Local and spatial factors determining HIV-1 protease substrate recognition.
Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) was used to address substrate recognition of HIV-1 protease in a well characterized structural context. By modifying the TS conformation while maintaining its enzymic activity, we investigated the influence of protein folding on protease-substrate recognition. A slight destabilization of the TS structure permitted the cleavage of a target site, which was resistant in the native TS. This result supports a dynamic interpretation of HIV-1 protease specificity. Exposure time of the potential cleavage site, which depends on the stability of the global conformation, must be compatible with the cleavage kinetics, which are determined by the local sequence. Cleavage specificity has been described as the consequence of cumulative interactions, globally favourable, between at least six amino acids around the cleavage site. To investigate influence of local sequence, we introduced insertions of variable lengths in two exposed loops of the TS. In both environments, insertion of only two amino acids could determine specific cleavage. We then inserted libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts.
Hazebrouck
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S
Genetique des Virus, ICGM-CNRS UPR 415, 22 rue Mechain, 75014 Paris, France.
Machtelinckx-Delmas
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Kupiec
J J
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Sonigo
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P
D000595:Amino Acid Sequence; D004151:Dipeptides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D015683:Gene Products, gag / Q000737:chemistry; D016655:HIV Core Protein p24 / Q000737:chemistry; D016333:HIV Protease / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus
D004151:Dipeptides; D015683:Gene Products, gag; D016655:HIV Core Protein p24; D011498:Protein Precursors; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus; C091950:pr160 gag-pol precursor protein, Human immunodeficiency virus 1; D013940:Thymidylate Synthase; D016333:HIV Protease