Characterization of a folding intermediate from HIV‐1 ribonuclease H

Kern, Günther; Handel, Tracy M.; Marqusee, Susan

doi:10.1002/pro.5560071014

Cited by 26 publications

(28 citation statements)

References 60 publications

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“…The dimer that we observed in solution most likely results from domain swapping at the helix region (Figure 3). Previously, the helix region was observed to be unstable in the folding intermediate of the RNH fragment (57), suggesting that the helix can be swapped during the folding process. Indeed, unfolding of the WT RNH monomer in 6 M urea followed by re-folding at very high protein concentration in NMR buffer (> 10 mM) yielded almost exclusively Fraction #1 (data not shown), in line with earlier observations for other domain swapped dimers (52).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of the Allosteric Inhibitor Interaction on the HIV‐1 Reverse Transcriptase RNase H Domain

et al. 2012

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HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical-shift changes of RNH with those of RNH dimer, in the presence and absence of Mg2+, were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the “substrate-handle region”). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong, el (2011) Chem. Biol. Drug Des. 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.

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Section: Discussionmentioning

confidence: 99%

Structural Basis of the Allosteric Inhibitor Interaction on the HIV‐1 Reverse Transcriptase RNase H Domain

et al. 2012

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“…Such a procedure assists conformational sampling and ensures that an enhanced representation of the solution state free energies is generated (29). Because the present restrained samplings enforce NMR observables (i.e., H/D exchange protection factors) that reflect fluctuations occurring in the millisecond timescale (and beyond) (21)(22)(23)(24), the resulting structural ensembles are particularly valuable for describing backbone dynamics relevant to the processes of protein folding and misfolding, which are those that affect the maintenance of protein solubility and are governed by events that typically occur in these timescales (34,35).…”

Section: Nmr Measurements Of Acpdro2mentioning

confidence: 99%

Experimental free energy surfaces reveal the mechanisms of maintenance of protein solubility

Simone

Dhulesia

Soldi

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The identification of the factors that enable normally folded proteins to remain in their soluble and functional states is crucial for a comprehensive understanding of any biological system. We have determined a series of energy landscapes of the acylphosphatase from Drosophila melanogaster under a variety of conditions by combining NMR measurements with restrained molecular dynamics simulations. We thus analyzed the differences in the structures, dynamics, and energy surfaces of the protein in its soluble state or in situations where it aggregates through conformational states that have native-like structure, folding stability, and enzymatic activity. The study identifies the nature of the energy barriers that under normal physiological conditions prevent the protein ensemble from populating dangerous aggregation-prone states. We found that such states, although similar to the native conformation, have altered surface charge distribution, alternative topologies of the β-sheet region, and modified solvent exposure of hydrophobic surfaces and aggregation-prone regions of the sequence. The identified barriers allow the protein to undergo functional dynamics while remaining soluble and without a significant risk of misfolding and aggregation into nonfunctional and potentially toxic species.protein misfolding | free energy barriers | avoidance of protein aggregation | molecular simulations

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“…They showed that the mutated form remained capable of adopting a folded structure similar to that of the isolated wild-type domain; however, a urea denaturation study indicated that it was 2 kcal/mol less stable than the longer construct. Given a previous report of the stability of the RH domain of 3.7 kcal/mol [66], the loss of the two N-terminal residues represents a destabilization of RH’ by >50%.…”

Section: Dimerization-induced Structural Changesmentioning

confidence: 99%

“…Multiple studies indicate that in the absence of ligands, the C-terminal helix E of RH is largely disordered, freeing as many as 22 residues on each subunit to act as linkers to an IN dimer [66,71,119,120,121]. Alternatively, although there are five disordered residues at the N-terminus of the p51 monomer (PDB ID: 4KSE, [16]), formation of a PR-RT linker would require additional unraveling of the N-termini.…”

Section: Maturation Within the Virionmentioning

confidence: 99%

Structural Maturation of HIV-1 Reverse Transcriptase—A Metamorphic Solution to Genomic Instability

London

2016

Viruses

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Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)—a critical enzyme of the viral life cycle—undergoes a complex maturation process, required so that a pair of p66 precursor proteins can develop conformationally along different pathways, one evolving to form active polymerase and ribonuclease H (RH) domains, while the second forms a non-functional polymerase and a proteolyzed RH domain. These parallel maturation pathways rely on the structural ambiguity of a metamorphic polymerase domain, for which the sequence–structure relationship is not unique. Recent nuclear magnetic resonance (NMR) studies utilizing selective labeling techniques, and structural characterization of the p66 monomer precursor have provided important insights into the details of this maturation pathway, revealing many aspects of the three major steps involved: (1) domain rearrangement; (2) dimerization; and (3) subunit-selective RH domain proteolysis. This review summarizes the major structural changes that occur during the maturation process. We also highlight how mutations, often viewed within the context of the mature RT heterodimer, can exert a major influence on maturation and dimerization. It is further suggested that several steps in the RT maturation pathway may provide attractive targets for drug development.

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Characterization of a folding intermediate from HIV‐1 ribonuclease H

Abstract: The RNase H domain from HIV-1 (HIV RNase H) encodes an essential retroviral activity.

Cited by 26 publications

References 60 publications

Structural Basis of the Allosteric Inhibitor Interaction on the HIV‐1 Reverse Transcriptase RNase H Domain

Structural Basis of the Allosteric Inhibitor Interaction on the HIV‐1 Reverse Transcriptase RNase H Domain

Experimental free energy surfaces reveal the mechanisms of maintenance of protein solubility

Structural Maturation of HIV-1 Reverse Transcriptase—A Metamorphic Solution to Genomic Instability

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