Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations.

Penicillin-binding proteins (PBPs) are the main targets for beta-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with beta-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr(338) --> Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser(389) --> Leu and Asn(514) --> His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for beta-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.     

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.     

The basolateral sorting signals of the thyrotropin and luteinizing hormone receptors: an unusual family of signals sharing an unusual distal intracellular localization, but unrelated in their structures

The mechanisms of the basolateral targeting of G protein-coupled receptors remain largely unknown. Mutagenesis experiments have allowed us to identify the basolateral sorting signals of the TSH and LH receptors expressed in Madin-Darby canine kidney cells and thyroid follicular FRT cells. Unexpectedly these signals (amino acids 731-746 and 672-689, respectively) share an unusual localization in the distal part of the intracellular domain of the receptors at a marked distance from the membrane. When grafted onto the p75-neurotropin receptor, these signals redirect this normally apically expressed protein to the basolateral cell surface. They are independent of the endocytosis signal. The basolateral sorting signals of TSH, LH, and FSH receptors do not exhibit primary sequence homology with each other or with any other known signal. Furthermore, circular dichroism studies show that the three signals exhibit distinct secondary structures. The TSH receptor has a stable helical structure, the LH receptor has both helix and beta-sheet structures, and the FSH receptor sorting signal has a main random coil structure. This means that even in closely-related receptors different secondary structures can be found for basolateral signals unrelated to internalization signals. This observation contrasts with what is known about basolateral signals related to internalization signals for which a common beta-turn structure has been described. Deletion of the basolateral sorting signals results in apical targeting of the receptors, suggesting the existence of apical sorting information. However, a soluble form of the TSH receptor, which harbors all N- and putative O-linked oligosaccharides, is secreted in a nonpolarized fashion. This implies that apical sorting information must be located elsewhere, either in the transmembrane or in the intracellular domains of the receptor.     

Homology of lysS and lysU, the two Escherichia coli genes encoding distinct lysyl-tRNA synthetase species.

In Escherichia coli, two distinct lysyl-tRNA synthetase species are encoded by two genes: the constitutive lysS gene and the thermoinducible lysU gene. These two genes have been isolated and sequenced. Their nucleotide and deduced amino acid sequences show 79% and 88% identity, respectively. Codon usage analysis indicates the lysS product being more efficiently translated than the lysU one. In addition, the lysS sequence exactly coincides with the sequence of herC, a gene which is part of the prfB-herC operon. In contrast to the recent proposal of Gampel and Tzagoloff (1989, Proc. Natl. Acad. Sci. USA 86, 6023-6027), the lysU sequence is distinct from the open reading frame located adjacent to frdA, although large homologies are shared by these two genes.     

Structural basis for membrane fusion by enveloped viruses.

Enveloped viruses such as HIV-1, influenza virus, and Ebola virus express a surface glycoprotein that mediates both cell attachment and fusion of viral and cellular membranes. The membrane fusion process leads to the release of viral proteins and the RNA genome into the host cell, initiating an infectious cycle. This review focuses on the HIV-1 gp41 membrane fusion protein and discusses the structural similarities of viral membrane fusion proteins from diverse families such as Retroviridae (HIV-1), Orthomyxoviridae (influenza virus), and Filoviridae (Ebola virus). Their structural organization suggests that they have all evolved to use a similar strategy to promote fusion of viral and cellular membranes. This observation led to the proposal of a general model for viral membrane fusion, which will be discussed in detail.     

Participation of the lone tryptophan residue of rat alpha-foetoprotein in its drug-binding sites. Comparison with rat serum albumin.

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.