed, a carbonyl oxygen contact with the putative oxyanion or ligand carbonyl oxygen appears in most complexes involving a beta-lactam recognizing enzyme. These observations may suggest that the high-energy intermediate for amide hydrolysis by beta-lactamases and related enzymes involves a hydroxyl and not an oxyanion, although the oxyanion form certainly cannot be discounted. The involvement of the main-chain carbonyl in ligand and transition-state recognition is a distinguishing feature between serine beta-lactamases and serine proteases, to which they are often compared. AmpC may use the interaction between the carbonyl of Ala318 and the carbonyl of the acylated enzyme to destabilize the ground-state intermediate, this destabilization energy might be relieved in the transition state by a hydroxyl hydrogen bond. The structure of the m-aminophenylboronic acid adduct also suggests several ways to improve the affinity of this class of inhibitor and points to the existence of several unusual binding-site-like features in the region of the AmpC catalytic site.     

Crystal structure of the DpnM DNA adenine methyltransferase from the DpnII restriction system of streptococcus pneumoniae bound to S-adenosylmethionine

BACKGROUND:. Methyltransferases (Mtases) catalyze the transfer of methyl groups from S-adenosylmethionine (AdoMet) to a variety of small molecular and macromolecular substrates. These enzymes contain a characteristic alpha/beta structural fold. Four groups of DNA Mtases have been defined and representative structures have been determined for three groups. DpnM is a DNA Mtase that acts on adenine N6 in the sequence GATC; the enzyme represents group alpha DNA Mtases, for which no structures are known. RESULTS:. The structure of DpnM in complex with AdoMet was determined at 1.80 A resolution. The protein comprises a consensus Mtase fold with a helical cluster insert. DpnM binds AdoMet in a similar manner to most other Mtases and the enzyme contains a hollow that can accommodate DNA. The helical cluster supports a shelf within the hollow that may recognize the target sequence. Modeling studies indicate a potential site for binding the target adenine, everted from the DNA helix. Comparison of the DpnM structure and sequences of group alpha DNA Mtases indicates that the group is a genetically related family. Structural comparisons show DpnM to be most similar to a small-molecule Mtase and then to macromolecular Mtases, although several dehydrogenases show greater similarity than one DNA Mtase. CONCLUSIONS:. DpnM, and by extension the DpnM family or group alpha Mtases, contains the consensus fold and AdoMet-binding motifs found in most Mtases. Structural considerations suggest that macromolecular Mtases evolved from small-molecule Mtases, with different groups of DNA Mtases evolving independently. Mtases may have evolved from dehydrogenases. Comparison of these enzymes indicates that in protein evolution, the structural fold is most highly conserved, then function and lastly sequence.     

Crystal structure and possible dimerization of the high-potential iron-sulfur protein from Chromatium purpuratum

The crystal structure of the high-potential iron-sulfur protein (HiPIP) isolated from Chromatium purpuratum is reported at 2.7 A resolution. The three HiPIP molecules in the asymmetric unit of the crystals form one and one-half dimers. Two molecules are related by a noncrystallographic symmetry rotation of approximately 175 degrees with negligible translation along the dyad axis. The third molecule in the asymmetric unit also forms a dimer with a second HiPIP molecule across the crystallographic 2-fold symmetry axis. The Fe4S4 clusters in both the crystallographic and noncrystallographic dimers are separated by approximately 13.0 A. Solution studies give mixed results regarding the oligomeric state of the C. purpuratum HiPIP. A comparison with crystal structures of HiPIPs from other species shows that HiPIP tends to associate rather nonspecifically about a conserved, relatively hydrophobic surface patch to form dimers.     

