The 1.25 A resolution refinement of the cholera toxin B-pentamer: evidence of peptide backbone strain at the receptor-binding site.

Crystals of the 61 kDa complex of the cholera toxin B-pentamer with the ganglioside GM1 receptor pentasaccharide diffract to near-atomic resolution. We have refined the crystallographic model for this complex using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.129 for all observed Bragg reflections in the resolution range 22 A to 1.25 A. Remarkably few residues show evidence of discrete conformational disorder. A notable exception is a minority conformation found for the Cys9 side-chain, which implies that the Cys9-Cys86 disulfide linkage is incompletely formed. In all five crystallographically independent instances, the peptide backbone in the region of the receptor-binding site shows evidence of strain, including unusual bond lengths and angles, and a highly non-planar (omega=153.7(7) degrees) peptide group between residues Gln49 and Val50. The location of well-ordered water molecules at the protein surface is notable reproduced among the five crystallographically independent copies of the peptide chain, both at the receptor-binding site and elsewhere. The 5-fold non-crystallographic symmetry of this complex allows an evaluation of the accuracy, reproducibility, and derived error estimates from refinement of large structures at near-atomic resolution. We find that blocked-matrix treatment of parameter covariance underestimates the uncertainty of atomic positions in the final model by approximately 10% relative to estimates based either on full-matrix inversion or on the 5-fold non-crystallographic symmetry.

Construction and characterization of versatile cloning vectors for efficient delivery of native foreign proteins to the periplasm of Escherichia coli.

Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones in Escherichia coli inhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic for E. coli, but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit of E. coli heat-labile enterotoxin LTIIb we succeeded for the first time in producing CT holotoxin in high yield in E. coli. Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm of E. coli. We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, using phoA from E. coli, we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using the pspA gene that encodes pneumococcal surface protein A from Streptococcus pneumoniae, we produced a 299-residue amino-terminal fragment of PspA in E. coli in large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure-function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm in E. coli.

Cyanobacteria carrying an smt-lux transcriptional fusion as biosensors for the detection of heavy metal cations.

The metal-responsive smt operator/promoter region of Synechococcus PCC7942 was fused to the luxCDABE genes of Vibrio fischeri. Plasmid DNA (pJLE23) carrying this fusion conferred metal ion-inducible luminescence to transformed cyanobacteria. Synechococcus PCC7942 (pJLE23) was sensitive to ZnCl2 concentrations within a range of 0.5-4 microM as demonstrated by induction of luminescence. Trace levels of CuSO24 and CdCl2 were also detected.

Expression of mouse metallothionein in the cyanobacterium Synechococcus PCC7942.

A cDNA encoding mouse metallothionein was cloned into the shuttle vector pUc303, creating a translational fusion with the bacterial chloramphenicol acetyltransferase gene. The resulting fusion protein has been expressed in the cyanobacterium Synechococcus PCC7942. Cyanobacterial transformants expressed mouse metallothionein-specific mRNA species as detected by RNA slot blots. In addition, the transformants expressed a unique cadmium ion-binding protein corresponding to the predicted size of the mouse metallothionein fusion protein. Expression of this fusion protein conferred a two- to five-fold increase in cadmium ion tolerance and accumulation on Synechococcus PCC7942.

Metalloregulation of the cyanobacterial smt locus: identification of SmtB binding sites and direct interaction with metals.

The smtB gene of Synechococcus PCC 7942 encodes a trans-acting repressor of the metal-regulated smtA gene that encodes a class II metallothionein. Recombinant SmtB has been expressed in Escherichia coli and purified. Electrophoretic mobility shift assays using recombinant SmtB or a protein extract from Synechococcus PCC 6301 reveal the concentration-dependent formation of three specific complexes with the smt operator/promoter. SmtB is also capable of direct interaction with metals as evidenced by 65Zn binding to the SmtB protein as well as the inhibition of repressor-DNA complex formation in the presence of various metal ions. Methylation interference analysis of such complexes identifies four protein contact points within the smt operator/promoter DNA. The points of contact appear to represent two pairs of binding sites, one pair in each of two inverted repeats (nt 548-563, 589-602). The contact points within each pair lie on opposing DNA strands and are separated by 10 bp, placing the repressor binding sites on opposite sides of the DNA helix. Based on electrophoretic mobility shift assays, methylation interference and molecular size calculations we propose that recombinant SmtB binds to the smt operator/promoter in multimeric fashion.