Lipid sensing apolipoprotein A-I for novel high -throughput lipidation assays

Apolipoprotein A-I (ApoA-I) is the primary protein component of high density lipoproteins (HDL). ApoA-I plays an important role in cholesterol metabolism by mediating the formation of nascent HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by the ATP-binding cassette A1 (ABCA1). Insufficient ABCA1 activity my lead to reduced HDL formation, reduced cholesterol efflux and the development of arteriosclerosis. The standard radioactive assay for measuring cholesterol transport to lipid-free ApoA-I has low through-put, poor dynamic range and fails to measure phospholipid transferred with cholesterol. We describe the development of two sensitive, non-radioactive high-throughput assays that report on the lipidation state of ApoA-I and may have applications for studying ABCA1 function and HDL metabolism: a homogenous assay based on the Time Resolved FRET (TR-FRET) and a discontinuous assay that uses the Epic. The TR-FRET assay employs a fluorescent ApoA-I where Cysteine is labeled with the FRET acceptor HiLyte-Fluor-647 and an N-terminal Biotin-AviTag is bound to the streptavidin-Terbium conjugate. When this ApoA-I was incorporated into recombinant HDL, TR-FRET decreased proportionally to the increase in the ratio of lipid to ApoA-I in agreement with the expansion of the surface area of lipids concomitant with the increase in separation of N-terminal and central regions of the protein and demonstrated that the HTRF assay was sensitive to the amount of lipid associated with ApoA-I. The Epic is a label-free platform that allows for the observation of direct biomolecular interactions via a resonant wavelength shift which is proportional to the mass bound to the surface. In the Epic assay, biotinylated ApoA-I was captured on streptavidin-coated sensor. The response was proportional to the amount of lipids associated with ApoA-I indicating that the assay could sense lipidation of ApoA-I.Peer reviewed: YesNRC publication: Ye

Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors

<div><p>Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.</p></div

HCV NS5B polymerase nonnucleoside inhibitors binding sites and NS5B constructs used in studies.

<p>(<b>A</b>) Thumb site I and thumb site II are located on the thumb domain (green); palm site I and palm site II are at the interface of the three domains, thumb, palm (blue) and fingers (red). GS-9669 inhibitor bound in the thumb site II pocket is shown in stick representation (grey, description of crystal structure of NS5B bound to thumb site II inhibitor GS-9669 will be published elsewhere). The active site is indicated by the cyan circle. The other main structural features shown are the C-terminal tail residues (magenta) which contact the β-loop (yellow). (<b>B</b>) 2D representation of domain structure of polymerase and C-terminal truncation sites Δ21, Δ39, Δ47, Δ55, as well as the β-loop deletion mutant Δ21-Δ8 (deleted residues shown in yellow) and LWF triple A mutant F550A/W551A/L553A. Δ55 is a tag free construct and all others contain C6-His. (<b>C</b>) Location of the mutations relative to the tertiary protein structure. (<b>D</b>) Close-up view of interface between LWF motif (magenta, stick representation) and β-loop (yellow) which is dominated by hydrophobic contacts on the surface of the protein.</p

Equilibrium dissociation constants (K_D) for binding of NNIs to Δ21 and fold-shifts in K_D for association to Δ55 and Δ21-Δ8 determined by SPR.

<p>^a Sensorgrams for the binding of NNIs to Δ21, Δ55 and Δ21-Δ8 along with a table of equilibrium and kinetic parameters of interaction are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084808#pone.0084808.s002" target="_blank">Figure S2</a> and Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084808#pone.0084808.s006" target="_blank">File S1</a>, respectively.</p><p>^b Fold shift in K_D for association of NNIs to Δ55 and Δ21-Δ8 was calculated relative to the K_D determined for binding towards Δ21.</p><p>^c Where K_D values could not be obtained either due to complex binding kinetics (Palm Site II inhibitor binding to Δ55 and Δ21-Δ8) or super-stoichiometric binding (Palm Site I inhibitor binding to Δ21-Δ8), numerical values have been replaced by n/a (not applicable).</p

IC₅₀ values for inhibition of RdRp activity of NS5B by Nuc (3′-d′CTP) and NNIs.

<p>Numbers represent mean and standard deviation of IC₅₀ values determined for each inhibitor against the set of NS5B constructs. Heatmap is colored according to IC₅₀ fold shift relative to Δ21 to show changes in inhibition profile for given NNI across NS5B mutant constructs. Thumb site II inhibitors begin to show significant loss of inhibitory potency as interactions between β-loop and C-terminal residues are disrupted by mutations in the interface (Δ21-AAA, truncations past Δ39 and Δ21-Δ39 mutations). Palm site I inhibitor is affected as well, which is explained by disruption of the inhibitor's interaction with the β-loop and C-terminal residues. Inhibition by thumb site I remains for the most part unaffected by NS5B mutations whereas inhibition by palm site II NNI is reduced due to decrease in binding to NS5B with truncated β-loop and/or C-terminal.</p