Steve Hagen for access to his circular dichroism spectrometer, Dr. parameters. Mutations that stabilize a closed-like conformation correlate with enzymes of lowered activity and with higher affinity for inhibitors, which is usually corroborated by a further increase in the fractional occupancy of the closed state upon addition of inhibitor or substrate-mimic. Cross resistance is found to correlate with combinations of mutations that increase the populace of the open-like conformations at the expense of the closed-like state while retaining native-like occupancy of the semi-open populace. These correlations suggest that at least three says are required in the conformational sampling model to establish the emergence of drug-resistance in HIV-1 PR. More importantly, these results shed light on a possible mechanism whereby mutations combine to impart drug resistance while maintaining catalytic activity. The inhibition of enzymes through small molecules that compete with a substrate for the active site is usually a common clinical method for effective treatment of disease. AZD4573 However, the development of drug resistance in rapidly proliferating cells or pathogenic organisms through selective pressure, where the incorporation of random genetic mutations prospects to the generation of an enzyme with amino acid substitutions, renders the drug molecule less effective.1, 2 The emergence of main mutations often results in a change in an amino acid whose structure interacts less favorably with the inhibitor by introducing steric hindrance or by removing essential molecular interactions, such as charge stabilization or van der Waals AZD4573 contacts.1, 2 In the case of competitive inhibition, these main mutations also tend to alter the interactions of the enzyme with the substrate or product, thus negatively impacting enzyme efficiency and compromising fitness. The observed pattern of continued evolutionary mutations shows that secondary mutations (also referred to as compensatory mutations) AZD4573 recover fitness, while maintaining drug-resistance.3 An understanding of the molecular mechanism by which the accumulation of mutations impart these effects is important for the rational design of future generations of drugs.4 In some enzymes, a clear rationale is illuminated through structural changes induced by the pattern of mutations, while in others, indirect effects such as changes in enzyme dynamics or protein-ligand dynamics are evoked.2 Here, we elucidate how shifts in equilibrium conformational sampling can act as an AZD4573 indirect mechanism by which drug-pressure accumulated mutations alter enzyme kinetics and inhibitor susceptibility. Some proteins are known to sample multiple conformations, where conversation with a ligand or an inhibitor just shifts the population to an already accessible state.5-7 Specifically, our work focuses on mutation induced changes in the fractional occupancy of the conformational sampling ensemble as an indirect mechanism for drug-resistance in HIV-1 protease (HIV-1 PR). HIV-1 PR, an aspartic protease that processes the and viral polypeptides, is an attractive target for AIDS antiviral therapy8 because of its central role in viral maturation.9 Protease inhibitors (PIs) that target HIV-1 PR prevent the formation of infectious virions by blocking viral replication. PIs bind in the protease active site where the two flexible -hairpin turns (aka codon-optimized subtype B HIV-1 PR (DNA 2.0) was cloned into pET-23a vector (Novagen) under the control of a T7 promoter. Seven stabilized (Q7K, L33I, L63I) and inactive (D25N) constructs (Bsi) with designed labeling sites (K55C) were made using the QuikChange site-directed mutagenesis kit by Stratagene: D30N, M36I, A71V, D30N/M36I, D30N/A71V, M36I/A71V, and D30N/M36I/A71V. Note that this procedure renders all mutations symmetrically applied to both subunits of the homodimer. Moreover, natural cysteine residues (C67 and C95) in these constructs are mutated to alanine to prevent non-specific disulfide bridge formation and to make sure site-specific labeling at C55. The C67A and C95A mutations have been utilized in numerous X-ray crystallography studies and do not alter kinetic parameters, protein stability or dimer dissociation compared to the unmutated sequence.47, 48 The fidelity of HIV-1 PR DNA sequences was confirmed by Sanger DNA sequencing (ICBR Genomics Facility, AZD4573 UF). Protein Expression, Purification, and Spin Labeling Protein expression, purification, and spin-labeling were carried out as previously explained20 with the following modification: the inclusion body resuspension PRSS10 buffer pH utilized for anion exchange depends upon the isoelectric point (pI) of a given construct. The buffer pH utilized for wild-type (WT) subtype B (Bsi), D30N, M36I, A71V, D30N/M36I, D30N/A71V, M36I/A71V, and D30N/M36I/A71V, respectively are as follows: 8.85, 9.00, 8.82, 8.80, 8.95, 8.98, 8.85, and 8.88. Methanethiosulfonate (MTSL) spin-label (Toronto Research Chemicals) was added in three to four-fold molar extra to 8 M HIV-1 PR homodimer in 10 mM Tris-HCl, pH 6.9, and the reaction is allowed to proceed in the dark for 12 hours at 25 C, 150 rpm. Homogeneous spin-labeling was verified via electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), as shown in the.
Steve Hagen for access to his circular dichroism spectrometer, Dr
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