Protein-ligand energetics assessed using deoxy and fluorodeoxy sugars in equilibrium binding and high resolution crystallographic studies.

Hydrogen bonding interactions are one of the most important single factors in protein-ligand interactions and molecular recognition. To probe the energetics of the interactions, we have analyzed the binding of 1-deoxy-, 2-deoxy- and 6-fluoro-6-deoxy- analogues of D-galactose (Gal) to a primary high-affinity periplasmic receptor for monosaccharide active transport. Kd values and atomic structures refined at 1.81 to 1.45 A resolution of the complexes have been determined and compared with those of Gal binding. With binding site residues and the bound modified sugars in nearly identical positions as found in the complex with Gal, the binding of 1-deoxy-Gal or 2-deoxy-Gal reflects the overall contribution of 1.8 kcal mol-1 per hydrogen bond (neutral-charge type) to the affinity of Gal. Neglected in these estimates is the contribution of van der Waals' forces that accompany the formation of hydrogen bonds with each sugar hydroxyl. Contrary to expectations, the 6-fluoro-6-deoxy analogue proved to be an inadequate probe of Gal OH6 as a hydrogen bond donor due to the binding of a new water molecule and structural changes arising from the electronegative fluoro group. This study sheds new light on the energetics of protein-ligand interactions and the use of engineered ligands in assessing these interactions.

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity.

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.

Stability of mutant type II dihydrofolate reductase proteins in suppressor strains.

In the course of study of a (Glu-58 to Gln-58) mutant type II dihydrofolate reductase (DHFR), it was found that the altered DHFR was poorly produced in vivo. Investigations with several common laboratory Escherichia coli strains including htpR and lon strains bearing plasmids expressing the Gln-58 DHFR indicated a correlation of rapid degradation with the presence of a sup+ phenotype. The supo strain MC1061(p3) was transformed with a series of plasmids containing the Gln-58 DHFR gene with and without an additional supF gene, and expression levels were compared. The supF+ constructs exhibited little accumulation of the Gln-58 DHFR, while reasonable levels were found in the supo cases. Experiments with extracts of plasmid-free sup+ and supo strains showed rapid degradation by certain strains compared to MC1061(p3) and this degradation was not dependent upon ATP. In another route to increasing the stability of labile DHFR derivatives, mutagenesis of a strain bearing a N-terminally shortened Gln-58 DHFR was performed. Selection and analysis of a trimethoprim-resistant stable mutant showed that this DHFR gene contained a triple repeat of leu-pro-ser in the enzymatically non-essential N-terminal portion of the protein.

Engineered interdomain disulfide in the periplasmic receptor for sulfate transport reduces flexibility. Site-directed mutagenesis and ligand-binding studies.

Using recombinant DNA techniques, an Escherichia coli periplasmic sulfate receptor or sulfate-binding protein involved in active transport has been overexpressed and characterized. This protein is essentially identical in size, sequence, antigenicity, and ligand affinity and specificity to the sulfate receptor from Salmonella typhimurium whose crystal structure has been refined at 2 A resolution. The dehydrated sulfate is bound in the deep cleft between the two lobes of the bilobate protein. Using the structure of the S. typhimurium as a guide, three site-directed mutants (Ser129Cys, Gly46Cys, and Ser129Cys/Gly46Cys) have been made. In the Cys129/Cys46 mutant the disulfide has been successfully introduced across the opening of the ligand-binding site cleft of the E. coli sulfate-binding protein. The dissociation of sulfate from the double mutant protein is very slow under oxidizing conditions and increases more than 200-fold when reducing agent is added. This effect is attributed to a loss of interdomain structural flexibility in the presence of the disulfide, and underscores the importance of protein conformational change in binding protein function.

A Pro to Gly mutation in the hinge of the arabinose-binding protein enhances binding and alters specificity. Sugar-binding and crystallographic studies.

The L-arabinose-binding protein (ABP) of Escherichia coli consists structurally of two distinct globular domains connected by a hinge of three separate peptide segments. Arabinose is bound and completely sequestered within the deep cleft between the two domains. With reduced affinity, ABP also binds D-galactose (approximately 2-fold reduction) and D-fucose (approximately 40-fold reduction). Experiments have been conducted to explore the role in sugar binding of the hinge connecting the two domains of ABP. To increase the flexibility of the hinge region, a glycine was substituted for a proline at position 254 by site-directed mutagenesis. Unexpectedly, this mutation resulted in the dramatic enhancement of galactose binding over that of arabinose. The affinity of the mutant ABP for galactose increased by over 20-fold, while that for arabinose and fucose remained relatively unchanged. We have measured association and dissociation rates of the Gly-254 ABP with L-arabinose, D-galactose, and D-fucose and have determined the crystallographic structure of the protein complexed with each of the three sugars. Both the ligand-binding kinetic measurements and structure analysis indicate that the altered specificity is due to an effective increase in the rigidity of the hinge in the closed conformation which is induced upon galactose binding. Stabilizing contacts are formed between the strands of the hinge in the Gly-254 ABP when galactose is bound which are not found in complexes with the other sugars or the liganded wild-type protein.

Synthesis and expression of a gene for a mini type II dihydrofolate reductase.

