Opposite allosteric mechanisms in TetR and CAP.

Regulation of the DNA binding affinity of an oligomeric protein can be considered to consist of an intrinsic component, in which the affinity of an individual DNA-binding domain is modulated in response to effector binding, and an extrinsic component, in which the relative position of the protein's two DNA-binding domains are altered so that they can or cannot contact both half-site operators simultaneously. We demonstrated directly that the TetR repressor utilizes an extrinsic mechanism and CAP, the catabolite activator protein, utilizes an intrinsic mechanism.

Impact of the enfuvirtide resistance mutation N43D and the associated baseline polymorphism E137K on peptide sensitivity and six-helix bundle structure.

Enfuvirtide (ENF), the first human immunodeficiency virus type 1 (HIV-1) fusion inhibitor approved for clinical use, acts by binding to gp41 heptad repeat 1 (HR1) and preventing its interaction with the viral HR2 region. Treatment-emergent resistance to ENF has been mapped to residues within HR1, and these mutations decrease its susceptibility to ENF and may reduce viral fitness and pathogenesis, although the mechanism for these effects is not clear. N43D, a common ENF resistance mutation, was found in in vitro assays to cause a 5-50-fold in antiviral activity.

Design of an engineered N-terminal HIV-1 gp41 trimer with enhanced stability and potency.

HIV fusion is mediated by a conformational transition in which the C-terminal region (HR2) of gp41 interacts with the N-terminal region (HR1) to form a six-helix bundle. Peptides derived from the HR1 form a well-characterized, trimeric coiled-coil bundle in the presence of HR2 peptides, but there is little structural information on the isolated HR1 trimer. Using protein design, we have designed synthetic HR1 peptides that form soluble, thermostable HR1 trimers.

Design of helical, oligomeric HIV-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus.

Enfuvirtide (ENF), the first approved fusion inhibitor (FI) for HIV, is a 36-aa peptide that acts by binding to the heptad repeat 1 (HR1) region of gp41 and preventing the interaction of the HR1 and HR2 domains, which is required for virus-cell fusion. Treatment-acquired resistance to ENF highlights the need to create FI therapeutics with activity against ENF-resistant viruses and improved durability. Using rational design, we have made a series of oligomeric HR2 peptides with increased helical structure and with exceptionally high HR1/HR2 bundle stability.