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t/data/cp1252/10482611.cp1252  view on Meta::CPAN

   Earlier genetic studies indicate that mutations in the Leu 568 and Trp
   571 residues abolish membrane fusion activity, although the mutant
   HIV-1 envelope proteins appear to have no other defects, including cell
   surface expression, gp160 precursor processing, and CD4 binding
   ([231]5). Our results indicate that these fusion-defective mutations
   destabilize the gp41 core structure although they still confer the
   six-helix bundle fold. Since the Leu 568 and Trp 571 residues form the
   right wall of a conserved coiled-coil cavity that provides a binding
   pocket for three C-terminal helices ([232]9), our data suggest that the
   fusion-defective mutations introduce structural perturbations in the
   cavity that weaken helical packing interactions in the six-helix
   complex and thus inhibit its formation.
   These fusion-defective mutations also exert striking effects on the
   inhibitory activity of N34(L6)C28; the L568A and W571R mutants exhibit
   5- to 16-fold-greater activity than the wild-type molecule. Several
   lines of evidence suggest that this enhanced inhibitory activity
   results from the synergistic inhibition of the N34 and C28 peptides in
   the mutant molecules. First, while the L568A and W571R trimers are
   stable, with T[m] values of 56 and 61°C, respectively, in PBS (pH 7.0)
   at a peptide concentration of 10 μM, L568A and W571R are predominantly
   unfolded at their IC[50]s (0.1 μM for L568A and 0.3 μM for W571R) under

t/data/good/10358003.utf8  view on Meta::CPAN

   not identical, positions are present in the GppNHp- or GppCp-bound
   complexes of Ras and Rac1 ([117]32, [118]40). A proton could be relayed
   from W^nuc to W^600 via O1G of the GTP γ-phosphate. This substituent
   does not otherwise participate in hydrogen bonds with the protein and
   corresponds to the thiol of GTPγS. The basicity of W^600may be enhanced
   by hydrogen bond formation with Glu^43, which is well conserved in Gα
   proteins with the exception of G[zα] where it is replaced with an
   asparagine residue. Glu^43 also forms a hydrogen-bonded ion pair with
   Arg^178. In this conformation, Arg^178 is restrained from interacting
   with the γ-phosphate of GTP. Transfer of a proton from W^nuc to W^600
   would tend to weaken this ion pair, releasing Arg^178 to stabilize the
   incipient pentacoordinate phosphoryl transition state. W^600 also
   blocks the side chain of Gln^204 from interacting with the
   pentacoordinate phosphate. Thus, until it diffuses from the active
   site, W^600 impedes the reorganization of the catalytic site that is
   required for transition state stabilization.

   Gln^204 is anchored in a noncatalytic conformation by hydrogen bonds to
   both W^nuc and Ser^206(Ser^206 is substituted by an Asp in G[αs],). In
   the ground state, Gln^204 could orient and perhaps activate W^nuc;
   however, to stabilize the transition state as represented by G protein

t/data/good/10358003.utf8  view on Meta::CPAN

   water and Ser^206. We suggest that Asn^128 of RGS4 displaces the side
   chain of Gln^204 from its “anti-catalytic” position in the ground
   state, freeing it to participate in stabilization of the transition
   state.

   Mutational analysis of RGS proteins supports this hypothesis. Mutation
   of Asn^131 in hRGSr (analogous to Asn^128 of RGS4) to either serine or
   glutamine resulted in a relatively small decrease in the k [cat] of
   G[tα] ([125]49). In addition, hRGSr in which Asn^131 was mutated to
   leucine or alanine also retains substantial stimulatory activity, and
   the loss of activity that was observed could be attributed to weakened
   binding of these mutants to G[tα]. Similar mutagenic studies have been
   performed with RGS4 ([126]50). Mutants of Asn^128analogous to those of
   hRGSr Asn^131 were modeled in the structure of the “RGS4·G[iα1]·GppNHp”
   complex. In all cases these residues were in steric conflict with
   Gln^204. These findings indicate that the bulk and binding of the
   residue at position 128 is important to the stimulatory activity of
   RGS4 although it is unlikely that it has a direct catalytic role in
   stimulation of GTPase activity ([127]49).

