Discovery of a pathway for terminal-alkyne amino acid biosynthesis.

Living systems can generate an enormous range of cellular functions, from mechanical infrastructure and signalling networks to enzymatic catalysis and information storage, using a notably limited set of chemical functional groups. This observation is especially notable when compared to the breadth of functional groups used as the basis for similar functions in synthetically derived small molecules and materials. The relatively small cross-section between biological and synthetic reactivity space forms the foundation for the development of bioorthogonal chemistry, in which the absence of a pair of reactive functional groups within the cell allows for a selective in situ reaction1-4. However, biologically 'rare' functional groups, such as the fluoro5, chloro6,7, bromo7,8, phosphonate9, enediyne10,11, cyano12, diazo13, alkene14 and alkyne15-17 groups, continue to be discovered in natural products made by plants, fungi and microorganisms, which offers a potential route to genetically encode the endogenous biosynthesis of bioorthogonal reagents within living organisms. In particular, the terminal alkyne has found broad utility via the Cu(I)-catalysed azide-alkyne cycloaddition 'click' reaction18. Here we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid in the bacterium Streptomyces cattleya. We found that L-lysine undergoes an unexpected reaction sequence that includes halogenation, oxidative C-C bond cleavage and triple bond formation through a putative allene intermediate. This pathway offers the potential for de novo cellular production of halo-, alkene- and alkyne-labelled proteins and natural products from glucose for a variety of downstream applications.

Phosphoaspartates in bacterial signal transduction.

Bacteria use a strategy referred to as two-component signal transduction to sense a variety of stimuli and initiate an appropriate response. Signal processing begins with proteins referred to as histidine kinases. In most cases, these are membrane-bound receptors that respond to environmental cues. Histidine kinases use ATP as a phosphodonor to phosphorylate a conserved histidine residue. Subsequent transfer of the phosphoryl group to a conserved aspartyl residue in the cognate response regulator results in an appropriate output. Recent structural studies of activated (phosphorylated) response regulators and their aspartate-bearing regulatory domains have provided insight into the links between the chemistry and biology of these ubiquitous regulatory proteins. Chemical aspects of their function appear to generalize broadly to enzymes that adopt a phosphoaspartate intermediate.

A novel engineered subtilisin BPN' lacking a low-barrier hydrogen bond in the catalytic triad.

The low-barrier hydrogen bond (LBHB) between the Asp and His residues of the catalytic triad in a serine protease was perturbed via the D32C mutation in subtilisin BPN' (Bacillus protease N'). This mutant enzyme catalyzes the hydrolysis of N-Suc-Ala-Ala-Pro-Phe-SBzl with a k(cat)/K(m) value that is only 8-fold reduced from that of the wild-type (WT) enzyme. The value of k(cat)/K(m) for the corresponding p-nitroanilide (pNA) substrate is only 50-fold lower than that of the WT enzyme (DeltaDeltaG++ = 2.2 kcal/mol). The pK(a) controlling the ascending limb of the pH versus k(cat)/K(m) profile is lowered from 7.01 (WT) to 6.53 (D32C), implying that any hydrogen bond replacing that between Asp32 and His64 of the WT enzyme most likely involves the neutral thiol rather than the thiolate form of Cys32. It is shown by viscosity variation that the reaction of WT subtilisin with N-Suc-Ala-Ala-Pro-Phe-SBzl is 50% (sucrose) to 100% (glycerol) diffusion-controlled, while that of the D32C construct is 29% (sucrose) to 76% (glycerol) diffusion-controlled. The low-field NMR resonance of 18 ppm that has been assigned to a proton shared by Asp32 and His64, and is considered diagnostic of a LBHB in the WT enzyme, is not present in D32C subtilisin. Thus, the LBHB is not an inherent requirement for substantial rate enhancement for subtilisin.

Crystal structure of activated CheY. Comparison with other activated receiver domains.

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.

