DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein.
ABSTRACT: The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.
Project description:We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.
Project description:The crystal structure of cyclic AMP-catabolite activator protein (CAP) from Escherichia coli containing cobalt(II) chloride and ammonium sulfate is reported at 1.97 Å resolution. Each of the two CAP subunits in the asymmetric unit binds one cobalt(II) ion, in each case coordinated by N-terminal domain residues His19, His21 and Glu96 plus an additional acidic residue contributed via a crystal contact. The three identified N-terminal domain cobalt-binding residues are part of a region of CAP that is important for transcription activation at class II CAP-dependent promoters. Sulfate anions mediate additional crystal lattice contacts and occupy sites corresponding to DNA backbone phosphate positions in CAP-DNA complex structures.
Project description:To understand structural and thermodynamic features of disulfides within an alpha-helix, a non-redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirty-five examples were found of intrahelical disulfides involving a CXXC motif between the N-Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The N-Cap residue for disulfide bonded CXXC motifs had average (phi,psi) values of (-112 +/- 25.2 degrees , 106 +/- 25.4 degrees ). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions N-Cap-3; 1,4; 7,10 in two helices of an Escherichia coli thioredoxin mutant lacking its active site disulfide (nSS Trx). In both helices, disulfides formed spontaneously during purification only at positions N-Cap-3. Mutant stabilities were characterized by chemical denaturation studies (in both oxidized and reduced states) and differential scanning calorimetry (oxidized state only). All oxidized as well as reduced mutants were destabilized relative to nSS Trx. All mutants were redox active, but showed decreased activity relative to wild-type thioredoxin. Such engineered disulfides can be used to probe helix start sites in proteins of unknown structure and to introduce redox activity into proteins. Conversely, a protein with CYS residues at positions N-Cap and 3 of an alpha-helix is likely to have redox activity.
Project description:The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.
Project description:A crucial element of many gene functions is protein-induced DNA bending. Computer-generated models of such bending have generally been derived by using a presumed bending angle for DNA. Here we describe a knowledge-based docking strategy for modeling the structure of bent DNA recognized by a major groove-inserting alpha-helix of proteins with a helix-turn-helix (HTH) motif. The method encompasses a series of molecular mechanics and dynamics simulations and incorporates two experimentally derived distance restraints: one between the recognition helix and DNA, the other between respective sites of protein and DNA involved in chemical modification-enabled nuclease scissions. During simulation, a DNA initially placed at a distance was "steered" by these restraints to dock with the binding protein and bends. Three prototype systems of dimerized HTH DNA binding were examined: the catabolite gene activator protein (CAP), the phage 434 repressor (Rep), and the factor for inversion stimulation (Fis). For CAP-DNA and Rep-DNA, the root mean square differences between model and x-ray structures in nonhydrogen atoms of the DNA core domain were 2.5 A and 1.6 A, respectively. An experimental structure of Fis-DNA is not yet available, but the predicted asymmetrical bending and the bending angle agree with results from a recent biochemical analysis.
Project description:The 2.2 A resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with cAMP and a 46-bp DNA fragment reveals a second cAMP molecule bound to each protein monomer. The second cAMP is in the syn conformation and is located on the DNA binding domain interacting with the helix-turn-helix, a beta-hairpin from the regulatory domain and the DNA (via water molecules). The presence of this second cAMP site resolves the apparent discrepancy between the NMR and x-ray data on the conformation of cAMP, and explains the cAMP concentration-dependent behaviors of the protein. In addition, this site's close proximity to mutations affecting transcriptional activation and its water-mediated interactions with a DNA recognition residue (E181) and DNA raise the possibility that this site has biological relevance.
Project description:The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.
Project description:BACKGROUND: Two drinking water systems at U.S. Marine Corps Base Camp Lejeune, North Carolina were contaminated with solvents during 1950s-1985. METHODS: We conducted a retrospective cohort mortality study of 4,647 civilian, full-time workers employed at Camp Lejeune during 1973-1985 and potentially exposed to contaminated drinking water. We selected a comparison cohort of 4,690 Camp Pendleton workers employed during 1973-1985 and unexposed to contaminated drinking water. Mortality follow-up period was 1979-2008. Cause-specific standardized mortality ratios utilized U.S. age-, sex-, race-, and calendar period-specific mortality rates as reference. We used survival analysis to compare mortality rates between Camp Lejeune and Camp Pendleton workers and assess the effects of estimated cumulative contaminant exposures within the Camp Lejeune cohort. Ground water contaminant fate/transport and distribution system models provided monthly estimated contaminant levels in drinking water serving workplaces at Camp Lejeune. The confidence interval (CI) indicated precision of effect estimates. RESULTS: Compared to Camp Pendleton, Camp Lejeune workers had mortality hazard ratios (HRs) >1.50 for kidney cancer (HR = 1.92, 95% CI: 0.58, 6.34), leukemias (HR = 1.59, 95% CI: 0.66, 3.84), multiple myeloma (HR = 1.84, 95% CI: 0.45, 7.58), rectal cancer (HR = 1.65, 95% CI: 0.36, 7.44), oral cavity cancers (HR = 1.93, 95% CI: 0.34, 10.81), and Parkinson's disease (HR = 3.13, 95% CI: 0.76, 12.81). Within the Camp Lejeune cohort, monotonic exposure-response relationships were observed for leukemia and vinyl chloride and PCE, with mortality HRs at the high exposure category of 1.72 (95% CI: 0.33, 8.83) and 1.82 (95% CI: 0.36, 9.32), respectively. Cumulative exposures were above the median for most deaths from cancers of the kidney, esophagus, rectum, prostate, and Parkinson's disease, but small numbers precluded evaluation of exposure-response relationships. CONCLUSION: The study found elevated HRs in the Camp Lejeune cohort for several causes of death including cancers of the kidney, rectum, oral cavity, leukemias, multiple myeloma, and Parkinson's disease. Only 14% of the Camp Lejeune cohort died by end of follow-up, producing small numbers of cause-specific deaths and wide CIs. Additional follow-up would be necessary to comprehensively assess drinking water exposure effects at the base.
