Involvement of Trp-284, Val-296, and Val-297 of the human delta-opioid receptor in binding of delta-selective ligands.

Given the high homology in amino acid sequence between the delta-opioid receptor and the two other types (mu and kappa), distinct residues in this receptor may confer its selectivity to some ligands. In order to identify molecular determinants in the human delta receptor responsible for the selectivity of delta-selective ligands, two different delta/mu chimeras were constructed. In the first one, the delta sequence from the top of transmembrane 5 to the C terminus was replaced by the equivalent mu sequence, and in the second one, 13 consecutive residues in the third extracellular loop region of the delta receptor were replaced by the mu counterpart. These two chimeras retained the ability to bind the nonselective bremazocine but completely lost the ability to bind different delta-selective ligands. These results suggested that the region of the third extracellular loop of the delta receptor is crucial for the type selectivity. Furthermore, an alanine scan was performed by site-directed mutagenesis of 20 amino acids located in or proximal to the third extracellular loop. Among all the point mutations, only mutations of Trp-284, Val-296, or Val-297 significantly decreased the binding of delta-selective ligands tested. Moreover, combined mutation of Trp-284, Val-296, and Val-297 considerably decreased the affinities of the receptor for delta-selective ligands compared with the single point mutations. These findings suggest that Trp-284, Val-296, and Val-297 are crucial residues involved in the delta receptor type selectivity.

Crossover site selection during recombination of polyomavirus replicons.

Two hybrid replicons containing polyomavirus (Py) genomes with large duplications of the viral late coding sequences were transfected into various permissive mouse cell lines. In all cell lines, either replicon yielded the sole amplifiable product expected from intramolecular homologous recombination, unit-length Py DNA (P155). In normal and in Py-transformed cells, such recombination was highly effective and involved sequences previously found to act as recombination hot spots (S repeats). In cells transformed by simian virus 40, however, these hot spots were inoperative in the generation of P155, which occurred with a reduced efficiency. These data confirm and extend earlier data indicating that the nature of products arising from recombination in Py replicons is tightly controlled by both cis- and trans-acting factors.

The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad.

General-base catalysis in the active site of serine proteases is carried out by the imidazole side chain of a histidine. During formation of the transition state, an adjacent carboxylic acid group stabilizes the positive charge that forms on the general-base catalyst and as a result contributes several orders of magnitude to the catalytic efficiency of these enzymes. In the recently discovered family of self-cleaving proteins exemplified by the LexA repressor of Escherichia coli, instead of the imidazole of a histidine, the active-site general-base catalyst was found to be the epsilon-amino of a lysine. The considerably higher capacity of the lysine side chain for proton acceptance raises interesting questions concerning the role of electrostatic interactions in the mechanism of proton transfer by this highly basic group. The negative charge elimination studies described here and their effects on the kmax and pK of LexA self-cleavage are consistent with a model in which electrostatic interactions between an acidic side chain and the general-base catalyst form a barrier to proton transfer. The implications are that the epsilon-amino group, unlike the imidazole group, is capable of effecting proton transfer without the intervention of a countercharge.

The same mammalian replicon yields distinct recombination products in different cell lines.

We have observed previously that some chimeric replicons inclusive of a partly duplicated polyomavirus (Py) genome yield unit-length Py DNA (P155) at high frequency when transfected into normal or Py-transformed mouse cells. We demonstrate here that one such replicon generates either P155 or illegitimate recombination products in other mouse cells, transformed by simian virus 40. Use of the polymerase chain reaction indicates that each of the illegitimate products carried a different deletion, but that all deletions mapped within a rather well defined portion of the precursor replicon. Thus, these products were organized as if two hotspots for recombination existed in the Py late-coding region, one being located within or near one of the duplicated sequences characteristic of the chimeric replicon. Since this particular hotspot has already been shown to be involved in the generation of P155, the data reported here could indicate that a single recombination mechanism can yield either homologous (P155) or illegitimate products. How the DNA interacts with certain proteins, such as papovavirus large tumor antigen, could explain why one or the other type of product is formed.