Mandy E. Blackburn

Drug Pressure Selected Mutations in HIV-1 Protease Alter Flap Conformations

The flap conformations of two drug-resistant HIV-1 protease constructs were characterized by molecular dynamic (MD) simulations and distance measurements with pulsed electron paramagnetic resonance (EPR) spectroscopy. MD simulations accurately regenerate the experimentally determined distance profiles and provide structural interpretations of the EPR data. The combined analyses show that the average conformation of the flaps, the range of flap opening and closing, and the flexibility of the flaps differ markedly in HIV-1PR as multiple mutations arise in response to antiviral therapy, providing structural insights into the mechanism of inhibitor resistance.

Monitoring Inhibitor-Induced Conformational Population Shifts in HIV-1 Protease by Pulsed EPR Spectroscopy

Double electron-electron resonance (DEER), a pulsed electron paramagnetic resonance (EPR) spectroscopy technique, was utilized to characterize conformational population shifts in HIV-1 protease (HIV-1PR) upon interaction with various inhibitors. Distances between spin-labeled sites in the flap region of HIV-1PR were determined, and detailed analyses provide population percentages for the ensemble flap conformations upon interaction with inhibitor or substrate. Comparisons are made between the percentage of the closed conformer seen with DEER and enzymatic inhibition constants, thermodynamic dissociation constants, and the number of hydrogen bonds identified in crystallographic complexes.

Solute effects on spin labels at an aqueous-exposed site in the flap region of HIV-1 protease.

The effects of solutes on spin-label mobility and protein conformation have been investigated with X-band continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy for spin labels attached to an aqueous-exposed site in the beta-hairpin flap region of HIV-1 protease. Specifically, we examined the effects of glycerol, sucrose, PEG3000, and Ficoll400 for four commonly used nitroxide spin labels and found that the largest perturbations to the EPR line shapes occur for solutions containing PEG3000 and glycerol. From comparisons of the spectral line shapes and distance distribution profiles of spin-labeled HIV-1 protease with and without inhibitor, it was concluded that solutes such as glycerol and PEG3000 alter the line shapes of the spin label in the beta-hairpin flaps of HIV-1 PR by modulation of spin-label mobility through changes in preferential interactions with the solutes. It is noteworthy that the high osmolality of the 40% glycerol solution did not alter the conformation of the flaps as determined from pulsed EPR distance measurements.

Inhibitor-induced Conformational Shifts and Ligand Exchange Dynamics for HIV-1 Protease Measured by Pulsed EPR and NMR Spectroscopy

Double electron-electron resonance (DEER) spectroscopy was utilized to investigate shifts in conformational sampling induced by nine FDA-approved protease inhibitors (PIs) and a nonhydrolyzable substrate mimic for human immunodeficiency virus type 1 protease (HIV-1 PR) subtype B, subtype C, and CRF_01 A/E. The ligand-bound subtype C protease has broader DEER distance profiles, but trends for inhibitor-induced conformational shifts are comparable to those previously reported for subtype B. Ritonavir, one of the strong-binding inhibitors for subtypes B and C, induces less of the closed conformation in CRF_01 A/E. (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra were acquired for each protease construct titrated with the same set of inhibitors. NMR (1)H-(15)N HSQC titration data show that inhibitor residence time in the protein binding pocket, inferred from resonance exchange broadening, shifting or splitting correlates with the degree of ligand-induced flap closure measured by DEER spectroscopy. These parallel results show that the ligand-induced conformational shifts resulting from protein-ligand interactions characterized by DEER spectroscopy of HIV-1 PR obtained at the cryogenic temperature are consistent with more physiological solution protein-ligand interactions observed by solution NMR spectroscopy.

Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron‐electron resonance (DEER) technique. This unit focuses on EPR‐based distance measurements by site‐directed spin labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV‐1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance‐distribution profiles. Experimental considerations, sample preparation, and error analysis for artifact suppression are also outlined herein. Curr. Protoc. Protein Sci. 74:17.17.1‐17.17.29.

Correlating Conformational Shift Induction with Altered Inhibitor Potency in a Multidrug Resistant HIV‑1 Protease Variant

Inhibitor-induced conformational ensemble shifts in a multidrug resistant HIV-1 protease variant, MDR769, are characterized by site-directed spin labeling double electron-electron resonance spectroscopy. For MDR769 compared to the native enzyme, changes in inhibitor IC(50) values are related to a parameter defined as |ΔC|, which is the relative change in the inhibitor-induced shift to the closed state. Specifically, a linear correlation is found between |ΔC| and the magnitude of the change in IC(50), provided that inhibitor binding is not too weak. Moreover, inhibitors that exhibit MDR769 resistance no longer induce a strong shift to a closed conformational ensemble as seen previously in the native enzyme.

422. Dominant-Negative Interference in the Pahenu2 Mouse Model of PKU: Effectiveness of Vectors Expressing Either Modified Forms of Phenylalanine Hydroxylase (PAH) or Ribozymes Plus a Hardened PAH mRNA

The common human genetic disorder Phenylketonuria (PKU) is primarily caused by defects in the enzyme phenylalanine hydroxylase (PAH). An animal model for PKU, the BTBR Pahenu2 mouse, has a missense mutation (F263S) that inactivates PAH; the mouse exhibits classic PKU, with elevated blood Phe levels, cognitive deficiencies, and maternal PKU syndrome. Like the Pahenu2 mouse, a majority of human PKU patients also have missense mutations. We have corrected both the serum Phe levels and maternal PKU in Pahenu2 mice using recombinant AAV vectors containing the mouse PAH gene. Although successful, unusually high vector doses were needed to normalize serum Phe levels. More critically, there was a gradual loss of therapeutic effect 20|[ndash]|24 weeks after vector delivery in many animals, particularly females.