Crystal structure of an activated response regulator bound to its target.

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

NMR structure of activated CheY.

The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains.

Development and use of a virtual NMR facility.

We have developed a "virtual NMR facility" (VNMRF) to enhance access to the NMR spectrometers in Pacific Northwest National Laboratory's Environmental Molecular Sciences Laboratory (EMSL). We use the term virtual facility to describe a real NMR facility made accessible via the Internet. The VNMRF combines secure remote operation of the EMSL's NMR spectrometers over the Internet with real-time videoconferencing, remotely controlled laboratory cameras, real-time computer display sharing, a Web-based electronic laboratory notebook, and other capabilities. Remote VNMRF users can see and converse with EMSL researchers, directly and securely control the EMSL spectrometers, and collaboratively analyze results. A customized Electronic Laboratory Notebook allows interactive Web-based access to group notes, experimental parameters, proposed molecular structures, and other aspects of a research project. This paper describes our experience developing a VNMRF and details the specific capabilities available through the EMSL VNMRF. We show how the VNMRF has evolved during a test project and present an evaluation of its impact in the EMSL and its potential as a model for other scientific facilities. All Collaboratory software used in the VNMRF is freely available from www.emsl.pnl.gov:2080/docs/collab.

Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators.

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.

Assignment of cytosine N3 resonances in nucleic acids via intrabase three-bond coupling to amino protons.

Coherences were observed between 15N3 of cytosine and its trans amino proton (H42) using a modified gradient-based heteronuclear single quantum coherence (HSQC) pulse sequence optimized for three-bond proton-nitrogen couplings. The method is demonstrated with a 22-nucleotide RNA fragment of the P5abc region of a group I intron uniformly labeled with 15N. Use of intraresidue 15N3-amino proton couplings to assign cytosine 15N3 signals complements the recently proposed JNN HNN COSY [Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293-8297] method of identifying hydrogen-bonded base pairs in RNA.

Solution structure of the DNA-binding domain of NtrC with three alanine substitutions.

The structure of the 20 kDa C-terminal DNA-binding domain of NtrC from Salmonella typhimurium (residues Asp380-Glu469) with alanine replacing Arg456, Asn457, and Arg461, was determined by NMR spectroscopy. NtrC is a homodimeric enhancer-binding protein that activates the transcription of genes whose products are required for nitrogen metabolism. The 91-residue C-terminal domain contains the determinants necessary for dimerization and DNA-binding of the full length protein. The mutant protein does not bind to DNA but retains many characteristics of the wild-type protein, and the mutant domain expresses at high yield (20 mg/l) in minimal medium. Three-dimensional (1)H/(13)C/(15)N triple-resonance, (1)H-(13)C-(13)C-(1)H correlation and (15)N-separated nuclear Overhauser effect (NOE) spectroscopy experiments were used to make backbone and side-chain (1)H,(15)N, and (13)C assignments. The structures were calculated using a total of 1580 intra and inter-monomer distance and hydrogen bond restraints (88 hydrogen bonds; 44 hydrogen bond restraints), and 88 phi dihedral restraints for residues Asp400 through Glu469 in both monomers. A total of 54 ambiguous restraints (intra or inter-monomer) involving residues close to the 2-fold symmetry axis were also included. Each monomer consists of four helical segments. Helices A (Trp402-Leu414) and B (Leu421-His440) join with those of another monomer to form an antiparallel four-helix bundle. Helices C (Gln446-Leu451) and D (Ala456-Met468) of each monomer adopt a classic helix-turn-helix DNA-binding fold at either end of the protein. The backbone rms deviation for the 28 best of 40 starting structures is 0.6 (+/-0.2) A. Structural differences between the C-terminal domain of NtrC and the homologous Factor for Inversion Stimulation are discussed.

Mapping of a protein-RNA kissing hairpin interface: Rom and Tar-Tar*.

