ABSTRACT
Application of protein engineering strategies to the redesign of the active site of subtilisin has successfully generated an efficient peptide ligase, subtiligase. The novel enzyme subtiligase has been shown to have many uses, from the total synthesis of RNase A to the semisynthesis of a variety of other proteins. Although the enzyme is in an early stage of development, it shows great promise. Subtiligase will certainly be a useful and important addition to the available strategies for the synthesis of proteins via segment condensation.
Subject(s)
Peptide Synthases , Proteins/chemical synthesis , Subtilisins , Amino Acid Substitution , Proteins/chemistryABSTRACT
Linear peptides derived from the membrane proximal region of the gp41 ectodomain are effective inhibitors of HIV type 1 (HIV-1)-mediated fusion events. These inhibitory peptides lack structure in solution, rendering mechanistic interpretation of their activity difficult. Using structurally constrained analogs of these molecules, we demonstrate that the peptides inhibit infectivity by adopting a helical conformation. Moreover, we show that a specific face of the helix must be exposed to block viral infectivity. Recent crystal structures show that the region of gp41 corresponding to the inhibitory peptides is helical and uses the analogous face to pack against a groove formed by an N-terminal coiled-coil trimer. Our results provide a direct link between the inhibition of HIV-1 infectivity by these peptides and the x-ray structures, and suggest that the conformation of gp41 observed by crystallography represents the fusogenic state. Other agents that block HIV-1 infectivity by binding to this groove may hold promise for the treatment of AIDS.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Envelope Protein gp41/pharmacology , HIV-1/drug effects , HIV-1/pathogenicity , Peptide Fragments/pharmacology , Amino Acid Sequence , Anti-HIV Agents/chemistry , Circular Dichroism , Crystallography, X-Ray , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/genetics , HIV Infections/drug therapy , HIV-1/genetics , Humans , In Vitro Techniques , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/virology , Membrane Fusion/drug effects , Membrane Fusion/physiology , Models, Biological , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Structure, Secondary , Structure-Activity Relationship , Virulence/drug effectsABSTRACT
Staphylococcal nuclease is an enzyme with enormous catalytic power, accelerating phosphodiester bond hydrolysis by a factor of 10(16) over the spontaneous rate. The mechanistic basis for this rate acceleration was investigated by substitution of the active site residues Glu43, Arg35, and Arg87 with unnatural amino acid analogs. Two Glu43 mutants, one containing the nitro analog of glutamate and the other containing homoglutamate, retained high catalytic activity at pH 9.9, but were less active than the wild-type enzyme at lower pH values. The x-ray crystal structure of the homoglutamate mutant revealed that the carboxylate side chain of this residue occupies a position and orientation similar to that of Glu43 in the wild-type enzyme. The increase in steric bulk is accommodated by a backbone shift and altered torsion angles. The nitro and the homoglutamate mutants display similar pH versus rate profiles, which differ from that of the wild-type enzyme. Taken together, these studies suggest that Glu43 may not act as a general base, as previously thought, but may play a more complex structural role during catalysis.
