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Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: Rethinking the “fireman's grip” hypothesis

Published online by Cambridge University Press:  05 October 2000

KVIDO STRISOVSKY
Affiliation:
Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Flemingovo n.2, 166 10 Praha 6, Czech Republic
UWE TESSMER
Affiliation:
Heinrich-Pette-Institut für experimentelle Virologie und Immunologie, Martinistr. 52, D-20251 Hamburg, Germany
JOSMAR LANGNER
Affiliation:
Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany
JAN KONVALINKA
Affiliation:
Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Flemingovo n.2, 166 10 Praha 6, Czech Republic
HANS-GEORG KRÄUSSLICH
Affiliation:
Heinrich-Pette-Institut für experimentelle Virologie und Immunologie, Martinistr. 52, D-20251 Hamburg, Germany
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Abstract

Aspartic proteinases share a conserved network of hydrogen bonds (termed “fireman's grip”), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the “fireman's grip,” Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the “fireman's grip” interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer–dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the “fireman's grip” is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the “fireman's grip” is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.

Type
Research Article
Copyright
2000 The Protein Society

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