An RNA 'kissing' complex is formed by the association of two hairpins via base pairing of their complementary loops. This sense-antisense RNA motif is used in the regulation of many cellular processes, including Escherichia coli ColE1 plasmid copy number. The RNA one modulator protein (Rom) acts as a co-regulator of ColE1 plasmid copy number by binding to the kissing hairpins and stabilizing their interaction. We have used heteronuclear two-dimensional NMR spectroscopy to map the interface between Rom and a kissing complex formed by the loop of the trans -activation response (Tar) element of immunodeficiency virus 1 (HIV-1) and its complement. The protein binding interface was obtained from changes in amide proton signals of uniformly 15N-labeled Rom with increasing concentrations of unlabeled Tar-Tar*. Similarly, the RNA-binding interface was obtained from changes in imino proton signals of uniformly 15N-labeled Tar with increasing concentrations of unlabeled Rom. Our results are in agreement with previous mutagenesis studies and provide additional information on Rom residues involved in RNA binding. The kissing hairpin interface with Rom leads to a model in which the protein contacts the minor groove of the loop-loop helix and, to a lesser extent, the major groove of the stems.

Structures of active site histidine mutants of IIIGlc, a major signal-transducing protein in Escherichia coli. Effects on the mechanisms of regulation and phosphoryl transfer.

IIIGlc (also called IIAGlc), a major signal-transducing protein in Escherichia coli, is also a phosphorylcarrier in glucose uptake. The crystal and NMR structures of IIIGlc show that His90, the phosphoryl acceptor, adjoins His75 in the active site. Glutamine was substituted for His-, giving H75QIIIGlc and H90QIIIGlc, respectively (Presper, K. A., Wong, C.-Y., Liu, L., Meadow, N. D., and Roseman, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4052-4055), but the mutants showed unexpected properties. H90QIIIGlc loses regulatory functions of IIIGlc, and the phosphoryltransfer rates between HPr/H75QIIIGlc are 200-fold less than HPr/IIIGlc (Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33440-33445). X-ray crystallography, differential scanning calorimetry, and NMR have now been used to determine the structures of the mutants (phospho-H75QIIIGlc was studied by NMR). The three methods gave completely consistent results. Except for the His to Gln substitutions, the only significant structural changes were in a few hydrogen bonds. H90QIIIGlc contains two structured water molecules (to Gln90), which could explain its inability to regulate glycerol kinase. Phospho-IIIGlc contains a chymotrypsin-like, hydrogen bond network (Thr73-His75-O--phosphoryl), whereas phospho-H75QIIIGlc contains only one bond (Gln75-O--phosphoryl). Hydrogen bonds play an essential role in a proposed mechanism for the phosphoryltransfer reaction.

Structure of the acid state of Escherichia coli ribonuclease HI.

Under acidic conditions Escherichia coli ribonuclease HI* (RNase H*) adopts a partially folded state with all of the properties of a molten globule. Using amide hydrogen exchange carried out under acid state conditions, followed by quenching and NMR detection on the native state, we have determined the residues that are responsible for the observed structure of the acid state. Although RNase H* is a mixed alpha + beta protein, a helical subdomain (helices A, D, and B) defines the structure of the acid state. This structure correlates with the rare higher energy conformations detected under native conditions and with data for the earliest intermediates populated in the kinetic folding pathway of the protein.

Yeast heat shock transcription factor N-terminal activation domains are unstructured as probed by heteronuclear NMR spectroscopy.

The structure and dynamics of the N-terminal activation domains of the yeast heat shock transcription factors of Kluyveromyces lactis and Saccharomyces cerevisiae were probed by heteronuclear 15N[1H] correlation and 15N[1H] NOE NMR studies. Using the DNA-binding domain as a structural reference, we show that the protein backbone of the N-terminal activation domain undergoes rapid, large-amplitude motions and is therefore unstructured. Difference CD data also show that the N-terminal activation domain remains random-coil, even in the presence of DNA. Implications for a "polypeptide lasso" model of transcriptional activation are discussed.

