Characterization of HIV-1 Reverse Transcriptase Substrate Specificity by Conformationally Sensitive Fluorescence
Characterization of HIV-1 Reverse Transcriptase Substrate Specificity by Conformationally Sensitive Fluorescence

Author: Matthew William Kellinger

Publisher:

Published: 2010

Total Pages: 390

ISBN-13:

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We have engineered a mutant of HIV Reverse Transcriptase that can be fluorescently labeled by covalent attachment of the environmentally sensitive fluorophore 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC). The result is a polymerase that is kinetically indistinguishable from the wild-type enzyme, but provides a signal to monitor changes in enzyme structure that result from conformational changes induced by substrate binding. Using this system, we have expanded the kinetic model governing nucleotide binding to include an enzymatic isomerization following initial nucleotide binding. In doing so, we define the role of induced-fit in nucleotide specificity and mismatch discrimination. Additionally, we have characterized the kinetics governing the specificity and discrimination of several widely administered Nucleotide Reverse Transcriptase Inhibitors (NRTI's) used to combat HIV infection including 3TC (Lamivudine), FTC (Emtricitabine), and AZT (Zidovudine) for the wild-type polymerase and mutants with clinical resistance to these compounds. Our findings resolve the apparent tighter binding of these inhibitor compounds compared to the correct nucleotide by showing that the affinity for the correct nucleotide is stronger than the inhibitors. The apparent weaker binding of the correct nucleotide is a result of a incomplete interpretation of binding data that fails to account for the importance of the reverse rate of the conformational change. The apparent Kd (Kd, app) measurements for correct nucleotide estimates Km rather than Kd because nucleotide binding does not reach equilibrium. The conformationally sensitive enzyme has also been used to characterize the kinetics governing DNA association. We show that DNA binding is governed by a two-step process where a fast initial association is followed by a second, slow isomerization that is off the pathway for nucleotide binding and incorporation. Finally, we have implemented single molecule techniques using fluorophore labeled nucleotides to study the effects of AZT incorporation on the DNA translocation dynamics of the polymerase. We find that primer termination with AZT results in DNA that fails to translocate, therefore occluding the next nucleotide from binding. This shift in translocation equilibrium exposes the newly formed phosphodiester bond to ATP- or pyrophosphate-mediated AZT excision; thereby rescuing productive polymerization. This finding represents the first kinetic measurement of DNA translocation by a polymerase.

Characterization of HIV-1 Reverse Transcriptase Drug Resistance Connection Subdomain Mutation N348I
Characterization of HIV-1 Reverse Transcriptase Drug Resistance Connection Subdomain Mutation N348I

Author: Matthew M. Schuckmann

Publisher:

Published: 2011

Total Pages: 98

ISBN-13:

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Connection subdomain mutations are a recently discovered class of reverse transcriptase (RT) drug resistance mutations which are positioned at some distance from previously described resistance mutations. The work presented in this thesis investigates drug resistance mechanisms conferred by HIV-1 RT connection subdomain mutation N348I. N348I was chosen as a representative connection subdomain mutation due to its clinical relevance to resistance against drugs belonging to both classes of HIV-1 RT inhibitors: the NRTIs and NNRTIs. N348I is the first, and currently only, clinically relevant HIV-1 RT single amino acid substitution mutation known to confer cross-resistance to drugs from both classes of RT inhibitors. I describe here a mechanism of HIV-1 RT N348I in vitro resistance against inhibition by the NNRTI nevirapine (NVP), where I show the mutant enzyme exhibits a decreased affinity towards inhibitor binding. Using pre-steady state kinetics techniques, I further show in detail how the N348I mutation on either of the heterodimer enzyme's subunits affects enzymatic activities. Interestingly, the mutation on either subunit decreases the rate of catalytic turnover for nucleotide incorporation reactions. The N348I mutant enzyme also displays an altered RNAse H activity, and I demonstrate that the N348I mutation on the p51 subunit provides the major contribution towards this altered activity. I also investigated RT N348I in vitro susceptibility to ddATP, the active form of the NRTI prodrug didanosine (ddI). Multiple mechanisms of NRTI resistance were studied, but significant levels of in vitro resistance to ddATP were not detected despite the fact that the N34I mutant virus has been shown to be resistant to ddI. Here I found that the N348I mutation does not negatively impact the steady-state kinetics of single nucleotide incorporation reactions, or the affinity for nucleotide substrate.

Kinetic Studies of HIV-1 Reverse Transcriptase Nucleotide Selectivity, Drug Resistance and RNase H Activity
Kinetic Studies of HIV-1 Reverse Transcriptase Nucleotide Selectivity, Drug Resistance and RNase H Activity

Author: An Li (Ph. D. in biochemistry)

Publisher:

Published: 2016

Total Pages: 456

ISBN-13:

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Mechanisms of nucleotide selectivity and drug resistance by HIV-1 Reverse Transcriptase (HIVRT) were examined using rapid kinetic methods and global data fitting. Thymidine analog resistance mutations provide only two-fold discrimination against the incorporation of AZT-triphosphate; therefore, it is generally believed that resistance arises from nucleotide excision, where ATP reacts with the 3’ terminal base to produce a dinucleotide tetraphosphate and a 3’-OH terminated primer capable of subsequent extension. Single turnover kinetic analysis with global data fitting revealed the intrinsic rate and equilibrium constants governing each step leading to resistance to chain terminators. Our data suggested the net resistance to AZT arises from nearly equal contributions involving discrimination during incorporation (2x), enhanced ATP-dependent excision (2-5x), reduced binding of the next correct nucleotide (2.6-5x) and more favored binding of the DNA primer in the N-site (5-20x). Chemistry is generally the only rate-limiting step for product formation during DNA polymerization with a DNA template, but with an RNA template we show that pyrophosphate (PPi) release was rate-limiting. Due to the slow PPi release step, the rate of reversal of chemistry could also be determined affording the first measurement of the equilibrium constants governing polymerization and the first complete free energy profile for HIVRT. Although PPi release is rate-limiting, nucleotide binding remains as the specificity-determining step. However, PPi release becomes exceedingly slow following mis-incorporation and thereby contributes to the specificity constant. Our data demonstrate that the fidelity of HIVRT has been underestimated by >20-fold in the past 20 years since the slow PPi release has been overlooked. The rate-limiting PPi release allows synchronization and coordination of the polymerase and RNase-H activities. Studies were undertaken to examine proposed coordination between the polymerase and RNase-H activities. Direct, simultaneous measurement of the two activities established a mechanism by which polymerization and RNase-H activities are coordinated by working independently at comparable rates, but with different efficiencies. In contrast to polymerization, RNase-H requires ~4–6 base pairs extending beyond the active site for optimal reactivity, providing fast but infrequent cleavage events. Consequently, the polymerase and RNase-H activities are seamlessly coordinated without any direct communication between the two sites.

Unfolding the HIV-1 Reverse Transcriptase RNase H Domain - how to Lose a Molecular Tug-of-war
Unfolding the HIV-1 Reverse Transcriptase RNase H Domain - how to Lose a Molecular Tug-of-war

Author:

Publisher:

Published: 2016

Total Pages: 13

ISBN-13:

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Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of the isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.