A 2-Hydroxyisoquinoline-1,3-Dione Active-Site RNase H Inhibitor Binds in Multiple Modes to HIV-1 Reverse Transcriptase.

The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC50] = 2.6 µM) and RNH functions (IC50 = 0.65 µM) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.

Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain.

This article communicates our study to elucidate the molecular determinants of weak Mg2+ interaction with the ribonuclease H (RNH) domain of HIV-1 reverse transcriptase in solution. As the interaction is weak (a ligand-dissociation constant >1 mM), nonspecific Mg2+ interaction with the protein or interaction of the protein with other solutes that are present in the buffer solution can confound the observed Mg2+-titration data. To investigate these indirect effects, we monitored changes in the chemical shifts of backbone amides of RNH by recording NMR 1H-15N heteronuclear single-quantum coherence spectra upon titration of Mg2+ into an RNH solution. We performed the titration under three different conditions: (1) in the absence of NaCl, (2) in the presence of 50 mM NaCl, and (3) at a constant 160 mM Cl- concentration. Careful analysis of these three sets of titration data, along with molecular dynamics simulation data of RNH with Na+ and Cl- ions, demonstrates two characteristic phenomena distinct from the specific Mg2+ interaction with the active site: (1) weak interaction of Mg2+, as a salt, with the substrate-handle region of the protein and (2) overall apparent lower Mg2+ affinity in the absence of NaCl compared to that in the presence of 50 mM NaCl. A possible explanation may be that the titrated MgCl2 is consumed as a salt and interacts with RNH in the absence of NaCl. In addition, our data suggest that Na+ increases the kinetic rate of the specific Mg2+ interaction at the active site of RNH. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein.

A combined NMR and computational approach to investigate peptide binding to a designed Armadillo repeat protein.

The specific recognition of peptide sequences by proteins plays an important role both in biology and in diagnostic applications. Here we characterize the relatively weak binding of the peptide neurotensin (NT) to the previously developed Armadillo repeat protein VG_328 by a multidisciplinary approach based on solution NMR spectroscopy, mutational studies, and molecular dynamics (MD) simulations, totaling 20µs for all MD runs. We describe assignment challenges arising from the repetitive nature of the protein sequence, and we present novel approaches to address them. Partial assignments obtained for VG_328 in combination with chemical shift perturbations allowed us to identify the repeats not involved in binding. Their subsequent elimination resulted in a reduced-size binder with very similar affinity for NT, for which near-complete backbone assignments were achieved. A binding mode suggested by automatic docking and further validated by explicit solvent MD simulations is consistent with paramagnetic relaxation enhancement data collected using spin-labeled NT. Favorable intermolecular interactions are observed in the MD simulations for the residues that were previously shown to contribute to binding in an Ala scan of NT. We further characterized the role of residues within the N-cap for protein stability and peptide binding. Our multidisciplinary approach demonstrates that an initial low-resolution picture for a low-micromolar-peptide binder can be refined through the combination of NMR, protein design, docking, and MD simulations to establish its binding mode, even in the absence of crystallographic data, thereby providing valuable information for further design.

Spontaneous self-assembly of engineered armadillo repeat protein fragments into a folded structure.

Repeat proteins are built of modules, each of which constitutes a structural motif. We have investigated whether fragments of a designed consensus armadillo repeat protein (ArmRP) recognize each other. We examined a split ArmRP consisting of an N-capping repeat (denoted Y), three internal repeats (M), and a C-capping repeat (A). We demonstrate that the C-terminal MA fragment adopts a fold similar to the corresponding part of the entire protein. In contrast, the N-terminal YM2 fragment constitutes a molten globule. The two fragments form a 1:1 YM2:MA complex with a nanomolar dissociation constant essentially identical to the crystal structure of the continuous YM3A protein. Molecular dynamics simulations show that the complex is structurally stable over a 1 µs timescale and reveal the importance of hydrophobic contacts across the interface. We propose that the existence of a stable complex recapitulates possible intermediates in the early evolution of these repeat proteins.

The p66 immature precursor of HIV-1 reverse transcriptase.

In contrast to the wealth of structural data available for the mature p66/p51 heterodimeric human immunodeficiency virus type 1 reverse transcriptase (RT), the structure of the homodimeric p66 precursor remains unknown. In all X-ray structures of mature RT, free or complexed, the processing site in the p66 subunit, for generating the p51 subunit, is sequestered into a ß-strand within the folded ribonuclease H (RNH) domain and is not readily accessible to proteolysis, rendering it difficult to propose a simple and straightforward mechanism of the maturation step. Here, we investigated, by solution NMR, the conformation of the RT p66 homodimer. Our data demonstrate that the RNH and Thumb domains in the p66 homodimer are folded and possess conformations very similar to those in mature RT. This finding suggests that maturation models which invoke a complete or predominantly unfolded RNH domain are unlikely. The present study lays the foundation for further in-depth mechanistic investigations at the atomic level.

Structural basis of the allosteric inhibitor interaction on the HIV-1 reverse transcriptase RNase H domain.

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 Mg(2+) , 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 et al. (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.

Human plasminogen kringle 3: solution structure, functional insights, phylogenetic landscape.

Human plasminogen kringle 3 (hPgn K3) domain contains most elements of the canonical lysine-binding site (LBS) found in other Pgn kringles. However, it does not exhibit affinity for either lysine or structurally related zwitterionic ligands. It has been shown that lysine-binding activity can be engineered via a Lys57 --> Asp mutation [Burgin, J., and Schaller, J. (2009) Cell. Mol. Life Sci. 55, 135]. Using a recombinant construct expressed in Escherichia coli, the three-dimensional solution structure of hPgn K3 was determined via NMR spectroscopy [heavy atom averaged rmsd = 0.35 +/- 0.07 A (backbone) and 0.75 +/- 0.12 A (all)]. The (1)H/(15)N heteronuclear single-quantum correlated (HSQC) spectra for both wild-type K3 and mutated [r(K57D)K3] structures are essentially identical, implying that the two structures are effectively isomorphous. The affinity of r(K57D)K3 for the lysine analogue trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) was investigated from ligand-induced NMR chemical shift perturbations, which enabled for mapping the binding site on the mutated domain surface. The equilibrium association constant, K(a), was determined to be approximately 5.23 +/- 0.03 mM(-1). Homology modeling combined with in silico docking of lysine-like zwitterionic ligands via AutoDock 4.0 supports functionality of the engineered (K57D)K3 LBS, whose electrostatic focal centers are defined by the Arg36/Arg71 cationic and Asp55/Asp57 anionic pairs. Comparison of K3-type sequences from different vertebrates, including kringles from hedgehog apolipoprotein(a) [Apo(a)] and Apo(a)-related (Arp) sequences, reveals that Lys57 is confined to the hPgn variant. Based on the likely phylogeny and ligand affinities of the homologous domains, it is suggested that the hPgn K3 is unique in that all other K3-type domains, including hedgehog Apo(a) and all Arp domains, except K3(1), are predicted to variously exhibit lysine-binding capability. In Arp K3(1) an Arg residue fills site 72, replacing the key aromatic residue found in other kringles, thus interfering with a requisite kringle-ligand hydrophobic interaction.