Refined solution structure and dynamics of the DNA-binding domain of the heat shock factor from Kluyveromyces lactis.

The solution structure of the 92 residue (11 kDa) winged helix-turn-helix DNA-binding domain from the kluyveromyces lactis heat shock factor was refined using a total of 932 NOE, 35 phi, 25 chi 1, 5 chi 2 and 44 hydrogen bond restraints. The overall root-mean-square deviation for structured regions was 0.75(+/- 0.15) A. The three-helix bundle and four-stranded beta-sheet are well defined with rmsd of 0.53(+/- 0.10) A and 0.60(+/- 0.17) A, respectively. Helix H2 is underwound and bent near Pro45. The angle between helix H2 and the proposed recognition helix H3 is 96(+/- 6) degrees. Detailed comparisons are made with the X-ray structure of this protein as well as other structural studies on HSF. Overall, the results are consistent with the earlier studies. Differences are related to protein-protein interactions in the crystal and dynamics in solution. Backbone dynamics was investigated via 15N relaxation. The average R1, R2 and NOE values for residues in segments of secondary structure were 1.9(+/- 0.9) s-1, 7.8(+/- 0.9) s-1 and 0.81(+/- 0.05), respectively. The correlation time based on these data was 5.6(+/- 0.4) ns. Motional order parameters were calculated by fitting the relaxation data to one of three models. Low-order parameters were found for residues that comprise the turn between helices H2 and H3 (residues Lys49 to Phe53), and most strikingly, the 16 residue wing (residues Val68 to Arg83). These data are consistent with the lack of long-range NOEs identified in these regions. The data provide a basis for comparison with results of the protein-DNA complex. The relationship between structure and function is discussed.

Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure.

A combination of circular dichroism spectroscopy, titration calorimetry, and optical melting has been used to investigate the association of the minor groove ligands netropsin and distamycin to the central A3T2 binding site of the DNA duplex d(CGCAAATTGGC).d(GCCAATTTGCG). For the complex with netropsin at 20 degrees C, a ligand/duplex stoichiometry of 1:1 was obtained with Kb approximately 4.3 x 10(7) M-1, delta Hb approximately -7.5 kcal mol-1, delta Sb approximately 9.3 cal K-1 mol-1, and delta Cp approximately 0. Previous NMR studies characterized the distamycin complex with A3T2 at saturation as a dimeric side-by-side complex. Consistent with this result, we found a ligand/duplex stoichiometry of 2:1. In the current study, the relative thermodynamic contributions of the two distamycin ligands in the formation of this side-by-side complex (2:1 Dst.A3T2) were evaluated and compared with the thermodynamic characteristics of netropsin binding. The association of the first distamycin molecule of the 2:1 Dst.A3T2 complex yielded the following thermodynamic profile: Kb approximately 3.1 x 10(7) M-1, delta Hb = -12.3 kcal mol-1, delta Sb = -8 cal K-1 mol-1, and delta Cp = -42 cal K-1 mol-1. The binding of the second distamycin molecule occurs with a lower Kb of approximately 3.3 x 10(6) M-1, a more favorable delta Hb of -18.8 kcal mol-1, a more unfavorable delta Sb of -34 cal K-1 mol-1, and a higher delta Cp of -196 cal K-1 mol-1. The latter term indicates an ordering of electrostricted and structural water molecules by the complexes. These results correlate well with the NMR titrations and are discussed in context of the solution structure of the 2:1 Dst.A3T2 complex.

Heteronuclear NMR pulse sequences applied to biomolecules.