Solution structure of the B-Myb DNA-binding domain: a possible role for conformational instability of the protein in DNA binding and control of gene expression

Double- and triple-resonance heteronuclear NMR spectroscopy have been used to determine the high-resolution solution structure of the minimal B-Myb DNA-binding domain (B-MybR2R3) and to characterize the specific complex formed with a synthetic DNA fragment corresponding to the Myb target site on the Myb-regulated gene tom-1. B-MybR2R3 is shown to consist of two independent protein domains (R2 and R3) joined by a short linker, which have strikingly different tertiary structures despite significant sequence similarities. In addition, the C-terminal region of B-Myb R2 is confirmed to have a poorly defined structure, reflecting the existence of multiple conformations in slow to intermediate exchange. This contrasts with the tertiary structure reported for c-MybR2R3, in which both R2 and R3 have the same fold and the C-terminal region of R2 forms a stable, well-defined helix [Ogata, K., et al. (1995) Nat. Struct. Biol. 2, 309-320]. The NMR data suggest there are extensive contacts between B-MybR2R3 and its DNA target site in the complex and are consistent with a significant conformational change in the protein on binding to DNA, with one possibility being the formation of a stable helix in the C-terminal region of R2. In addition, conformational heterogeneity identified in R2 of B-MybR2R3 bound to the tom-1-A target site may play an important role in the control of gene expression by Myb proteins.     

Solution structure of human intestinal fatty acid binding protein: implications for ligand entry and exit

The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy. Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra (NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each, and two short alpha-helices that connect the beta-strands A and B. The interior of the protein consists of a water-filled cavity between the two beta-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP. The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.     

Crystal structure of beta-D-cellotetraose hemihydrate with implications for the structure of cellulose II

The crystal structure of beta-D-cellotetraose shows the same molecule packing as cellulose II, with two antiparallel molecules in the unit cell: For cellulose II, the orientation of the C6-O6 bonds has been described as gauche-trans and trans-gauche, respectively, for the two antiparallel molecules, which otherwise have identical conformations. In contrast, in beta-D-cellotetraose all C6-O6 bonds are gauche-trans, but the conformations of the two antiparallel molecules are different. Energy minimization and molecular dynamics studies suggest that the structure of cellulose II should be reinvestigated in light of these findings.     

Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent

The Hsp90 chaperone is required for the activation of several families of eukaryotic protein kinases and nuclear hormone receptors, many of which are protooncogenic and play a prominent role in cancer. The geldanamycin antibiotic has antiproliferative and antitumor effects, as it binds to Hsp90, inhibits the Hsp90-mediated conformational maturation/refolding reaction, and results in the degradation of Hsp90 substrates. The structure of the geldanamycin-binding domain of Hsp90 (residues 9-232) reveals a pronounced pocket, 15 A deep, that is highly conserved across species. Geldanamycin binds inside this pocket, adopting a compact structure similar to that of a polypeptide chain in a turn conformation. This, and the pocket's similarity to substrate-binding sites, suggest that the pocket binds a portion of the polypeptide substrate and participates in the conformational maturation/refolding reaction.     

RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations

Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.     

Protein footprinting approach to mapping DNA binding sites of two archaeal homing enzymes: evidence for a two-domain protein structure

The archaeal intron-encoded homing enzymes I-PorI and I-DmoI belong to a family of endonucleases that contain two copies of a characteristic LAGLIDADG motif. These endonucleases cleave their intron- or intein- alleles site-specifically, and thereby facilitate homing of the introns or inteins which encode them. The protein structure and the mechanism of DNA recognition of these homing enzymes is largely unknown. Therefore, we examined these properties of I-PorI and I-DmoI by protein footprinting. Both proteins were susceptible to proteolytic cleavage within regions that are equidistant from each of the two LAGLIDADG motifs. When complexed with their DNA substrates, a characteristic subset of the exposed sites, located in regions immediately after and 40-60 amino acids after each of the LAGLIDADG motifs, were protected. Our data suggest that the enzymes are structured into two, tandemly repeated, domains, each containing both the LAGLIDADG motif and two putative DNA binding regions. The latter contains a potentially novel DNA binding motif conserved in archaeal homing enzymes. The results are consistent with a model where the LAGLIDADG endonucleases bind to their non-palindromic substrates as monomeric enzymes, with each of the two domains recognizing one half of the DNA substrate.     

Crystal structure of heat-labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit

Cholera toxin (CT) produced by Vibrio cholerae and heat-labile enterotoxin (LT-I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP-ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti-diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT-I are known to be relatively temperature sensitive. To improve the thermostability of LT-I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT-I has been determined to 2.0 A resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT-I by 6 degrees C. The enhancement in thermostability could make this disulfide bond variant of LT-I of considerable interest for the design of enterotoxin-based vaccines.