   The evidence presented is consistent with a self-inhibited or

t/data/good/10398587.utf8  view on Meta::CPAN

   in the wild-type protein. On the other hand, the mutation of Met159 to
   Thr seems to eliminate an unfavorable interaction between the C^ε atom
   of Met159 and one of the oxygen atoms of the diazaborine sulfonyl
   group, which may explain the increased sensitivity of this mutant to
   diazaborines. The third mutation, Phe203Leu, is located on the opposite
   side of the binding pocket relative to the other two mutations and
   leads to a sixfold increase in the MIC compared to the wild-type
   protein with a similar effect on the diazaborines. The side-chain of
   Phe203 seems to be important for the formation of the inner surface of
   the binding pocket and participates in hydrophobic interactions with
   both types of inhibitors. These interactions could be weakened in the
   mutant leading to a decreased binding affinity of either inhibitor.
   Very recently, mutations in InhA from M. smegmatisleading to resistance
   against triclosan have been identified [[122]McMurry et al 1999]. Two
   of the mutated residues, Met161 and Met103, superimpose or are in close
   proximity to Met159 in EnvM and might have the same effect as the
   Met159Thr mutation. The Ala124Val mutation in InhA is not located in
   the putative triclosan-binding site and its structural effect can not
   be explained with our current model.
     __________________________________________________________________

t/data/macroman/10358003.macroman  view on Meta::CPAN

   not identical, positions are present in the GppNHp- or GppCp-bound
   complexes of Ras and Rac1 ([117]32, [118]40). A proton could be relayed
   from W^nuc to W^600 via O1G of the GTP γ-phosphate. This substituent
   does not otherwise participate in hydrogen bonds with the protein and
   corresponds to the thiol of GTPγS. The basicity of W^600may be enhanced
   by hydrogen bond formation with Glu^43, which is well conserved in Gα
   proteins with the exception of G[zα] where it is replaced with an
   asparagine residue. Glu^43 also forms a hydrogen-bonded ion pair with
   Arg^178. In this conformation, Arg^178 is restrained from interacting
   with the γ-phosphate of GTP. Transfer of a proton from W^nuc to W^600
   would tend to weaken this ion pair, releasing Arg^178 to stabilize the
   incipient pentacoordinate phosphoryl transition state. W^600 also
   blocks the side chain of Gln^204 from interacting with the
   pentacoordinate phosphate. Thus, until it diffuses from the active
   site, W^600 impedes the reorganization of the catalytic site that is
   required for transition state stabilization.

   Gln^204 is anchored in a noncatalytic conformation by hydrogen bonds to
   both W^nuc and Ser^206(Ser^206 is substituted by an Asp in G[αs],). In
   the ground state, Gln^204 could orient and perhaps activate W^nuc;
   however, to stabilize the transition state as represented by G protein

t/data/macroman/10358003.macroman  view on Meta::CPAN

   water and Ser^206. We suggest that Asn^128 of RGS4 displaces the side
   chain of Gln^204 from its Òanti-catalyticÓ position in the ground
   state, freeing it to participate in stabilization of the transition
   state.

   Mutational analysis of RGS proteins supports this hypothesis. Mutation
   of Asn^131 in hRGSr (analogous to Asn^128 of RGS4) to either serine or
   glutamine resulted in a relatively small decrease in the k [cat] of
   G[tα] ([125]49). In addition, hRGSr in which Asn^131 was mutated to
   leucine or alanine also retains substantial stimulatory activity, and
   the loss of activity that was observed could be attributed to weakened
   binding of these mutants to G[tα]. Similar mutagenic studies have been
   performed with RGS4 ([126]50). Mutants of Asn^128analogous to those of
   hRGSr Asn^131 were modeled in the structure of the ÒRGS4áG[iα1]áGppNHpÓ
   complex. In all cases these residues were in steric conflict with
   Gln^204. These findings indicate that the bulk and binding of the
   residue at position 128 is important to the stimulatory activity of
   RGS4 although it is unlikely that it has a direct catalytic role in
   stimulation of GTPase activity ([127]49).

   The evidence presented is consistent with a self-inhibited or