The Type II dihydrofolate reductases (DHFR) are resistant to the folate analogs, trimethoprim and methotrexate. The monomer is very small (MW 9,000) and has no structural homology with other known DHFR types. A dhfr structural gene was synthesized which incorporates many unique restriction sites (Nco I, Nhe I, Pvu I, Hind III, Sma I, Bgl II, Xho I, and Ban I) within the coding sequence. This gene encodes a small DHFR (68 amino acids) which is 10 amino acids shorter at the amino-terminus than natural Type II DHFRs. The last 60 residues of the synthetically encoded protein are identical in sequence to R388 DHFR. The enzyme is functional and relatively stable, as evidenced by trimethoprim resistance conferred to cells expressing the synthetic gene. The gene was cloned onto a high-copy-number plasmid, pPV7SYN5, in which a trp-lac promoter drives transcription of both the dhfr gene and the primer for plasmid replication (RNA II). High levels of the small DHFR are accumulated in stationary phase cultures of MC1061(p3) containing pPV7SYN5 without the addition of IPTG.

The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene.

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.

The glyceraldehyde-3-phosphate dehydrogenase gene family in the nematode, Caenorhabditis elegans: isolation and characterization of one of the genes.

The isolation and genomic sequence of one of possibly four glyceraldehyde-3-phosphate dehydrogenase genes in the nematode, Caenorhabditis elegans is presented. The complete nucleotide sequence of the coding as well as the noncoding flanking regions of this gene has been determined. The deduced amino-acid sequence agrees with the sequence of typical glyceraldehyde-3-phosphate dehydrogenase enzymes and its molecular weight of 36,235 agrees with its size determined previously (Yarbrough, P. and Hecht, R. (1984) J. Biol. Chem. 259, 14711-14720). That this isolated gene encodes a nematode glyceraldehyde-3-phosphate dehydrogenase is additionally confirmed by demonstrating its immunoreactivity to an anti-nematode glyceraldehyde-3-phosphate dehydrogenase antibody after its expression as a fusion protein with dihydrofolate reductase. Codon utilization follows a pattern typical of other expressed nematode genes. The gene is split by two introns that are highly conserved in comparison to other introns observed in C. elegans. The placement of one of these introns is conserved with respect to the chicken glyceraldehyde-3-phosphate dehydrogenase gene. Within the 5' flanking sequence homology to actin and the homology 2 block of the major myosin gene (unc-54) is noted. It is of interest that the 3' flanking region contains a CAAAT box, followed by a TATAAT box, before an open reading frame of a closely linked gene that also contains a small AT-rich intron with the nematode consensus splice junction.

Use of bacterial DHFR-II fusion proteins to elicit specific antibodies.

Plasmids containing the coding region of the type II dihydrofolate reductase (DHFR) specified by R388 have been used to alter the amino acid (aa) sequence at the C-terminus of this protein. These plasmids have a unique cloning site in the C-terminal portion of the 78-aa coding region. Insertions of DNA fragments into this site produced plasmids that code for proteins with 6- to 80-aa extensions. The vectors were constructed to terminate translation in all three phases beyond the position of insertion of foreign DNA. Random DNA fragments from the major sperm protein (MSP) gene of Caenorhabditis elegans produced by DNase I cleavage were inserted into these vectors. Cell extracts from colonies containing MSP sequences were examined by gel electrophoresis and immunoblotting. One of the hybrid DHFR-MSP proteins was isolated and antibody was prepared to it. This antibody preparation reacted with MSP in immunoblots of purified MSP and whole cell extracts of the worm. A rapid purification procedure for the DHFR is presented.

Construction and characterization of pBR322-derived plasmids with deletions of the RNA I region.

A region upstream from the origin of replication in ColE1-type plasmids has been shown to be necessary for replication. Two RNA transcripts are produced from this area, RNA II, which yields the primer for DNA polymerase initiation at the origin and RNA I, which is complementary to the 5' end of RNA II and acts to inhibit primer formation. We have constructed plasmids which do not possess the nucleotide sequence for RNA I, or the normal 5' terminus and promoter of RNA II. The RNA II analog, in these plasmids, is believed to be synthesized by the readthrough transcription of the upstream trimethoprim-resistant dihydrofolate reductase (DHFR) gene at a level comparable to that produced by the tryptophan promoter. These plasmids have a copy number of about tenfold higher than that of pBR322 during logarithmic growth and are compatible with other ColE1-type plasmids. These plasmids are stably maintained in several strains when selective pressure is present and the plasmids are stably maintained during exponential growth in W3110 strains without selective pressure. In all strains examined, the dimeric form of the plasmid was lost from cells much more rapidly than those containing the monomeric form.

Role of DNA regions flanking the tryptophan promoter of Escherichia coli. I. Insertion of synthetic oligonucleotides.

To examine the effect of altering the nucleotide sequence near the promoter on its activity, pKO-1 vector derivatives have been constructed which allow insertion of DNA fragments at specified sites upstream or downstream from the trp promoter. Oligonucleotides that might be expected to alter the melting properties, or have a tendency to form a distinctive nonstandard structure were introduced. These oligonucleotides had the repeating dinucleotide sequences GC, AT or AG. Sequence analysis of the inserts and studies of the relative galactokinase expression from the altered plasmids indicated that changes upstream from the trp promoter at -39 or beyond had little effect on trp promoter activity, whereas changes at +2 or farther downstream produced up to two-fold increases in gene expression, as compared to the control plasmid.