Current concepts in heteronuclear multidimensional NMR spectroscopy are reviewed. Methods to improve the sensitivity and the efficiency of data collection include constant time, compression through the overlap of chemical shift evolution and dephasing and rephasing periods, and dual or time-shared evolution. Two classes of three-dimensional and four-dimensional triple-resonance experiments applied to proteins are considered. The first class correlates 1H, 15N, and 13C signals of the protein backbone. The second class correlates both backbone and side-chain signals. Application of triple resonance to RNA is also discussed. Heteronuclear cross polarization (HCP) is considered as an alternative to INEPT transfer, and its application to nucleic acids is presented. Finally, two methods of employing pulsed field gradients (PFGs) are reviewed.

Solution structure of the DNA-binding domain of the heat shock transcription factor determined by multidimensional heteronuclear magnetic resonance spectroscopy.

The solution structure of the 92-residue DNA-binding domain of the heat shock transcription factor from Kluyveromyces lactis has been determined using multidimensional NMR methods. Three-dimensional (3D) triple resonance, 1H-13C-13C-1H total correlation spectroscopy, and 15N-separated total correlation spectroscopy-heteronuclear multiple quantum correlation experiments were used along with various 2D spectra to make nearly complete assignments for the backbone and side-chain 1H, 15N, and 13C resonances. Five-hundred eighty-three NOE constraints identified in 3D 13C- and 15N-separated NOE spectroscopy (NOESY)-heteronuclear multiple quantum correlation spectra and a 4-dimensional 13C/13C-edited NOESY spectrum, along with 35 phi, 9 chi 1, and 30 hydrogen bond constraints, were used to calculate 30 structures by hybrid distance geometry/stimulated annealing protocol, of which 24 were used for structural comparison. The calculations revealed that a 3-helix bundle packs against a small 4-stranded antiparallel beta-sheet. The backbone RMS deviation (RMSD) for the family of structures was 1.03 +/- 0.19 A with respect to the average structure. The topology is analogous to that of the C-terminal domain of the catabolite gene activator protein and appears to be in the helix-turn-helix family of DNA-binding proteins. The overall fold determined by the NMR data is consistent with recent crystallographic work on this domain (Harrison CJ, Bohm AA, Nelson HCM, 1994, Science 263:224) as evidenced by RMSD between backbone atoms in the NMR and X-ray structures of 1.77 +/- 0.20 A. Several differences were identified some of which may be due to protein-protein interactions in the crystal.

Effect of delay in radiation in the combined modality treatment of breast cancer.

PURPOSE: To study how the timing of radiation influences local control, overall survival, and disease-free survival in patients being treated with chemotherapy and radiation for a local-regional carcinoma of the breast. METHODS AND MATERIALS: Over a ten year period, 105 patients received chemotherapy and radiation treatments for a local-regional breast cancer. The population was divided into two groups based on the timing of their radiation treatments. Forty-eight patients began their radiation within 6 months of their diagnosis (early radiation group). Fifty-seven patients had a delay in their radiation for 6 or greater months in order to first maximize chemotherapy treatments (delayed radiation group). There were no significant differences between the two groups with respect to nodal status, stage of the primary, estrogen receptor status, patient age, or type of surgery performed. The only prognostic parameter that was imbalanced was that of a greater percentage of patients with close or positive margins in the early radiation group compared to the delayed radiation group (69% versus 38%, p < 0.02). RESULTS: Comparisons of local control, overall survival, and disease-free survival between the early radiation patients and delayed radiation patients all favored the early radiation group. Respective 8-year actuarial rates were: local control--98% vs. 76%, p = 0.004; overall survival--80% vs. 52%, p = 0.016; disease-free survival--71% vs. 48%, p = 0.008. The differences continued to be significant in a multivariate Cox regression model comparison: p = 0.011, p = 0.050, p = 0.009. There was only one death from intercurrent disease, so that overall survival approximated cause-specific survival to an accurate degree. CONCLUSIONS: In patients requiring chemotherapy and radiation treatments for a local-regional breast cancer, a delay in the initiation of radiation for a period of 6 months or greater from diagnosis resulted in a higher local failure rate. Furthermore, this higher local failure rate was associated with an increased rate of distant metastases and a decreased overall survival rate.