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A positively charged side chain at position 154 on the beta8-alphaE loop of HIV-1 RT is required for stable ternary complex formation.

ABSTRACT: Lys154 is the only positively charged residue located in the VLPQGWK motif on the beta8-alphaE loop at the junction of the fingers and palm subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Some of the conserved residues in this motif are critical for RT function, while others have been shown to confer nucleoside drug resistance and fidelity to the enzyme. In order to understand the functional implication of this positively charged residue, we carried out site-directed mutagenesis at position 154 and biochemically characterized the mutant enzymes. Mutants carrying negatively charged side chains (K154D and K154E) were severely impaired in their polymerase function, while those with hydrophobic side chains (K154A and K154I) were moderately affected. Analysis of the binary complexes formed by these mutants revealed that all the mutant derivatives retained their ability to form an enzyme template primer (E-TP) binary complex similar to the wild-type enzyme. In contrast, their ability to form stable E-TP-dNTP ternary complexes varied greatly and was dependent on the nature of the side chain at position 154. The conservative Lys-->Arg mutant was not affected in its ability to form a stable ternary complex, while those carrying non-polar or negatively charged side chains were significantly impaired. The apparent K(d [dNTP]) values for these non-conservative mutants were approximately 16- to 400-fold higher than the wild-type enzyme, indicating that a positively charged side chain at position 154 may be required for efficient formation of a stable ternary complex. Interestingly, all the mutant derivatives of Lys154 were completely resistant to a nucleoside analog inhibitor, 3'-dideoxy 3'-thiacytidine (3TC), implying that Lys154 may play a role in conferring 3TC sensitivity to HIV-1 RT. These findings are discussed in the context of the binary and ternary complex crystal structures of HIV-1 RT.

SUBMITTER: Sharma B

PROVIDER: S-EPMC212807 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

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A positively charged side chain at position 154 on the beta8-alphaE loop of HIV-1 RT is required for stable ternary complex formation.

Sharma Bechan B Kaushik Neerja N Upadhyay Alok A Tripathi Snehlata S Singh Kamalendra K Pandey Virendra N VN

Nucleic acids research 20030901 17

Lys154 is the only positively charged residue located in the VLPQGWK motif on the beta8-alphaE loop at the junction of the fingers and palm subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Some of the conserved residues in this motif are critical for RT function, while others have been shown to confer nucleoside drug resistance and fidelity to the enzyme. In order to understand the functional implication of this positively charged residue, we carried out site-d ...[more]

PMID: 12930968

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Project description:Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-?) and interferon gamma (IFN-?). While TNF-?/IFN-? treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes.

Dataset Information

A positively charged side chain at position 154 on the beta8-alphaE loop of HIV-1 RT is required for stable ternary complex formation.

Publications

A positively charged side chain at position 154 on the beta8-alphaE loop of HIV-1 RT is required for stable ternary complex formation.

Similar Datasets

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