Project description:Collagen is an abundant, triple-helical protein comprising three strands of the repeating sequence: Xaa-Yaa-Gly. (2S)-Proline and (2S,4R)-4-hydroxyproline (Hyp) are common in the primary structure of collagen. Here, we use nonnatural proline derivatives to reveal determinants of collagen stability. Specifically, we report high-yielding syntheses of (2S,4S)-4-chloroproline (clp) and (2S,4R)-4-chloroproline (Clp). We find that the molecular structure of Ac-Clp-OMe in the solid state is virtually identical to that of Ac-Hyp-OMe. In contrast, the conformational properties of Ac-clp-OMe are similar to those of Ac-Pro-OMe. Ac-Clp-OMe has a stronger preference for a trans amide bond than does Ac-Pro-OMe, whereas Ac-clp-OMe has a weaker preference. (Pro-Clp-Gly)(10) forms triple helices that are significantly more stable than those of (Pro-Pro-Gly)(10). Triple helices of (clp-Pro-Gly)(10) have stability similar to those of (Pro-Pro-Gly)(10). Unlike (Pro-Clp-Gly)(10) and (clp-Pro-Gly)(10), (clp-Clp-Gly)(10) does not form a stable triple helix, presumably due to a deleterious steric interaction between proximal chlorines on different strands. These data, which are consistent with previous work on 4-fluoroprolines and 4-methylprolines, support the importance of stereoelectronic and steric effects in the stability of the collagen triple helix and provide another means to modulate that stability. (
Project description:BACKGROUND: Two drinking water systems at U.S. Marine Corps Base Camp Lejeune, North Carolina were contaminated with solvents during 1950s-1985. METHODS: We conducted a retrospective cohort mortality study of Marine and Naval personnel who began service during 1975-1985 and were stationed at Camp Lejeune or Camp Pendleton, California during this period. Camp Pendleton's drinking water was uncontaminated. Mortality follow-up was 1979-2008. Standardized Mortality Ratios were calculated using U.S. mortality rates as reference. We used survival analysis to compare mortality rates between Camp Lejeune (N = 154,932) and Camp Pendleton (N = 154,969) cohorts and assess effects of cumulative exposures to contaminants within the Camp Lejeune cohort. Models estimated monthly contaminant levels at residences. Confidence intervals (CIs) indicated precision of effect estimates. RESULTS: There were 8,964 and 9,365 deaths respectively, in the Camp Lejeune and Camp Pendleton cohorts. Compared to Camp Pendleton, Camp Lejeune had elevated mortality hazard ratios (HRs) for all cancers (HR = 1.10, 95% CI: 1.00, 1.20), kidney cancer (HR = 1.35, 95% CI: 0.84, 2.16), liver cancer (HR = 1.42, 95% CI: 0.92, 2.20), esophageal cancer (HR = 1.43 95% CI: 0.85, 2.38), cervical cancer (HR = 1.33, 95% CI: 0.24, 7.32), Hodgkin lymphoma (HR = 1.47, 95% CI: 0.71, 3.06), and multiple myeloma (HR = 1.68, 95% CI: 0.76, 3.72). Within the Camp Lejeune cohort, monotonic categorical cumulative exposure trends were observed for kidney cancer and total contaminants (HR, high cumulative exposure = 1.54, 95% CI: 0.63, 3.75; log10 ? = 0.06, 95% CI: -0.05, 0.17), Hodgkin lymphoma and trichloroethylene (HR, high cumulative exposure = 1.97, 95% CI: 0.55, 7.03; ? = 0.00005, 95% CI: -0.00003, 0.00013) and benzene (HR, high cumulative exposure = 1.94, 95% CI: 0.54, 6.95; ? = 0.00203, 95% CI: -0.00339, 0.00745). Amyotrophic Lateral Sclerosis (ALS) had HR = 2.21 (95% CI: 0.71, 6.86) at high cumulative vinyl chloride exposure but a non-monotonic exposure-response relationship (? = 0.0011, 95% CI: 0.0002, 0.0020). CONCLUSION: The study found elevated HRs at Camp Lejeune for several causes of death including cancers of the kidney, liver, esophagus, cervix, multiple myeloma, Hodgkin lymphoma and ALS. CIs were wide for most HRs. Because <6% of the cohort had died, long-term follow-up would be necessary to comprehensively assess effects of drinking water exposures at the base.